document_id
int32
185
2.68k
context
stringlengths
2.88k
70.8k
question
stringlengths
11
194
is_impossible
bool
1 class
id
int32
225
5.32k
answers
sequence
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
Whose was the first reported case?
false
4,193
{ "text": [ "a 60 year old man from Bisha in the KSA" ], "answer_start": [ 3211 ] }
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 are recommended when URT sampling is to be conducted?
false
4,245
{ "text": [ "an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash" ], "answer_start": [ 22719 ] }
1,582
Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .
What percentage of the patients were between 20 and 30 years old in this study?
false
5,199
{ "text": [ "52%" ], "answer_start": [ 9888 ] }
2,684
1918 Influenza: the Mother of All Pandemics Jeffery K. Taubenberger" and David M. Morens1- The “Spanish" influenza pandemic of 1918—1919, which caused :50 million deaths worldwide, remains an ominous warning to public health. Many questions about its origins, its unusual epidemiologic features, and the basis of its pathogenicity remain unanswered. The public health implications of the pandemic therefore remain in doubt even as we now grapple with the feared emergence of a pandemic caused by H5N1 or other virus. However, new information about the 1918 virus is emerging, for example, sequencing of the entire genome from archival autopsy tis- sues. But, the viral genome alone is unlikely to provide answers to some critical questions. Understanding the 1918 pandemic and its implications for future pandemics requires careful experimentation and in-depth historical analysis. ”Curiouser and curiouser/ ” criedAlice Lewis Carroll, Alice’s Adventures in Wonderland, 1865 An estimated one third of the world’s population (or z500 million persons) were infected and had clinical- ly apparent illnesses (1,2) during the 191871919 influenza pandemic. The disease was exceptionally severe. Case- fatality rates were >2.5%, compared to <0.1% in other influenza pandemics (3,4). Total deaths were estimated at z50 million (577) and were arguably as high as 100 mil- lion (7). The impact of this pandemic was not limited to 191871919. All influenza A pandemics since that time, and indeed almost all cases of influenza A worldwide (except- ing human infections from avian Viruses such as H5N1 and H7N7), have been caused by descendants of the 1918 Virus, including “drifted” H1N1 Viruses and reassorted H2N2 and H3N2 Viruses. The latter are composed of key genes from the 1918 Virus, updated by subsequently-incor— porated avian influenza genes that code for novel surface *Armed Forces Institute of Pathology, Rockville, Maryland, USA; and TNational Institutes of Health, Bethesda, Maryland, USA proteins, making the 1918 Virus indeed the “mother” of all pandemics. In 1918, the cause of human influenza and its links to avian and swine influenza were unknown. Despite clinical and epidemiologic similarities to influenza pandemics of 1889, 1847, and even earlier, many questioned whether such an explosively fatal disease could be influenza at all. That question did not begin to be resolved until the 1930s, when closely related influenza Viruses (now known to be H1N1 Viruses) were isolated, first from pigs and shortly thereafter from humans. Seroepidemiologic studies soon linked both of these viruses to the 1918 pandemic (8). Subsequent research indicates that descendants of the 1918 Virus still persists enzootically in pigs. They probably also circulated continuously in humans, undergoing gradual antigenic drift and causing annual epidemics, until the 1950s. With the appearance of a new H2N2 pandemic strain in 1957 (“Asian flu”), the direct H1N1 Viral descen- dants 0f the 1918 pandemic strain disappeared from human circulation entirely, although the related lineage persisted enzootically in pigs. But in 1977, human H1N1 Viruses suddenly “reemerged” from a laboratory freezer (9). They continue to circulate endemically and epidemically. Thus in 2006, 2 major descendant lineages of the 1918 H1N1 Virus, as well as 2 additional reassortant lineages, persist naturally: a human epidemic/endemic H1N1 line- age, a porcine enzootic H1N1 lineage (so-called classic swine flu), and the reassorted human H3N2 Virus lineage, which like the human H1N1 Virus, has led to a porcine H3N2 lineage. None of these Viral descendants, however, approaches the pathogenicity of the 1918 parent Virus. Apparently, the porcine H1N1 and H3N2 lineages uncom- monly infect humans, and the human H1N1 and H3N2 lin- eages have both been associated with substantially lower rates ofillness and death than the virus of 1918. In fact, cur- rent H1N1 death rates are even lower than those for H3N2 lineage strains (prevalent from 1968 until the present). H1N1 Viruses descended from the 1918 strain, as well as H3N2 Viruses, have now been cocirculating worldwide for 29 years and show little evidence of imminent extinction. Trying To Understand What Happened By the early 1990s, 75 years of research had failed to answer a most basic question about the 1918 pandemic: why was it so fatal? No Virus from 1918 had been isolated, but all of its apparent descendants caused substantially milder human disease. Moreover, examination of mortality data from the 1920s suggests that within a few years after 1918, influenza epidemics had settled into a pattern of annual epidemicity associated with strain drifting and sub- stantially lowered death rates. Did some critical Viral genet- ic event produce a 1918 Virus of remarkable pathogenicity and then another critical genetic event occur soon after the 1918 pandemic to produce an attenuated H1N1 Virus? In 1995, a scientific team identified archival influenza autopsy materials collected in the autumn of 1918 and began the slow process of sequencing small Viral RNA fragments to determine the genomic structure of the causative influenza Virus (10). These efforts have now determined the complete genomic sequence of 1 Virus and partial sequences from 4 others. The primary data from the above studies (11717) and a number of reviews covering different aspects of the 1918 pandemic have recently been published ([8720) and confirm that the 1918 Virus is the likely ancestor of all 4 of the human and swine H1N1 and H3N2 lineages, as well as the “extinct” H2N2 lineage. No known mutations correlated with high pathogenicity in other human or animal influenza Viruses have been found in the 1918 genome, but ongoing studies to map Virulence factors are yielding interesting results. The 1918 sequence data, however, leave unanswered questions about the ori- gin of the Virus (19) and about the epidemiology of the pandemic. When and Where Did the 1918 Influenza Pandemic Arise? Before and after 1918, most influenza pandemics developed in Asia and spread from there to the rest of the world. Confounding definite assignment of a geographic point of origin, the 1918 pandemic spread more or less simultaneously in 3 distinct waves during an z12-month period in 191871919, in Europe, Asia, and North America (the first wave was best described in the United States in March 1918). Historical and epidemiologic data are inade- quate to identify the geographic origin of the Virus (21), and recent phylogenetic analysis of the 1918 Viral genome does not place the Virus in any geographic context ([9). Although in 1918 influenza was not a nationally reportable disease and diagnostic criteria for influenza and pneumonia were vague, death rates from influenza and pneumonia in the United States had risen sharply in 1915 and 1916 because of a major respiratory disease epidemic beginning in December 1915 (22). Death rates then dipped slightly in 1917. The first pandemic influenza wave appeared in the spring of 1918, followed in rapid succes- sion by much more fatal second and third waves in the fall and winter of 191871919, respectively (Figure 1). Is it pos- sible that a poorly-adapted H1N1 Virus was already begin- ning to spread in 1915, causing some serious illnesses but not yet sufficiently fit to initiate a pandemic? Data consis- tent with this possibility were reported at the time from European military camps (23), but a counter argument is that if a strain with a new hemagglutinin (HA) was caus- ing enough illness to affect the US national death rates from pneumonia and influenza, it should have caused a pandemic sooner, and when it eventually did, in 1918, many people should have been immune or at least partial- ly immunoprotected. “Herald” events in 1915, 1916, and possibly even in early 1918, if they occurred, would be dif- ficult to identify. The 1918 influenza pandemic had another unique fea- ture, the simultaneous (or nearly simultaneous) infection of humans and swine. The Virus of the 1918 pandemic like- ly expressed an antigenically novel subtype to which most humans and swine were immunologically naive in 1918 (12,20). Recently published sequence and phylogenetic analyses suggest that the genes encoding the HA and neu- raminidase (NA) surface proteins of the 1918 Virus were derived from an avianlike influenza Virus shortly before the start of the pandemic and that the precursor Virus had not circulated widely in humans or swine in the few decades before (12,15, 24). More recent analyses of the other gene segments of the Virus also support this conclu- sion. Regression analyses of human and swine influenza sequences obtained from 1930 to the present place the ini- tial circulation of the 1918 precursor Virus in humans at approximately 191571918 (20). Thus, the precursor was probably not circulating widely in humans until shortly before 1918, nor did it appear to have jumped directly from any species of bird studied to date (19). In summary, its origin remains puzzling. Were the 3 Waves in 1918—1 919 Caused by the Same Virus? If So, How and Why? Historical records since the 16th century suggest that new influenza pandemics may appear at any time of year, not necessarily in the familiar annual winter patterns of interpandemic years, presumably because newly shifted influenza Viruses behave differently when they find a uni- versal or highly susceptible human population. Thereafter, confronted by the selection pressures of population immu- nity, these pandemic Viruses begin to drift genetically and eventually settle into a pattern of annual epidemic recur- rences caused by the drifted Virus variants. Figure 1. Three pandemic waves: weekly combined influenza and pneumonia mortality, United Kingdom, 1918—1919 (21). In the 1918-1919 pandemic, a first or spring wave began in March 1918 and spread unevenly through the United States, Europe, and possibly Asia over the next 6 months (Figure 1). Illness rates were high, but death rates in most locales were not appreciably above normal. A sec- ond or fall wave spread globally from September to November 1918 and was highly fatal. In many nations, a third wave occurred in early 1919 (21). Clinical similari- ties led contemporary observers to conclude initially that they were observing the same disease in the successive waves. The milder forms of illness in all 3 waves were identical and typical of influenza seen in the 1889 pandem- ic and in prior interpandemic years. In retrospect, even the rapid progressions from uncomplicated influenza infec- tions to fatal pneumonia, a hallmark of the 191871919 fall and winter waves, had been noted in the relatively few severe spring wave cases. The differences between the waves thus seemed to be primarily in the much higher fre- quency of complicated, severe, and fatal cases in the last 2 waves. But 3 extensive pandemic waves of influenza within 1 year, occurring in rapid succession, with only the briefest of quiescent intervals between them, was unprecedented. The occurrence, and to some extent the severity, of recur- rent annual outbreaks, are driven by Viral antigenic drift, with an antigenic variant Virus emerging to become domi- nant approximately every 2 to 3 years. Without such drift, circulating human influenza Viruses would presumably disappear once herd immunity had reached a critical threshold at which further Virus spread was sufficiently limited. The timing and spacing of influenza epidemics in interpandemic years have been subjects of speculation for decades. Factors believed to be responsible include partial herd immunity limiting Virus spread in all but the most favorable circumstances, which include lower environ- mental temperatures and human nasal temperatures (bene- ficial to thermolabile Viruses such as influenza), optimal humidity, increased crowding indoors, and imperfect ven- tilation due to closed windows and suboptimal airflow. However, such factors cannot explain the 3 pandemic waves of 1918-1919, which occurred in the spring-sum- mer, summer—fall, and winter (of the Northern Hemisphere), respectively. The first 2 waves occurred at a time of year normally unfavorable to influenza Virus spread. The second wave caused simultaneous outbreaks in the Northern and Southern Hemispheres from September to November. Furthermore, the interwave peri- ods were so brief as to be almost undetectable in some locales. Reconciling epidemiologically the steep drop in cases in the first and second waves with the sharp rises in cases of the second and third waves is difficult. Assuming even transient postinfection immunity, how could suscep- tible persons be too few to sustain transmission at 1 point, and yet enough to start a new explosive pandemic wave a few weeks later? Could the Virus have mutated profoundly and almost simultaneously around the world, in the short periods between the successive waves? Acquiring Viral drift sufficient to produce new influenza strains capable of escaping population immunity is believed to take years of global circulation, not weeks of local circulation. And hav- ing occurred, such mutated Viruses normally take months to spread around the world. At the beginning of other “off season” influenza pan- demics, successive distinct waves within a year have not been reported. The 1889 pandemic, for example, began in the late spring of 1889 and took several months to spread throughout the world, peaking in northern Europe and the United States late in 1889 or early in 1890. The second recurrence peaked in late spring 1891 (more than a year after the first pandemic appearance) and the third in early 1892 (21 ). As was true for the 1918 pandemic, the second 1891 recurrence produced of the most deaths. The 3 recur- rences in 1889-1892, however, were spread over >3 years, in contrast to 191871919, when the sequential waves seen in individual countries were typically compressed into z879 months. What gave the 1918 Virus the unprecedented ability to generate rapidly successive pandemic waves is unclear. Because the only 1918 pandemic Virus samples we have yet identified are from second-wave patients ([6), nothing can yet be said about whether the first (spring) wave, or for that matter, the third wave, represented circulation of the same Virus or variants of it. Data from 1918 suggest that persons infected in the second wave may have been pro- tected from influenza in the third wave. But the few data bearing on protection during the second and third waves after infection in the first wave are inconclusive and do lit- tle to resolve the question of whether the first wave was caused by the same Virus or whether major genetic evolu- tionary events were occurring even as the pandemic exploded and progressed. Only influenza RNAipositive human samples from before 1918, and from all 3 waves, can answer this question. What Was the Animal Host Origin of the Pandemic Virus? Viral sequence data now suggest that the entire 1918 Virus was novel to humans in, or shortly before, 1918, and that it thus was not a reassortant Virus produced from old existing strains that acquired 1 or more new genes, such as those causing the 1957 and 1968 pandemics. On the con- trary, the 1918 Virus appears to be an avianlike influenza Virus derived in toto from an unknown source (17,19), as its 8 genome segments are substantially different from contemporary avian influenza genes. Influenza Virus gene sequences from a number offixed specimens ofwild birds collected circa 1918 show little difference from avian Viruses isolated today, indicating that avian Viruses likely undergo little antigenic change in their natural hosts even over long periods (24,25). For example, the 1918 nucleoprotein (NP) gene sequence is similar to that ofviruses found in wild birds at the amino acid level but very divergent at the nucleotide level, which suggests considerable evolutionary distance between the sources of the 1918 NP and of currently sequenced NP genes in wild bird strains (13,19). One way of looking at the evolutionary distance of genes is to com- pare ratios of synonymous to nonsynonymous nucleotide substitutions. A synonymous substitution represents a silent change, a nucleotide change in a codon that does not result in an amino acid replacement. A nonsynonymous substitution is a nucleotide change in a codon that results in an amino acid replacement. Generally, a Viral gene sub- jected to immunologic drift pressure or adapting to a new host exhibits a greater percentage of nonsynonymous mutations, while a Virus under little selective pressure accumulates mainly synonymous changes. Since little or no selection pressure is exerted on synonymous changes, they are thought to reflect evolutionary distance. Because the 1918 gene segments have more synony- mous changes from known sequences of wild bird strains than expected, they are unlikely to have emerged directly from an avian influenza Virus similar to those that have been sequenced so far. This is especially apparent when one examines the differences at 4-fold degenerate codons, the subset of synonymous changes in which, at the third codon position, any of the 4 possible nucleotides can be substituted without changing the resulting amino acid. At the same time, the 1918 sequences have too few amino acid difierences from those of wild-bird strains to have spent many years adapting only in a human or swine intermedi- ate host. One possible explanation is that these unusual gene segments were acquired from a reservoir of influenza Virus that has not yet been identified or sampled. All of these findings beg the question: where did the 1918 Virus come from? In contrast to the genetic makeup of the 1918 pandem- ic Virus, the novel gene segments of the reassorted 1957 and 1968 pandemic Viruses all originated in Eurasian avian Viruses (26); both human Viruses arose by the same mech- anismireassortment of a Eurasian wild waterfowl strain with the previously circulating human H1N1 strain. Proving the hypothesis that the Virus responsible for the 1918 pandemic had a markedly different origin requires samples of human influenza strains circulating before 1918 and samples of influenza strains in the wild that more closely resemble the 1918 sequences. What Was the Biological Basis for 1918 Pandemic Virus Pathogenicity? Sequence analysis alone does not ofier clues to the pathogenicity of the 1918 Virus. A series of experiments are under way to model Virulence in Vitro and in animal models by using Viral constructs containing 1918 genes produced by reverse genetics. Influenza Virus infection requires binding of the HA protein to sialic acid receptors on host cell surface. The HA receptor-binding site configuration is different for those influenza Viruses adapted to infect birds and those adapted to infect humans. Influenza Virus strains adapted to birds preferentially bind sialic acid receptors with 01 (273) linked sugars (27729). Human-adapted influenza Viruses are thought to preferentially bind receptors with 01 (2%) link- ages. The switch from this avian receptor configuration requires of the Virus only 1 amino acid change (30), and the HAs of all 5 sequenced 1918 Viruses have this change, which suggests that it could be a critical step in human host adaptation. A second change that greatly augments Virus binding to the human receptor may also occur, but only 3 of5 1918 HA sequences have it (16). This means that at least 2 H1N1 receptor-binding vari- ants cocirculated in 1918: 1 with high—affinity binding to the human receptor and 1 with mixed-affinity binding to both avian and human receptors. No geographic or chrono- logic indication eXists to suggest that one of these variants was the precursor of the other, nor are there consistent dif- ferences between the case histories or histopathologic fea- tures of the 5 patients infected with them. Whether the Viruses were equally transmissible in 1918, whether they had identical patterns of replication in the respiratory tree, and whether one or both also circulated in the first and third pandemic waves, are unknown. In a series of in Vivo experiments, recombinant influen- za Viruses containing between 1 and 5 gene segments of the 1918 Virus have been produced. Those constructs bearing the 1918 HA and NA are all highly pathogenic in mice (31). Furthermore, expression microarray analysis performed on whole lung tissue of mice infected with the 1918 HA/NA recombinant showed increased upregulation of genes involved in apoptosis, tissue injury, and oxidative damage (32). These findings are unexpected because the Viruses with the 1918 genes had not been adapted to mice; control experiments in which mice were infected with modern human Viruses showed little disease and limited Viral replication. The lungs of animals infected with the 1918 HA/NA construct showed bronchial and alveolar epithelial necrosis and a marked inflammatory infiltrate, which suggests that the 1918 HA (and possibly the NA) contain Virulence factors for mice. The Viral genotypic basis of this pathogenicity is not yet mapped. Whether pathogenicity in mice effectively models pathogenicity in humans is unclear. The potential role of the other 1918 pro- teins, singularly and in combination, is also unknown. Experiments to map further the genetic basis of Virulence of the 1918 Virus in various animal models are planned. These experiments may help define the Viral component to the unusual pathogenicity of the 1918 Virus but cannot address whether specific host factors in 1918 accounted for unique influenza mortality patterns. Why Did the 1918 Virus Kill So Many Healthy Young Ad ults? The curve of influenza deaths by age at death has histor- ically, for at least 150 years, been U-shaped (Figure 2), exhibiting mortality peaks in the very young and the very old, with a comparatively low frequency of deaths at all ages in between. In contrast, age-specific death rates in the 1918 pandemic exhibited a distinct pattern that has not been documented before or since: a “W—shaped” curve, similar to the familiar U-shaped curve but with the addition of a third (middle) distinct peak of deaths in young adults z20410 years of age. Influenza and pneumonia death rates for those 1534 years of age in 191871919, for example, were 20 times higher than in previous years (35). Overall, near- ly half of the influenza—related deaths in the 1918 pandem- ic were in young adults 20410 years of age, a phenomenon unique to that pandemic year. The 1918 pandemic is also unique among influenza pandemics in that absolute risk of influenza death was higher in those <65 years of age than in those >65; persons <65 years of age accounted for >99% of all excess influenza—related deaths in 191871919. In com- parison, the <65-year age group accounted for 36% of all excess influenza—related deaths in the 1957 H2N2 pandem- ic and 48% in the 1968 H3N2 pandemic (33). A sharper perspective emerges when 1918 age-specific influenza morbidity rates (21) are used to adj ust the W- shaped mortality curve (Figure 3, panels, A, B, and C [35,37]). Persons 65 years of age in 1918 had a dispro- portionately high influenza incidence (Figure 3, panel A). But even after adjusting age-specific deaths by age-specif— ic clinical attack rates (Figure 3, panel B), a W—shaped curve with a case-fatality peak in young adults remains and is significantly different from U-shaped age-specific case- fatality curves typically seen in other influenza years, e.g., 192871929 (Figure 3, panel C). Also, in 1918 those 5 to 14 years of age accounted for a disproportionate number of influenza cases, but had a much lower death rate from influenza and pneumonia than other age groups. To explain this pattern, we must look beyond properties of the Virus to host and environmental factors, possibly including immunopathology (e.g., antibody-dependent infection enhancement associated with prior Virus exposures [38]) and exposure to risk cofactors such as coinfecting agents, medications, and environmental agents. One theory that may partially explain these findings is that the 1918 Virus had an intrinsically high Virulence, tem- pered only in those patients who had been born before 1889, e.g., because of exposure to a then-circulating Virus capable of providing partial immunoprotection against the 1918 Virus strain only in persons old enough (>35 years) to have been infected during that prior era (35). But this the- ory would present an additional paradox: an obscure pre- cursor Virus that left no detectable trace today would have had to have appeared and disappeared before 1889 and then reappeared more than 3 decades later. Epidemiologic data on rates of clinical influenza by age, collected between 1900 and 1918, provide good evi- dence for the emergence of an antigenically novel influen- za Virus in 1918 (21). Jordan showed that from 1900 to 1917, the 5- to 15-year age group accounted for 11% of total influenza cases, while the >65-year age group accounted for 6 % of influenza cases. But in 1918, cases in Figure 2. “U-” and “W—” shaped combined influenza and pneumo- nia mortality, by age at death, per 100,000 persons in each age group, United States, 1911—1918. Influenza- and pneumonia- specific death rates are plotted for the interpandemic years 1911—1917 (dashed line) and for the pandemic year 1918 (solid line) (33,34). Incidence male per 1 .nao persunslage group Mortality per 1.000 persunslige group + Case—fataiity rale 1918—1919 Case fatalily par 100 persons ill wilh P&I pel age group Figure 3. Influenza plus pneumonia (P&l) (combined) age-specific incidence rates per 1,000 persons per age group (panel A), death rates per 1,000 persons, ill and well combined (panel B), and case-fatality rates (panel C, solid line), US Public Health Service house-to-house surveys, 8 states, 1918 (36). A more typical curve of age-specific influenza case-fatality (panel C, dotted line) is taken from US Public Health Service surveys during 1928—1929 (37). the 5 to 15-year-old group jumped to 25% of influenza cases (compatible with exposure to an antigenically novel Virus strain), while the >65-year age group only accounted for 0.6% of the influenza cases, findings consistent with previously acquired protective immunity caused by an identical or closely related Viral protein to which older per- sons had once been exposed. Mortality data are in accord. In 1918, persons >75 years had lower influenza and pneumonia case-fatality rates than they had during the prepandemic period of 191171917. At the other end of the age spectrum (Figure 2), a high proportion of deaths in infancy and early childhood in 1918 mimics the age pat- tern, if not the mortality rate, of other influenza pandemics. Could a 1918-like Pandemic Appear Again? If So, What Could We Do About It? In its disease course and pathologic features, the 1918 pandemic was different in degree, but not in kind, from previous and subsequent pandemics. Despite the extraordi- nary number of global deaths, most influenza cases in 1918 (>95% in most locales in industrialized nations) were mild and essentially indistinguishable from influenza cases today. Furthermore, laboratory experiments with recombi- nant influenza Viruses containing genes from the 1918 Virus suggest that the 1918 and 1918-like Viruses would be as sensitive as other typical Virus strains to the Food and Drug Administrationiapproved antiinfluenza drugs riman- tadine and oseltamivir. However, some characteristics of the 1918 pandemic appear unique: most notably, death rates were 5 7 20 times higher than expected. Clinically and pathologically, these high death rates appear to be the result of several factors, including a higher proportion of severe and complicated infections of the respiratory tract, rather than involvement of organ systems outside the normal range of the influenza Virus. Also, the deaths were concentrated in an unusually young age group. Finally, in 1918, 3 separate recurrences of influenza followed each other with unusual rapidity, resulting in 3 explosive pandemic waves within a year’s time (Figure 1). Each of these unique characteristics may reflect genetic features of the 1918 Virus, but understand- ing them will also require examination of host and envi- ronmental factors. Until we can ascertain which of these factors gave rise to the mortality patterns observed and learn more about the formation of the pandemic, predictions are only educated guesses. We can only conclude that since it happened once, analogous conditions could lead to an equally devastating pandemic. Like the 1918 Virus, H5N1 is an avian Virus (39), though a distantly related one. The evolutionary path that led to pandemic emergence in 1918 is entirely unknown, but it appears to be different in many respects from the cur- rent situation with H5N1. There are no historical data, either in 1918 or in any other pandemic, for establishing that a pandemic “precursor” Virus caused a highly patho- genic outbreak in domestic poultry, and no highly patho- genic avian influenza (HPAI) Virus, including H5N1 and a number of others, has ever been known to cause a major human epidemic, let alone a pandemic. While data bearing on influenza Virus human cell adaptation (e.g., receptor binding) are beginning to be understood at the molecular level, the basis for Viral adaptation to efficient human-to- human spread, the chief prerequisite for pandemic emer- gence, is unknown for any influenza Virus. The 1918 Virus acquired this trait, but we do not know how, and we cur- rently have no way of knowing whether H5N1 Viruses are now in a parallel process of acquiring human-to-human transmissibility. Despite an explosion of data on the 1918 Virus during the past decade, we are not much closer to understanding pandemic emergence in 2006 than we were in understanding the risk of H1N1 “swine flu” emergence in 1976. Even with modern antiviral and antibacterial drugs, vaccines, and prevention knowledge, the return of a pan- demic Virus equivalent in pathogenicity to the Virus of 1918 would likely kill >100 million people worldwide. A pandemic Virus with the (alleged) pathogenic potential of some recent H5N1 outbreaks could cause substantially more deaths. Whether because of Viral, host or environmental fac- tors, the 1918 Virus causing the first or ‘spring’ wave was not associated with the exceptional pathogenicity of the second (fall) and third (winter) waves. Identification of an influenza RNA-positive case from the first wave could point to a genetic basis for Virulence by allowing differ- ences in Viral sequences to be highlighted. Identification of pre-1918 human influenza RNA samples would help us understand the timing of emergence of the 1918 Virus. Surveillance and genomic sequencing of large numbers of animal influenza Viruses will help us understand the genet- ic basis of host adaptation and the extent of the natural reservoir of influenza Viruses. Understanding influenza pandemics in general requires understanding the 1918 pan- demic in all its historical, epidemiologic, and biologic aspects. Dr Taubenberger is chair of the Department of Molecular Pathology at the Armed Forces Institute of Pathology, Rockville, Maryland. His research interests include the molecular patho- physiology and evolution of influenza Viruses. Dr Morens is an epidemiologist with a long-standing inter- est in emerging infectious diseases, Virology, tropical medicine, and medical history. Since 1999, he has worked at the National Institute of Allergy and Infectious Diseases. References 1. Frost WH. Statistics of influenza morbidity. Public Health Rep. 19203558497. 2. Bumet F, Clark E. Influenza: a survey ofthe last 50 years in the light of modern work on the Virus of epidemic influenza. Melbourne: MacMillan; 1942. 3. Marks G, Beatty WK. Epidemics. New York: Scribners, 1976. 4. Rosenau MJ, Last JM. Maxcy-Rosenau preventative medicine and public health. New York: Appleton-Century-Crofts; 1980. 5. Crosby A. America’s forgotten pandemic. Cambridge (UK): Cambridge University Press;1989. 6. Patterson KD, Pyle GF. The geography and mortality of the 1918 influenza pandemic. Bull Hist Med. 1991;65:4–21. 7. Johnson NPAS, Mueller J. Updating the accounts: global mortality of the 1918–1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105–15. 8. Shope RE. The incidence of neutralizing antibodies for swine influenza virus in the sera of human beings of different ages. J Exp Med. 1936;63:669–84. 9. Kendal AP, Noble GR, Skehel JJ, Dowdle WR. Antigenic similarity of influenza A (H1N1) viruses from epidemics in 1977–1978 to “Scandinavian” strains isolated in epidemics of 1950–1951. Virology. 1978;89:632–6. 10. Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 11. Basler CF, Reid AH, Dybing JK, Janczewski TA, Fanning TG, Zheng H, et al. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A 2001;98:2746–51. 12. Reid AH, Fanning TG, Hultin JV, Taubenberger JK. Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene. Proc Natl Acad Sci U S A 1999;96:1651–6. 13. Reid AH, Fanning TG, Janczewski TA, Lourens RM, and Taubenberger JK. Novel origin of the 1918 pandemic influenza virus nucleoprotein gene segment. J Virol. 2004;78:12462–70. 14. Reid AH, Fanning TG, Janczewski TA, McCall S, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus matrix gene segment. J Virol. 2002;76:10717–23. 15. Reid AH, Fanning TG, Janczewski TA, Taubenberger JK. Characterization of the 1918 “Spanish” influenza virus neuraminidase gene. Proc Natl Acad Sci U S A 2000;97:6785–90. 16. Reid AH, Janczewski TA, Lourens RM, Elliot AJ, Daniels RS, Berry CL, et al. 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis. 2003;9:1249–53. 17. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature. 2005;437:889–93. 18. Reid AH, Taubenberger JK. The 1918 flu and other influenza pandemics: “over there” and back again. Lab Invest. 1999;79:95–101. 19. Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol. 2004;2:909–14. 20. Taubenberger JK, Reid AH, Fanning TG. The 1918 influenza virus: a killer comes into view. Virology. 2000;274:241–5. 21. Jordan E. Epidemic influenza: a survey. Chicago: American Medical Association, 1927. 22. Capps J, Moody A. The recent epidemic of grip. JAMA. 1916;67:1349–50. 33. Oxford JS, Sefton A, Jackson R, Innes W, Daniels RS, Johnson NP. World War I may have allowed the emergence of “Spanish” influenza. Lancet Infect Dis. 2002;2:111–4. 24. Fanning TG, Slemons RD, Reid AH, Janczewski TA, Dean J, Taubenberger JK. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J Virol. 2002;76:7860–2. 25. Reid AH, Fanning TG, Slemons RD, Janczewski TA, Dean J, Taubenberger JK. Relationship of pre-1918 avian influenza HA and NP sequences to subsequent avian influenza strains. Avian Dis. 2003;47:921–5. 26. Bean W, Schell M, Katz J, Kawaoka Y, Naeve C, Gorman O, et al. Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts. J Virol. 1992;66:1129–38. 27. Weis W, Brown JH, Cusack S, Paulson JC, Skehel JJ, Wiley DC. Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid. Nature. 1988;333:426–31. 28. Gambaryan AS, Tuzikov AB, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, et al. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6′-sialyl(N-acetyllactosamine). Virology. 1997;232: 345–50. 29. Matrosovich M, Gambaryan A, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, et al. Avian influenza A viruses differ from human viruses by recognition of sialyloigosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 1997;233:224–34. 30. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, et al. A single amino acid substitution in the 1918 influenza virus hemagglutinin changes the receptor binding specificity. J Virol. 2005;79:11533–6. 31. Kobasa D, Takada A, Shinya K, Hatta M, Halfmann P, Theriault S, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 2004;431:703–7. 32. Kash JC, Basler CF, Garcia-Sastre A, Carter V, Billharz R, Swayne DE, et al. Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus. J Virol. 2004;78:9499–511. 33. Grove RD, Hetzel AM. Vital statistics rates in the United States: 1940–1960. Washington: US Government Printing Office, 1968. 34. Linder FE, Grove RD. Vital statistics rates in the United States: 1900–1940. Washington: US Government Printing Office, 1943. 35. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K. Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 1998;178:53–60. 36. Frost WH. The epidemiology of influenza. Public Health Rep. 1919;34:1823–61. 37. Collins SD. Age and sex incidence of influenza and pneumonia morbidity and mortality in the epidemic of 1928-1929 with comparative data for the epidemic of 1918–1919. Public Health Rep. 1931;46:1909–37. 38. Majde JA. Influenza: Learn from the past. ASM News. 1996;62:514. 39. Peiris JS, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JM, et al. Re-emergence of fatal human influenza A subtype H5N1 disease. Lancet. 2004;363:617–9. Address for correspondence: Jeffery K. Taubenberger, Department of Molecular Pathology, Armed Forces Institute of Pathology, 1413 Research Blvd, Bldg 101, Rm 1057, Rockville, MD 20850-3125, USA; fax. 301-295-9507; email: [email protected] The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.
Did the Spanish Influenza or Swine flu or the H1N1 virus disappear in humans for some time?
false
1,065
{ "text": [ "descendants of the 1918\nVirus still persists enzootically in pigs. They probably also\ncirculated continuously in humans, undergoing gradual\nantigenic drift and causing annual epidemics, until the\n1950s. With the appearance of a new H2N2 pandemic\nstrain in 1957 (“Asian flu”), the direct H1N1 Viral descen-\ndants 0f the 1918 pandemic strain disappeared from human\ncirculation entirely, although the related lineage persisted\nenzootically in pigs." ], "answer_start": [ 2670 ] }
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&rsquo;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?
false
2,199
{ "text": [ "s are spherical, enveloped, and the largest of positive-strand RNA v" ], "answer_start": [ 1930 ] }
2,486
Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review https://doi.org/10.3390/jcm9030623 SHA: 9b0c87f808b1b66f2937d7a7acb524a756b6113b Authors: Pang, Junxiong; Wang, Min Xian; Ang, Ian Yi Han; Tan, Sharon Hui Xuan; Lewis, Ruth Frances; Chen, Jacinta I. Pei; Gutierrez, Ramona A.; Gwee, Sylvia Xiao Wei; Chua, Pearleen Ee Yong; Yang, Qian; Ng, Xian Yi; Yap, Rowena K. S.; Tan, Hao Yi; Teo, Yik Ying; Tan, Chorh Chuan; Cook, Alex R.; Yap, Jason Chin-Huat; Hsu, Li Yang Date: 2020 DOI: 10.3390/jcm9030623 License: cc-by Abstract: Rapid diagnostics, vaccines and therapeutics are important interventions for the management of the 2019 novel coronavirus (2019-nCoV) outbreak. It is timely to systematically review the potential of these interventions, including those for Middle East respiratory syndrome-Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS)-CoV, to guide policymakers globally on their prioritization of resources for research and development. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Supplementary strategies through Google Search and personal communications were used. A total of 27 studies fulfilled the criteria for review. Several laboratory protocols for confirmation of suspected 2019-nCoV cases using real-time reverse transcription polymerase chain reaction (RT-PCR) have been published. A commercial RT-PCR kit developed by the Beijing Genomic Institute is currently widely used in China and likely in Asia. However, serological assays as well as point-of-care testing kits have not been developed but are likely in the near future. Several vaccine candidates are in the pipeline. The likely earliest Phase 1 vaccine trial is a synthetic DNA-based candidate. A number of novel compounds as well as therapeutics licensed for other conditions appear to have in vitro efficacy against the 2019-nCoV. Some are being tested in clinical trials against MERS-CoV and SARS-CoV, while others have been listed for clinical trials against 2019-nCoV. However, there are currently no effective specific antivirals or drug combinations supported by high-level evidence. Text: Since mid-December 2019 and as of early February 2020, the 2019 novel coronavirus (2019-nCoV) originating from Wuhan (Hubei Province, China) has infected over 25,000 laboratory-confirmed cases across 28 countries with about 500 deaths (a case-fatality rate of about 2%). More than 90% of the cases and deaths were in China [1] . Based on the initial reported surge of cases in Wuhan, the majority were males with a median age of 55 years and linked to the Huanan Seafood Wholesale Market [2] . Most of the reported cases had similar symptoms at the onset of illness such as fever, cough, and myalgia or fatigue. Most cases developed pneumonia and some severe and even fatal respiratory diseases such as acute respiratory distress syndrome [3] . The 2019 novel coronavirus (2019-nCoV), a betacoronavirus, forms a clade within the subgenus sarbecovirus of the Orthocoronavirinae subfamily [4] . The severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also betacoronaviruses that are zoonotic in origin and have been linked to potential fatal illness during the outbreaks in 2003 and 2012, respectively [5, 6] . Based on current evidence, pathogenicity for 2019-nCoV is about 3%, which is significantly lower than SARS-CoV (10%) and MERS-CoV (40%) [7] . However, 2019-nCoV has potentially higher transmissibility (R0: 1.4-5.5) than both SARS-CoV (R0: [2] [3] [4] [5] and MERS-CoV (R0: <1) [7] . With the possible expansion of 2019-nCoV globally [8] and the declaration of the 2019-nCoV outbreak as a Public Health Emergency of International Concern by the World Health Organization, there is an urgent need for rapid diagnostics, vaccines and therapeutics to detect, prevent and contain 2019-nCoV promptly. There is however currently a lack of understanding of what is available in the early phase of 2019-nCoV outbreak. The systematic review describes and assesses the potential rapid diagnostics, vaccines and therapeutics for 2019-nCoV, based in part on the developments for MERS-CoV and SARS-CoV. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies examining the diagnosis, therapeutic drugs and vaccines for Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and the 2019 novel coronavirus (2019-nCoV), in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. There were two independent reviewers each focusing on SARS, MERS, and 2019-nCoV, respectively. A third independent reviewer was engaged to resolve any conflicting article of interest. We used the key words "SARS", "coronavirus", "MERS", "2019 Novel coronavirus", "Wuhan virus" to identify the diseases in the search strategy. The systematic searches for diagnosis, therapeutic drugs and vaccines were carried out independently and the key words "drug", "therapy", "vaccine", "diagnosis", "point of care testing" and "rapid diagnostic test" were used in conjunction with the disease key words for the respective searches. Examples of search strings can be found in Table S1 . We searched for randomized controlled trials (RCTs) and validation trials (for diagnostics test) published in English, that measured (a) the sensitivity and/or specificity of a rapid diagnostic test or a point-of-care testing kit, (b) the impact of drug therapy or (c) vaccine efficacy against either of these diseases with no date restriction applied. For the 2019-nCoV, we searched for all in vitro, animal, or human studies published in English between 1 December 2019 and 6 February 2020, on the same outcomes of interest. In addition, we reviewed the references of retrieved articles in order to identify additional studies or reports not retrieved by the initial searches. Studies that examined the mechanisms of diagnostic tests, drug therapy or vaccine efficacy against SARS, MERS and 2019-nCoV were excluded. A Google search for 2019-nCoV diagnostics (as of 6 February 2020; Table S2 ) yielded five webpage links from government and international bodies with official information and guidelines (WHO, Europe CDC, US CDC, US FDA), three webpage links on diagnostic protocols and scientific commentaries, and five webpage links on market news and press releases. Six protocols for diagnostics using reverse transcriptase polymerase chain reaction (RT-PCR) from six countries were published on WHO's website [9] . Google search for 2019-nCoV vaccines yielded 19 relevant articles. With the emergence of 2019-nCoV, real time RT-PCR remains the primary means for diagnosing the new virus strain among the many diagnostic platforms available ( [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ; Table S3 ). Among the 16 diagnostics studies selected, one study discussed the use of RT-PCR in diagnosing patients with 2019-nCoV [11] ( Table 1 ). The period and type of specimen collected for RT-PCR play an important role in the diagnosis of 2019-nCoV. It was found that the respiratory specimens were positive for the virus while serum was negative in the early period. It has also suggested that in the early days of illness, patients have high levels of virus despite the mild symptoms. Apart from the commonly used RT-PCR in diagnosing MERS-CoV, four studies identified various diagnostic methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), RT-insulated isothermal PCR (RT-iiPCR) and a one-step rRT-PCR assay based on specific TaqMan probes. RT-LAMP has similar sensitivity as real time RT-PCR. It is also highly specific and is used to detect MERS-CoV. It is comparable to the usual diagnostic tests and is rapid, simple and convenient. Likewise, RT-iiPCR and a one-step rRT-PCR assay have also shown similar sensitivity and high specificity for MER-CoV. Lastly, one study focused on the validation of the six commercial real RT-PCR kits, with high accuracy. Although real time RT-PCR is a primary method for diagnosing MERS-CoV, high levels of PCR inhibition may hinder PCR sensitivity (Table 1) . There are eleven studies that focus on SARS-CoV diagnostic testing (Table 1) . These papers described diagnostic methods to detect the virus with the majority of them using molecular testing for diagnosis. Comparison between the molecular test (i.e RT-PCR) and serological test (i.e., ELISA) showed that the molecular test has better sensitivity and specificity. Hence, enhancements to the current molecular test were conducted to improve the diagnosis. Studies looked at using nested PCR to include a pre-amplification step or incorporating N gene as an additional sensitive molecular marker to improve on the sensitivity (Table 1 ). In addition, there are seven potential rapid diagnostic kits (as of 24 January 2020; Table 2 ) available on the market for 2019-nCoV. Six of these are only for research purposes. Only one kit from Beijing Genome Institute (BGI) is approved for use in the clinical setting for rapid diagnosis. Most of the kits are for RT-PCR. There were two kits (BGI, China and Veredus, Singapore) with the capability to detect multiple pathogens using sequencing and microarray technologies, respectively. The limit of detection of the enhanced realtime PCR method was 10 2 -fold higher than the standard real-time PCR assay and 10 7fold higher than conventional PCR methods In the clinical aspect, the enhanced realtime PCR method was able to detect 6 cases of SARS-CoV positive samples that were not confirmed by any other assay [25] • The real time PCR has a threshold sensitivity of 10 genome equivalents per reaction and it has a good reproducibility with the inter-assay coefficients of variation of 1.73 to 2.72%. • 13 specimens from 6 patients were positive with viral load range from 362 to 36,240,000 genome equivalents/mL. The real-time RT-PCR reaction was more sensitive than the nested PCR reaction, as the detection limit for the nested PCR reaction was about 10 3 genome equivalents in the standard cDNA control. [34] Real-time reverse-transcription PCR (rRT-PCR); RNA-dependent RNA polymerase (RdRp); open reading frame 1a (ORF1a); Loop-mediated isothermal amplification (LAMP); enzyme-linked immunosorbent assay (ELISA); immunofluorescent assay (IFA); immunochromatographic test (ICT); nasopharyngeal aspirate (NPA). With the emergence of 2019-nCoV, there are about 15 potential vaccine candidates in the pipeline globally (Table 3 ), in which a wide range of technology (such as messenger RNA, DNA-based, nanoparticle, synthetic and modified virus-like particle) was applied. It will likely take about a year for most candidates to start phase 1 clinical trials except for those funded by Coalition for Epidemic Preparedness Innovations (CEPI). However, the kit developed by the BGI have passed emergency approval procedure of the National Medical Products Administration, and are currently used in clinical and surveillance centers of China [40] . Of the total of 570 unique studies on 2019-nCoV, SARS CoV or MERS-CoV vaccines screened, only four were eventually included in the review. Most studies on SARS and MERS vaccines were excluded as they were performed in cell or animal models ( Figure 1 ). The four studies included in this review were Phase I clinical trials on SARS or MERS vaccines (Table 4 ) [44] [45] [46] [47] . There were no studies of any population type (cell, animal, human) on the 2019-nCoV at the point of screening. The published clinical trials were mostly done in United States except for one on the SARS vaccine done in China [44] . All vaccine candidates for SARS and MERS were reported to be safe, well-tolerated and able to trigger the relevant and appropriate immune responses in the participants. In addition, we highlight six ongoing Phase I clinical trials identified in the ClinicalTrials.gov register ( [48, 49] ); Table S4 ) [50] [51] [52] . These trials are all testing the safety and immunogenicity of their respective MERS-CoV vaccine candidates but were excluded as there are no results published yet. The trials are projected to complete in December 2020 (two studies in Russia [50, 51] ) and December 2021 (in Germany [52] ). Existing literature search did not return any results on completed 2019-nCoV trials at the time of writing. Among 23 trials found from the systematic review (Table 5) , there are nine clinical trials registered under the clinical trials registry (ClinicalTrials.gov) for 2019-nCoV therapeutics [53] [54] [55] [56] [57] [58] [59] [60] [61] . Of which five studies on hydroxychloroquine, lopinavir plus ritonavir and arbidol, mesenchymal stem cells, traditional Chinese medicine and glucocorticoid therapy usage have commenced recruitment. The remaining four studies encompass investigation of antivirals, interferon atomization, darunavir and cobicistat, arbidol, and remdesivir usage for 2019-nCoV patients (Table 5) . Seroconversion measured by S1-ELISA occurred in 86% and 94% participants after 2 and 3 doses, respectively, and was maintained in 79% participants up to study end at week 60. Neutralising antibodies were detected in 50% participants at one or more time points during the study, but only 3% maintained neutralisation activity to end of study. T-cell responses were detected in 71% and 76% participants after 2 and 3 doses, respectively. There were no differences in immune responses between dose groups after 6 weeks and vaccine-induced humoral and cellular responses were respectively detected in 77% and 64% participants at week 60. [47] Molecules developed by the university scientists inhibit two coronavirus enzymes and prevent its replication. The discovered drug targets are said to be more than 95% similar to enzyme targets found on the SARS virus. Researchers note that identified drugs may not be available to address the ongoing outbreak but they hope to make it accessible for future outbreaks. [85] Besides the six completed randomized controlled trials (RCT) selected from the systematic review (Table 6) , there is only one ongoing randomized controlled trial targeted at SARS therapeutics [92] . The studies found from ClinicalTrials.gov have not been updated since 2013. While many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir or ribavirin only, there has yet to be well-designed clinical trials investigating their usage. Three completed randomized controlled trials were conducted during the SARS epidemic-3 in China, 1 in Taiwan and 2 in Hong Kong [93] [94] [95] [96] [97] . The studies respectively investigated antibiotic usage involving 190 participants, combination of western and Chinese treatment vs. Chinese treatment in 123 participants, integrative Chinese and Western treatment in 49 patients, usage of a specific Chinese medicine in four participants and early use of corticosteroid in 16 participants. Another notable study was an open non-randomized study investigating ribavirin/lopinavir/ritonavir usage in 152 participants [98] . One randomized controlled trial investigating integrative western and Chinese treatment during the SARS epidemic was excluded as it was a Chinese article [94] . There is only one ongoing randomized controlled trial targeted at MERS therapeutics [99] . It investigates the usage of Lopinavir/Ritonavir and Interferon Beta 1B. Likewise, many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir/ribavirin, interferon, and convalescent plasma usage. To date, only one trial has been completed. One phase 1 clinical trial investigating the safety and tolerability of a fully human polyclonal IgG immunoglobulin (SAB-301) was found in available literature [46] . The trial conducted in the United States in 2017 demonstrated SAB-301 to be safe and well-tolerated at single doses. Another trial on MERS therapeutics was found on ClinicalTrials.gov-a phase 2/3 trial in the United States evaluating the safety, tolerability, pharmacokinetics (PK), and immunogenicity on coadministered MERS-CoV antibodies REGN3048 & REGN3051 [100]. Rapid diagnostics plays an important role in disease and outbreak management. The fast and accurate diagnosis of a specific viral infection enables prompt and accurate public health surveillance, prevention and control measures. Local transmission and clusters can be prevented or delayed by isolation of laboratory-confirmed cases and their close contacts quarantined and monitored at home. Rapid diagnostic also facilitates other specific public health interventions such as closure of high-risk facilities and areas associated with the confirmed cases for prompt infection control and environmental decontamination [11, 101] . Laboratory diagnosis can be performed by: (a) detecting the genetic material of the virus, (b) detecting the antibodies that neutralize the viral particles of interest, (c) detecting the viral epitopes of interest with antibodies (serological testing), or (d) culture and isolation of viable virus particles. The key limitations of genetic material detection are the lack of knowledge of the presence of viable virus, the potential cross-reactivity with non-specific genetic regions and the short timeframe for accurate detection during the acute infection phase. The key limitations of serological testing is the need to collect paired serum samples (in the acute and convalescent phases) from cases under investigation for confirmation to eliminate potential cross-reactivity from non-specific antibodies from past exposure and/or infection by other coronaviruses. The limitation of virus culture and isolation is the long duration and the highly specialized skills required of the technicians to process the samples. All patients recovered. Significantly shorted time from the disease onset to the symptom improvement in treatment (5.10 ± 2.83 days) compared to control group (7.62 ± 2.27 days) (p < 0.05) No significant difference in blood routine improvement, pulmonary chest shadow in chest film improvement and corticosteroid usgae between the 2 groups. However, particularly in the respect of improving clinical symptoms, elevating quality of life, promoting immune function recovery, promoting absorption of pulmonary inflammation, reducing the dosage of cortisteroid and shortening the therapeutic course, treatment with integrative chinese and western medicine treatment had obvious superiority compared with using control treatment alone. Single infusions of SAB-301 up to 50 mg/kg appear to be safe and well-tolerated in healthy participants. [46] Where the biological samples are taken from also play a role in the sensitivity of these tests. For SARS-CoV and MERS-CoV, specimens collected from the lower respiratory tract such as sputum and tracheal aspirates have higher and more prolonged levels of viral RNA because of the tropism of the virus. MERS-CoV viral loads are also higher for severe cases and have longer viral shedding compared to mild cases. Although upper respiratory tract specimens such as nasopharyngeal or oropharyngeal swabs can be used, they have potentially lower viral loads and may have higher risk of false-negatives among the mild MERS and SARS cases [102, 103] , and likely among the 2019-nCoV cases. The existing practices in detecting genetic material of coronaviruses such as SARS-CoV and MERS-CoV include (a) reverse transcription-polymerase chain reaction (RT-PCR), (b) real-time RT-PCR (rRT-PCR), (c) reverse transcription loop-mediated isothermal amplification (RT-LAMP) and (d) real-time RT-LAMP [104] . Nucleic amplification tests (NAAT) are usually preferred as in the case of MERS-CoV diagnosis as it has the highest sensitivity at the earliest time point in the acute phase of infection [102] . Chinese health authorities have recently posted the full genome of 2019-nCoV in the GenBank and in GISAID portal to facilitate in the detection of the virus [11] . Several laboratory assays have been developed to detect the novel coronavirus in Wuhan, as highlighted in WHO's interim guidance on nCoV laboratory testing of suspected cases. These include protocols from other countries such as Thailand, Japan and China [105] . The first validated diagnostic test was designed in Germany. Corman et al. had initially designed a candidate diagnostic RT-PCR assay based on the SARS or SARS-related coronavirus as it was suggested that circulating virus was SARS-like. Upon the release of the sequence, assays were selected based on the match against 2019-nCoV upon inspection of the sequence alignment. Two assays were used for the RNA dependent RNA polymerase (RdRP) gene and E gene where E gene assay acts as the first-line screening tool and RdRp gene assay as the confirmatory testing. All assays were highly sensitive and specific in that they did not cross-react with other coronavirus and also human clinical samples that contained respiratory viruses [11] . The Hong Kong University used two monoplex assays which were reactive with coronaviruses under the subgenus Sarbecovirus (consisting of 2019-nCoV, SARS-CoV and SARS-like coronavirus). Viral RNA extracted from SARS-CoV can be used as the positive control for the suggested protocol assuming that SARS has been eradicated. It is proposed that the N gene RT-PCR can be used as a screening assay while the Orf1b assay acts as a confirmatory test. However, this protocol has only been evaluated with a panel of controls with the only positive control SARS-CoV RNA. Synthetic oligonucleotide positive control or 2019-nCoV have yet to be tested [106] . The US CDC shared the protocol on the real time RT-PCR assay for the detection of the 2019-nCoV with the primers and probes designed for the universal detection of SARS-like coronavirus and the specific detection of 2019-nCoV. However, the protocol has not been validated on other platforms or chemistries apart from the protocol described. There are some limitations for the assay. Analysts engaged have to be trained and familiar with the testing procedure and result interpretation. False negative results may occur due to insufficient organisms in the specimen resulting from improper collection, transportation or handling. Also, RNA viruses may show substantial genetic variability. This could result in mismatch between the primer and probes with the target sequence which can diminish the assay performance or result in false negative results [107] . Point-of-care test kit can potentially minimize these limitations, which should be highly prioritized for research and development in the next few months. Serological testing such as ELISA, IIFT and neutralization tests are effective in determining the extent of infection, including estimating asymptomatic and attack rate. Compared to the detection of viral genome through molecular methods, serological testing detects antibodies and antigens. There would be a lag period as antibodies specifically targeting the virus would normally appear between 14 and 28 days after the illness onset [108] . Furthermore, studies suggest that low antibody titers in the second week or delayed antibody production could be associated with mortality with a high viral load. Hence, serological diagnoses are likely used when nucleic amplification tests (NAAT) are not available or accessible [102] . Vaccines can prevent and protect against infection and disease occurrence when exposed to the specific pathogen of interest, especially in vulnerable populations who are more prone to severe outcomes. In the context of the current 2019-nCoV outbreak, vaccines will help control and reduce disease transmission by creating herd immunity in addition to protecting healthy individuals from infection. This decreases the effective R0 value of the disease. Nonetheless, there are social, clinical and economic hurdles for vaccine and vaccination programmes, including (a) the willingness of the public to undergo vaccination with a novel vaccine, (b) the side effects and severe adverse reactions of vaccination, (c) the potential difference and/or low efficacy of the vaccine in populations different from the clinical trials' populations and (d) the accessibility of the vaccines to a given population (including the cost and availability of the vaccine). Vaccines against the 2019-nCoV are currently in development and none are in testing (at the time of writing). On 23 January 2020, the Coalition for Epidemic Preparedness Innovations (CEPI) announced that they will fund vaccine development programmes with Inovio, The University of Queensland and Moderna, Inc respectively, with the aim to test the experimental vaccines clinically in 16 weeks (By June 2020). The vaccine candidates will be developed by the DNA, recombinant and mRNA vaccine platforms from these organizations [109] . Based on the most recent MERS-CoV outbreak, there are already a number of vaccine candidates being developed but most are still in the preclinical testing stage. The vaccines in development include viral vector-based vaccine, DNA vaccine, subunit vaccine, virus-like particles (VLPs)-based vaccine, inactivated whole-virus (IWV) vaccine and live attenuated vaccine. The latest findings for these vaccines arebased on the review by Yong et al. (2019) in August 2019 [110] . As of the date of reporting, there is only one published clinical study on the MERS-CoV vaccine by GeneOne Life Science & Inovio Pharmaceuticals [47] . There was one SARS vaccine trial conducted by the US National Institute of Allergy and Infectious Diseases. Both Phase I clinical trials reported positive results, but only one has announced plans to proceed to Phase 2 trial [111] . Due to the close genetic relatedness of SARS-CoV (79%) with 2019-nCoV [112] , there may be potential cross-protective effect of using a safe SARS-CoV vaccine while awaiting the 2019-nCoV vaccine. However, this would require small scale phase-by-phase implementation and close monitoring of vaccinees before any large scale implementation. Apart from the timely diagnosis of cases, the achievement of favorable clinical outcomes depends on the timely treatment administered. ACE2 has been reported to be the same cell entry receptor used by 2019-nCoV to infect humans as SARS-CoV [113] . Hence, clinical similarity between the two viruses is expected, particularly in severe cases. In addition, most of those who have died from MERS-CoV, SARS-CoV and 2019-nCoV were advance in age and had underlying health conditions such as hypertension, diabetes or cardiovascular disease that compromised their immune systems [114] . Coronaviruses have error-prone RNA-dependent RNA polymerases (RdRP), which result in frequent mutations and recombination events. This results in quasispecies diversity that is closely associated with adaptive evolution and the capacity to enhance viral-cell entry to cause disease over time in a specific population at-risk [115] . Since ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, coronaviruses are likely to infect the upper respiratory and gastrointestinal tract and this may influence the type of therapeutics against 2019-nCoV, similarly to SAR-CoV. However, in the years following two major coronavirus outbreaks SARS-CoV in 2003 and MERS-CoV in 2012, there remains no consensus on the optimal therapy for either disease [116, 117] . Well-designed clinical trials that provide the gold standard for assessing the therapeutic measures are scarce. No coronavirus protease inhibitors have successfully completed a preclinical development program despite large efforts exploring SARS-CoV inhibitors. The bulk of potential therapeutic strategies remain in the experimental phase, with only a handful crossing the in vitro hurdle. Stronger efforts are required in the research for treatment options for major coronaviruses given their pandemic potential. Effective treatment options are essential to maximize the restoration of affected populations to good health following infections. Clinical trials have commenced in China to identify effective treatments for 2019-nCoV based on the treatment evidence from SARS and MERS. There is currently no effective specific antiviral with high-level evidence; any specific antiviral therapy should be provided in the context of a clinical study/trial. Few treatments have shown real curative action against SARS and MERS and the literature generally describes isolated cases or small case series. Many interferons from the three classes have been tested for their antiviral activities against SARS-CoV both in vitro and in animal models. Interferon β has consistently been shown to be the most active, followed by interferon α. The use of corticosteroids with interferon alfacon-1 (synthetic interferon α) appeared to have improved oxygenation and faster resolution of chest radiograph abnormalities in observational studies with untreated controls. Interferon has been used in multiple observational studies to treat SARS-CoV and MERS-CoV patients [116, 117] . Interferons, with or without ribavirin, and lopinavir/ritonavir are most likely to be beneficial and are being trialed in China for 2019-nCoV. This drug treatment appears to be the most advanced. Timing of treatment is likely an important factor in effectiveness. A combination of ribavirin and lopinavir/ritonavir was used as a post-exposure prophylaxis in health care workers and may have reduced the risk of infection. Ribavirin alone is unlikely to have substantial antiviral activities at clinically used dosages. Hence, ribavirin with or without corticosteroids and with lopinavir and ritonavir are among the combinations employed. This was the most common agent reported in the available literature. Its efficacy has been assessed in observational studies, retrospective case series, retrospective cohort study, a prospective observational study, a prospective cohort study and randomized controlled trial ranging from seven to 229 participants [117] . Lopinavir/ritonavir (Kaletra) was the earliest protease inhibitor combination introduced for the treatment of SARS-CoV. Its efficacy was documented in several studies, causing notably lower incidence of adverse outcomes than with ribavirin alone. Combined usage with ribavirin was also associated with lower incidence of acute respiratory distress syndrome, nosocomial infection and death, amongst other favorable outcomes. Recent in vitro studies have shown another HIV protease inhibitor, nelfinavir, to have antiviral capacity against SARS-CoV, although it has yet to show favorable outcomes in animal studies [118] . Remdesivir (Gilead Sciences, GS-5734) nucleoside analogue in vitro and in vivo data support GS-5734 development as a potential pan-coronavirus antiviral based on results against several coronaviruses (CoVs), including highly pathogenic CoVs and potentially emergent BatCoVs. The use of remdesivir may be a good candidate as an investigational treatment. Improved mortality following receipt of convalescent plasma in various doses was consistently reported in several observational studies involving cases with severe acute respiratory infections (SARIs) of viral etiology. A significant reduction in the pooled odds of mortality following treatment of 0.25 compared to placebo or no therapy was observed [119] . Studies were however at moderate to high risk of bias given their small sample sizes, allocation of treatment based on the physician's discretion, and the availability of plasma. Factors like concomitant treatment may have also confounded the results. Associations between convalescent plasma and hospital length of stay, viral antibody levels, and viral load respectively were similarly inconsistent across available literature. Convalescent plasma, while promising, is likely not yet feasible, given the limited pool of potential donors and issues of scalability. Monoclonal antibody treatment is progressing. SARS-CoV enters host cells through the binding of their spike (S) protein to angiotensin converting enzyme 2 (ACE2) and CD209L [118] . Human monoclonal antibodies to the S protein have been shown to significantly reduce the severity of lung pathology in non-human primates following MERS-CoV infection [120] . Such neutralizing antibodies can be elicited by active or passive immunization using vaccines or convalescent plasma respectively. While such neutralizing antibodies can theoretically be harvested from individuals immunized with vaccines, there is uncertainty over the achievement of therapeutic levels of antibodies. Other therapeutic agents have also been reported. A known antimalarial agent, chloroquine, elicits antiviral effects against multiple viruses including HIV type 1, hepatitis B and HCoV-229E. Chloroquine is also immunomodulatory, capable of suppressing the production and release of factors which mediate the inflammatory complications of viral diseases (tumor necrosis factor and interleukin 6) [121] . It is postulated that chloroquine works by altering ACE2 glycosylation and endosomal pH. Its anti-inflammatory properties may be beneficial for the treatment of SARS. Niclosamide as a known drug used in antihelminthic treatment. The efficacy of niclosamide as an inhibitor of virus replication was proven in several assays. In both immunoblot analysis and immunofluorescence assays, niclosamide treatment was observed to completely inhibit viral antigen synthesis. Reduction of virus yield in infected cells was dose dependent. Niclosamide likely does not interfere in the early stages of virus attachment and entry into cells, nor does it function as a protease inhibitor. Mechanisms of niclosamide activity warrant further investigation [122] . Glycyrrhizin also reportedly inhibits virus adsorption and penetration in the early steps of virus replication. Glycyrrhizin was a significantly potent inhibitor with a low selectivity index when tested against several pathogenic flaviviruses. While preliminary results suggest production of nitrous oxide (which inhibits virus replication) through induction of nitrous oxide synthase, the mechanism of Glycyrrhizin against SARS-CoV remains unclear. The compound also has relatively lower toxicity compared to protease inhibitors like ribavirin [123] . Inhibitory activity was also detected in baicalin [124] , extracted from another herb used in the treatment of SARS in China and Hong Kong. Findings on these compounds are limited to in vitro studies [121] [122] [123] [124] . Due to the rapidly evolving situation of the 2019-nCoV, there will be potential limitations to the systematic review. The systematic review is likely to have publication bias as some developments have yet to be reported while for other developments there is no intention to report publicly (or in scientific platforms) due to confidentiality concerns. However, this may be limited to only a few developments for review as publicity does help in branding to some extent for the company and/or the funder. Furthermore, due to the rapid need to share the status of these developments, there may be reporting bias in some details provided by authors of the scientific articles or commentary articles in traditional media. Lastly, while it is not viable for any form of quality assessment and metaanalysis of the selected articles due to the limited data provided and the heterogeneous style of reporting by different articles, this paper has provided a comprehensive overview of the potential developments of these pharmaceutical interventions during the early phase of the outbreak. This systematic review would be useful for cross-check when the quality assessment and meta-analysis of these developments are performed as a follow-up study. Rapid diagnostics, vaccines and therapeutics are key pharmaceutical interventions to limit transmission of respiratory infectious diseases. Many potential developments on these pharmaceutical interventions for 2019-nCoV are ongoing in the containment phase of this outbreak, potentially due to better pandemic preparedness than before. However, lessons from MERS-CoV and SARS-CoV have shown that the journeys for these developments can still be challenging moving ahead. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1 : Example of full search strategy in Pubmed, Table S2 : Google Search: 2019-nCoV diagnostics, Table S3 : Summary of diagnostic assays developed for 2019-nCoV, Table S4
What are potential vaccines based on?
false
3,642
{ "text": [ "messenger RNA, DNA-based, nanoparticle, synthetic and modified virus-like particle)" ], "answer_start": [ 10922 ] }
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?
false
4,215
{ "text": [ "based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (LRT) involvement." ], "answer_start": [ 6732 ] }
1,576
Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521148/ SHA: ee14de143337eec0e9708f8139bfac2b7b8fdd27 Authors: Wang, Ning; Luo, Chuming; Liu, Haizhou; Yang, Xinglou; Hu, Ben; Zhang, Wei; Li, Bei; Zhu, Yan; Zhu, Guangjian; Shen, Xurui; Peng, Cheng; Shi, Zhengli Date: 2019-04-24 DOI: 10.3390/v11040379 License: cc-by Abstract: Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission. Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China. Bat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] . Viral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] . To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%. Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus sequence. The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder (https://www.ncbi.nlm.nih.gov/ orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform (http://services.cbu.uib.no/tools/kaks) with default parameters [27] . The protein homology detection was analyzed using HHpred (https://toolkit.tuebingen.mpg.de/#/tools/hhpred) with default parameters [28] . A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing. Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, www.atcc.org). The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag. The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] . Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant. HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] . The complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904. The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location. The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history. The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction. To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test). NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ). To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) . In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving. Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV. Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure. Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway. Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments. Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/11/4/379/s1, Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3
What is the length of the replicase gene ORF1ab?
false
3,684
{ "text": [ "20.4 kb" ], "answer_start": [ 12855 ] }
1,689
Chikungunya: A Potentially Emerging Epidemic? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860491/ SHA: f7c3160bef4169d29e2a8bdd79dd6e9056d4774c Authors: Thiboutot, Michelle M.; Kannan, Senthil; Kawalekar, Omkar U.; Shedlock, Devon J.; Khan, Amir S.; Sarangan, Gopalsamy; Srikanth, Padma; Weiner, David B.; Muthumani, Karuppiah Date: 2010-04-27 DOI: 10.1371/journal.pntd.0000623 License: cc-by Abstract: Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts. Text: Chikungunya virus (CHIKV), a mosquito-borne pathogen listed by National Institute of Allergy and Infectious Diseases (NIAID) as a Category C Priority Pathogen that causes Chikungunya fever (CHIKF), has been spreading throughout Asia, Africa, and parts of Europe in recent times [1, 2, 3] . CHIKV is an arthropod-borne virus (arbovirus) and is transmitted to humans primarily by Aedes aegypti, the infamous yellow fever propagator [4, 5] . CHIKV infection is marked by severe joint pain, contorting its victims into unusual postures [6] . The disease gets its name from the Kimakonde vernacular language of Tanzania and Mozambique, and the word chikungunya means ''that which contorts or bends up'' and translates in Swahili to ''the illness of the bended walker'' [7, 8, 9] . In Africa, CHIKV is maintained in a sylvatic cycle among forest-dwelling Aedes spp. mosquitoes, wild primates, squirrels, birds, and rodents ( Figure 1 ) [10] . In Asia, the disease is vectored by Ae. aegypti and Ae. albopictus [11] . Transmission in Asia occurs in an urban cycle whereby the mosquito spreads the disease from an infected human to an uninfected human, following an epidemiological pattern similar to dengue fever [12] . The 2005-2006 epidemic of CHIKV in La Reunion islands in the Indian Ocean, spurred the discovery of a new vector species, Ae. albopictus [5] . Wrecking over one-third of the island's population, this epidemic peaked its devastation between January and February 2006, when over 46,000 cases came into light every week, including 284 deaths [5, 13] . Ae. albopictus is common in urban areas of the United States and is already flourishing in 36 states, raising grave concerns to the immunologically naive populace of the United States [14] . Accordingly, this review elaborately details the epidemiology and global expansion of CHIKV, describes its clinical features and pathogenesis and its symptoms and complications, and finally nominates a possible vaccine approach against CHIKV infection. CHIKV has been isolated into three genotypes based on phylogenetic studies. These genotypes, based on the gene sequences of an Envelope protein (E1), are Asian, East/Central/ South African, and West African [4, 11, 15] . Using phylogenetic models, Cherian et al. estimate that the Asian genotype of CHIKV emerged between 50 and 310 y ago, and the West and East African genotypes diverged between 100 and 840 y ago [15] . Since then, CHIKV has come a long way, with several mutations incorporated, and has continued to wreak epidemics in several regions. Recent activities of CHIKV include the Indian epidemic in 2005-2006, which was followed by a sudden explosion of cases in 2007. An estimated 1.3 million people across 13 states were reported to be infected in India [12, 16] , and CHIKV was also widespread in Malaysia, Sri Lanka, and Indonesia [17] . In July-August of 2007, CHIKV was reported in Italy, probably brought in by travelers from CHIKV-prone regions of India, Africa, and Indian Ocean islands such as Mauritius, Madagascar, and Seychelles. Few of the Italian isolates were found to have evolved from the Kerala isolate, which was associated with a A226V shift in E1 gene that represents a successful evolutionary adaptation in the mosquito vector similar to the ones observed in Reunion Island [2, 18, 19] . In recent times, with an increase in global travel, the risk for spreading CHIKV to non-endemic regions has heightened [1] . Several travelers have brought CHIKV home with them after visiting areas with actively infected populations [12, 20] . Such cases have been documented in European countries, Australia, Asia, and the United States [8, 21] . The United States has already reported at least twelve cases of travel-associated CHIKV, while France has reported 850 cases, and the United Kingdom 93 [8, 14] . Beyond this, CHIKV-infected travelers have also been diagnosed in Australia, Belgium, Canada, Czech Republic, French Guiana, Germany, Hong Kong, Italy, Japan, Kenya, Malaysia, Martinique, Norway, Switzerland, and Sri Lanka [21] . Some travelers were viremic, worrying public health officials about the spread of CHIKV to new areas [1, 8] . The incubation time for CHIKV is relatively short, requiring only 2-6 d with symptoms usually appearing 4-7 d post-infection [22] . Vazeille et al. detected CHIKV in the salivary glands of Ae. albopictus only 2 d after infection [5] . Upon infection, CHIKF tends to present itself in two phases. The first stage is acute, while the second stage, experienced by most but not all, is persistent, causing disabling polyarthritis. Characteristics of the acute phase include an abrupt onset of fever, arthralgia, and in some cases, maculopapular rash [6, 23] . The acute phase causes such intense joint and muscular pain that makes movement very difficult and prostrates its victims [6, 20] . Ninety-five percent of infected adults are symptomatic after infection, and of these, most become disabled for weeks to months as a result of decreased dexterity, loss of mobility, and delayed reaction. Eighteen months after disease onset, 40% of patients are found to still have anti-CHIKV IgM [6, 18, 23, 24] . The chronic stage of CHIKF is characterized by polyarthralgia that can last from weeks to years beyond the acute stage [6] . CHIKV has been shown to attack fibroblasts, explaining the involvement of muscles, joints, and skin connective tissues. The high number of nociceptive nerve endings found within the joints and muscle connective tissues can explain pain associated with CHIKF [25, 26] . More than 50% of patients who suffer from severe CHIKF are over 65 y old, and more than 33% of them die. Most adults who suffer from severe CHIKF have underlying medical conditions [6, 24, 27] . The other group that is disproportionately affected by severe CHIKV is children. Other complications associated with CHIKV, from most common to least common, include respiratory failure, cardiovascular decompensation, meningoencephalitis, severe acute hepatitis, severe cutaneous effects, other central nervous system problems, and kidney failure [6, 18, 20, 23, 24, 26, 27] . CHIKV undertakes a complex replication cycle upon host infection (Figure 2 ), which makes its genome susceptible to mutations [28, 29] . For instance, Ae. aegypti, responsible for epidemics in Kenya, Comoros, and Seychelles, carried CHIKV with an alanine in the 226 position of the E1 gene (E1-A226) [4, 18] . However, when the virus struck La Reunion Islands, a decline in population of Ae. aegypti, due to massive dichlorodiphenyltrichloroethane usage and dearth of Ae. albopictus species' www.plosntds.org population, resulted in an ecological pressure, favoring replacement of alanine at position 226 with valine (E1-A226V) [5] . This mutation allowed CHIKV's secondary vector species, Ae. albopictus, to supplement Ae. aegypti as its primary vector [5] . Within a year, the E1-A226V mutation was present in La Reunion Island, and Ae. albopictus apparently vectored the large epidemic infecting 34% of La Reunion Island's population [5] . All of the CHIKV strains isolated from Mayotte carried the E1-A226V mutation, and the mutation was also found in Madagascar in 2007 [5] . The E1-A226V mutation was not present at the beginning of the Indian Ocean Islands outbreak (before September 2005). However, more than 90% of later viral strains found there had incorporated the mutation (December-March 2006), indicating a genotype switch during the winter season [5, 18, 20] . The E1-A226V mutation also enabled an increase in infectivity of Ae. albopictus when compared to its infectivity of Ae. aegypti [4, 11, 18, 30] , and with several factors taken together, Ae. albopictus has become the new preferred and more lethal vector for CHIKV [4, 5, 11] . In fact, Tsetsarkin et al. found that a Green Fluorescent Protein tagged E1-A226V virus was 100 times more infective to Ae. albopictus than it was to Ae. aegypti [4] . In all the Indian Ocean Islands, Ae. albopictus became the main vector for CHIKV within 1-2 y after CHIKV was introduced to the region [31] . Of note is that Ae. aegypti has most likely been established in North America for over 300 y, while Ae. albopictus has been in many areas of the US, since 1985, primarily in Florida [32] and since then has expanded its range in the country. Reiskind et al. set out to determine if Ae. aegypti and Ae. albopictus mosquitoes captured in Florida were susceptible to CHIKV infection by a La Reunion isolate [32] . Each mosquito tested was highly susceptible to infection by a full-length infectious clone of the La Réunion Island isolate, CHIKV LR2006 OPY1 strain. Even though the Ae. albopictus strains were more susceptible to infection, overall ecology and differences in human biting patterns need to be studied further Characteristically, there are two rounds of translation: (+) sense genomic RNA (49S9 = 11.7 kb) acts directly as mRNA and is partially translated (59 end) to produce non-structural proteins (nsp's). These proteins are responsible for replication and formation of a complementary (2) strand, the template for further (+) strand synthesis. Subgenomic mRNA (26 S = 4.1 kb) replication occurs through the synthesis of full-length (2) intermediate RNA, which is regulated by nsp4 and p123 precursor in early infection and later by mature nsp's. Translation of the newly synthesized sub-genomic RNA results in production of structural proteins such as Capsid and protein E2-6k-E1 (from 39 end of genome). Assembly occurs at the cell surface, and the envelope is acquired as the virus buds from the cell and release and maturation almost simultaneous occurred. Replication occurs in the cytoplasm and is very rapid (,4 h) [28, 29] . doi:10.1371/journal.pntd.0000623.g002 www.plosntds.org to gain a more accurate understanding of a potential CHIKV epidemic in the US [32] . During the 7 d preceding birth, no human mother has been reported to transmit the disease vertically. However, about 50% of newborns delivered while the mother was infected with CHIKV contracted the disease from their mother, despite the method of delivery. Furthermore, there have been instances of CHIKV transmission from mother to fetus causing congenital illness and fetal death [33] . During the 2005-2006 La Reunion Island outbreaks, Ramful et al. discovered that mothers could transmit CHIKV to their progeny during the perinatal period (Day 24 to Day +1) [33, 34] , and it is associated with a high degree of morbidity. By mean Day 4 of life, all of the neonates were symptomatic for CHIKV, exhibiting common CHIKF symptoms. Six neonates were confirmed to have contracted CHIKV and developed mengoencephalitis. Of those mothers who, during the La Reunion Island epidemic, were infected long before delivery, only three fetal deaths were reported [12, 33] . Ramful et al. theorized that motherto-child transmission most likely happens transplacentally shortly before delivery [33] . A similar study by Gerardin et al. reported nineteen cases of neonatal infection associated with intrapartum maternal viremia that progressed to develop encephalitis owing to vertical transmission from infected mothers [34] . Clinical and epidemiological similarities with dengue fever make CHIKV diagnosis difficult, which may lead physicians to misdiagnose CHIKV as dengue fever; therefore, the incidence of CHIKV may actually be higher than currently believed (Table 1 ) [6, 12, 35] . The amount of time elapsed since disease onset is the most critical parameter when choosing a diagnostic test. CHIKV can be detected and isolated by culturing with mosquito cells (C6/36), Vero cells (mammalian), or in mice [26] . However, this method can take at least a week and only achieves a high sensitivity during the viremic phase, which usually only lasts up to 48 h after the bite. Five days post-infection, the viral isolation approach has a low sensitivity but is still the preferred method for detecting the CHIKV strain [12, 26, 31, 35] . RT-PCR on the other hand is a faster and more sensitive method that can be used within the first week of disease onset [26] , and it is currently the most sensitive method for detecting and quantifying viral mRNA [4, 36] . Classic serological detection, by assays such as ELISA [37] , immunofluorescence [5, 38] , complement binding, and haemagglutination inhibition [39] , constitutes the second diagnostic tool used for biological diagnosis of CHIKV infection. These proven techniques are useful for detection of Antigen in mosquitoes during epidemiological studies. These assays detect virus-specific IgM and IgG, however the sensitivity and specificity of these assays has been poorly characterized. Viral competence, or the potential of viral infection and transmission, is an important parameter that can be quantified by ELISA, viral culture, and PCR. A study by Ng et al. showed biomarkers indicative of severe CHIKV infection [40] . They found decreased levels of RANTES and increased levels of Interleukin-6 (IL-6) and Interleukin-1b (IL-1b) that could be sued for CHIKV detection in patients as indicators of CHIKV-driven cytokine storm. Couderc et al. demonstrate another cytokine, type-I IFN, as a key player in the progression to CHIKV infection [26] . Using an IFN-a/b null mouse model, they demonstrated evidence of muscles, joints, and skin as privileged CHIKV targets, which is consistent with human pathology. Although Ng et al. concluded that RANTES levels were significantly suppressed in severe CHIKF patients [40] , interestingly, an increase in levels of RANTES has been observed in dengue infection [41] . Since the symptoms of CHIKF mimic those of dengue fever, results obtained from this study strongly suggest that RANTES could be a potential distinctive biomarker that differentiates between these two clinically similar diseases. There are no approved antiviral treatments currently available for CHIKV [1, 3, 12, 42] . Currently, CHIKF is treated symptomatically, usually with non-steroidal anti-inflammatory drugs or steroids, bed rest, and fluids. Movement and mild exercise are thought to decrease stiffness and morning arthralgia, but heavy exercise may exacerbate rheumatic symptoms. Corticosteroids may be used in cases of debilitating chronic CHIKV infection. There is a debate about the appropriateness of chloroquine as treatment for unresolved, non-steroidal anti-inflammatory drugresistant arthritis [43] . A study showed that viral production was www.plosntds.org drastically reduced at 16 h post-infection after treatment with 100 mM dec-RVKR-cmk (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone), a furine inhibitor [42, 44] . Chloroquine acted by raising the pH, blocking low pH-dependent entry of virus into the cell. It is important to note that dec-RVKR-cmk or chloroquine only inhibited viral spreading from cell to cell, not CHIKV replication once it had entered the cell [43] . However, most would agree that the best weapon against CHIKV is prevention. A live CHIKV vaccine developed by the United States reached phase II clinical trial encompassing 59 healthy volunteers [45] . Eight percent of the volunteers experienced transient arthralgia, while 98% of the volunteers had seroconversion [45] . However, live CHIKV vaccines are still questionable. One cannot discount the risk of a live vaccine possibly inducing chronic rheumatism. Also, there is the question as to whether widespread use among the public could trigger mosquito transmission or lead to chronic infection or viral reversion [1] . An alternative approach would be to produce a chimeric vaccine against CHIKV. Wang et al. developed a chimeric alphavirus vaccine that is uniformly attenuated and does not cause reactogenicity in mice [3] . Three different versions of this vaccine were made using three different backbone vectors: Venezuelan equine encephalitis virus (VEEV) attenuated vaccine strain T-83, naturally attenuated eastern equine encephalitis virus (EEEV), and attenuated Sindbis virus (SINV). In short, CHIKV structural proteins were engineered into the backbones of the aforementioned vaccines to produce the chimeras [3] . These chimeras were found to stimulate a strong humoral immunity, and even at doses of 5.3-5.8 log 10 PFU, they did not trigger reactogenicity. When vaccinated mice were challenged with CHIKV, neither adult nor neonatal mice gained weight, had fever, or displayed signs of neurological illness. Upon comparison of the chimeras with the Army181/25 vaccine, the Army vaccine resulted in higher levels of viremia and replication in the joints of neonatal mice. Because the joints are known targets of CHIKV, Wang et al. noted their vaccine might avoid the negative reactogenic side effects of the Army vaccine. After being subcutaneously vaccinated with 5.3-5.8 log 10 PFU of the chimeric vaccines, mice produced strong neutralizing antibody titers. The VEEV and EEEV chimeras yielded higher neutralizing antibody titers than the SINV chimera without being more virulent. On top of this, the VEEV and EEEV CHIKV chimeras seemed to be more immunogenic than the Army vaccine despite the chimeras' lower viremia and replication in the joints of neonatal mice [3] . Tiwari et al. [46] adopted a different strategy using formalin inactivated CHIKV in combination with alhydrogel (Aluminum Hydroxide) as an adjuvant. This study clearly suggests that this vaccine elicits both humoral and cell-mediated immune responses in mice, providing its immunogenic potential. A recent study by Couderc et al. [47] showed passive immunization as a potential treatment for CHIKV infection. Using purified immunoglobulin extracted from convalescent CHIKV patients, they demonstrated effective neutralizing activity against CHIKV infection both in vitro and in vivo. This thereby establishes a potential preventive and therapeutic approach to combat CHIKV infection. Pathogenesis studies conducted with related alpha virus, like RRV, have shown the role of macrophages in persistence on infection [48] . They also demonstrated the role of RRV-specific CD8 T cells in clearing viral load in infected patients, thereby warranting similar investigations with CHIKV and the importance of investigating a cell-mediated immune response-based vaccine against CHIKV [49] . There are always certain risks associated with live attenuated or inactivated viral vaccines [50] . One way to avoid these potential problems is to construct a consensus-based DNA vaccine. DNA based vaccines have an improved safety profile as compared to live or attenuated vaccines [51, 52] . A consequence of CHIKV's rapid evolution is difficulty in constructing a vaccine that will be able to Figure 3 . Levels of CHIKV-specific IgG in mice immunized with CHIKV vaccines. Each group of C57BL/6 mice (n = 5) was immunized with 12.5 mg of pVax1 control vector or CHIKV vaccine plasmids as indicated at 0 and 2 wk. Mice were bled 2 wk after each immunization, and each group's serum pool was diluted to 1:100 and 1:500 for reaction with specific vaccine constructs. Serum was incubated for 1 h at 37uC on 96-well plates coated with 2 mg/ml of respective CHIKV peptides, and antibody was detected using anti-mouse IgG-HRP and OD was measured at 405 nm. doi:10.1371/journal.pntd.0000623.g003 www.plosntds.org effectively protect large populations from multiple strains of the virus. One of the strengths of DNA consensus vaccines is its ability to induce cross-reactive immune responses against the three distinct phylogenetic groups of CHIKV. Also DNA-based vaccines can be produced more rapidly than protein-based vaccines. Recently, Muthumani et al. constructed a vaccine that was shown to induce both humoral and cellular immunity in vivo in 3-4-wk-old female C57/BL6 mice [49] . These mice were immunized using an in vivo electroporation method to deliver the vaccine into the quadriceps muscle. The consensus construct was designed against E1, E2, and the core protein capsid. To design the construct, they aligned 21 sequences of CHIKV isolated between 1952 and 2006, using strains from differing countries, including La Reunion Island. The most common nucleotide among the sequences was chosen at each position to be used in the consensus construct, taking care not to alter the reading frame. They conducted codon and RNA optimization, added a strong Kozak sequence, and substituted signal peptide with an immunoglobulin E leader sequence to improve vaccine efficacy. After immunizing the mice, spleens were harvested along with serum and tested to determine antibody titer. After three immunizations, consensus E1, E2, and C vaccines were shown to induce T-cell immune responses leading to strong IFN-c responses and proliferation in C57/BL6 mice. Furthermore, when compared with control mice, immunized mice had higher total IgG levels as well as higher anti-E1 specific, anti-E2 specific, and anti-C specific IgG antibodies, suggesting a strong humoral immune response ( Figure 3 ) and also specificity for the antigens encoded in the vaccine constructs ( Figure 4 ). Because of its promising results and the need for a safer vaccine, this consensus DNA vaccine deserves further investigation. Determining longevity of protective effects of the vaccine and persistence of antibody and IFN-c responses could be the next step of investigation. Challenged studies of immunized mice must also be carried out. CHIKV mosquito-borne disease has caused massive outbreaks for at least half a century but is no longer confined to the www.plosntds.org developing nations. It began to encroach into the boundaries of the developing world. As a result, the NIAID has designated CHIKV as a Category C pathogen alongside the influenza and SARS-CoV viruses [3] . Realization of the potential severity of this disease is exigent; for instance, if used as a biological weapon, the world economy could be severely crippled; if enough members of the armed forces were to become infected during a military deployment, military operations could be significantly affected. Efforts to monitor the disease will only provide minimal warning in a global society, and steps to prevent the morbidity and mortality associated with pandemic are imperative [21, 31] . Despite the gravity of its infectious potency and the fear of it being a potential biological weapon, there is currently no vaccine for CHIKV infections. Live attenuated vaccine trials were carried out in 2000, but funding for the project was discontinued. Newer approaches such as DNA vaccines appear promising over conventional strategies like live attenuated or inactivated virus and thus call for further investigation. Recent advances such electroporation delivery and incorporation of adjuvants has boosted DNA vaccine efficacy [51, 53] . Despite the low antibody response to DNA vaccines, other numerous advantages have overshadowed these minor drawbacks (Table 2) , the most important one being the ability to induce both humoral and cellular immune responses [51, 54] . Judging by recent success, such as the immunogenic construct developed by Muthumani et al., DNA vaccines could play a major role in combating CHIKV [49] . Vaccines are literally a critical component of CHIKV disease control and therefore research in this area is highly encouraged. The dramatic spread of dengue viruses (DENV) throughout tropical America since 1980 via the same vectors and human hosts underscores the risk to public health in the Americas. The adverse events associated with the current live vaccine are well documented [55] . Realizing these drawbacks, earnest efforts should be taken to develop new strategies to forestall further spread and complications.
What is the status of Asian CHIKV since its emergence?
false
2,492
{ "text": [ "has come a long way, with several mutations incorporated, and has continued to wreak epidemics in several regions" ], "answer_start": [ 3640 ] }
1,660
Hantaviruses in the Americas and Their Role as Emerging Pathogens https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/ SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101 Authors: Hjelle, Brian; Torres-Pérez, Fernando Date: 2010-11-25 DOI: 10.3390/v2122559 License: cc-by Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] . The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖). Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: (1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] . (2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] . (3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil). Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] . Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] . Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease.
What leads to death in most fatal cases of HCPS?
false
4,556
{ "text": [ "not hypoxemia, due to the prominent pulmonary edema" ], "answer_start": [ 17472 ] }
650
Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776769/ Tsukamoto, Takashi; Li, Xianglan; Morita, Hiromi; Minowa, Takashi; Aizawa, Tomoyasu; Hanagata, Nobutaka; Demura, Makoto 2013-09-18 DOI:10.1371/journal.pone.0075831 License:cc-by Abstract: Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation. Text: The interferon-induced transmembrane (IFITM) protein family (also known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino-and carboxyl-terminal polypeptide sequences (Figure 1-A) . The IFITM proteins are evolutionarily conserved in vertebrates [2] . Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10). These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3] [4] [5] , cell-to-cell adhesion (IFITM1) [6] [7] [8] , antiviral activity (IFITM1-3) [9] [10] [11] [12] [13] [14] [15] [16] [17] , and bone formation (IFITM5) [18] [19] [20] [21] [22] , although the detailed functions of IFITM6, 7, and 10 are unknown at present. In particular, IFITM3 has been a target of intensive studies on its activity against influenza A (H1N1) virus infection and internalization [9] [10] [11] [12] [13] [14] . In 2010, Dr. Yount and co-workers reported that the antiviral activity of IFITM3 is dependent on S-palmitoylation on the protein [10] . The S-palmitoylation [23] is a post-translational modification on proteins by C 16 saturated-fatty acids (palmitic acids) covalently attached to certain cysteine residues via a thioester linkage (Figure 1-B) . The modification is reversibly catalyzed by protein acyltransferases and acylprotein thioesterases, and confers unique properties to the protein, such as membrane binding and targeting, immunoreactivity, Amino-acid sequence alignment of IFITM5, IFITM1, IFITM2, and IFITM3 derived from mice. The conserved residues are highlighted in black. The three conserved cysteines are highlighted in red and numbered based on the sequence of IFITM5 (top) and IFITM3 (bottom). The residues unique in IFITM5 are highlighted in gray. The first and the second transmembrane domains, the extracellular sequences, and the cytoplasmic loop are indicated by arrows and denoted as TM1 and TM2, EC, and the CP loop, respectively. The TM domains were predicted by SOSUI. The aspartates at the C-terminal region in IFITM5 are shown in blue. B) The schematic illustration of the protein S-palmitoylation. The C 16 -palmitic acid is attached to cysteine via a thioester linkage. The palmitoylation and depalmitoylation are catalyzed by protein acyltransferases and acylprotein thioesterases, respectively. In this study, hydroxylamine, NH 2 OH, was used to reduce the thioester linkage. C) The amino acid sequence identity (similarity) among IFITM5, IFITM1, IFITM2, and IFITM3 is summarized. doi: 10.1371/journal.pone.0075831.g001 and protein-protein interaction. The authors revealed that IFITM3 is S-palmitoylated on three membrane proximal cysteines, Cys71 and Cys72 in the first transmembrane (TM1) domain, and Cys105 in the CP loop (Figure 1-A) [10] . In addition, IFITM3 lacking the S-palmitoylation is not clustered in the cell membrane and significantly diminishes the antiviral activity. Moreover, the cysteines in IFITM2, Cys70, Cys71, and Cys104 are also palmitoylated in the same manner, which affects the intracellular localization [24] . A resent study has revealed that murine IFITM1 has four cysteine residues (Cys49, Cys50, Cys83, and Cys103) for the S-palmitoylation, which is required for the antiviral activity and the protein stability [25] . The other IFITM family members also possess these cysteines (Figure 1-A) , and thus the role of the Spalmitoylation on the cysteines should be significant for the functions of IFITM proteins. Here, we focused on IFITM5, which is also known as bonerestricted IFITM-like (BRIL) protein [18] . Among the IFITM family proteins, IFITM5 is unique. (i) Expression of IFITM5: Unlike the other IFITM family proteins, the expression of IFITM5 is not induced by interferons because the region upstream of the ifitm5 gene lacks the interferon regulatory elements [26] . Furthermore, the expression of IFITM5 is mostly restricted to osteoblast cells [18, 19, 27] , while the other IFITM proteins are expressed ubiquitously (ii). Amino-acid sequence similarity: The amino acid sequence of IFITM5 is relatively dissimilar to IFITM1-3 proteins (~ 65% similarity), while IFITM1-3 proteins share ~ 85% similarity with each other (Figure 1 -C). In addition, IFITM5 has an aspartate-rich domain in the C-terminal region, which could be involved in calcium binding (Figure 1 -A) [26] . (iii) Role of IFITM5 in bone formation: The expression of IFITM5 is associated with mineralization during the bone formation process in osteoblast cells [18] [19] [20] [21] . Previous studies have confirmed the expression of IFITM5 in bone tissues in mice, rats, humans and tammar wallabies [2] . The ifitm5-gene knockout mice have smaller bones [19] . Moreover, the knockdown of the ifitm5 gene by small hairpin RNA induces a decrease in bone nodule formation, whereas overexpression of the gene in UMR106 cells has been shown to increase calcium uptake and bone nodule formation [18] . (iv) Role of IFITM5 for immune activity: Recent studies have revealed that IFITM5 interacts with the FK506-binding protein 11 (FKBP11) to form IFITM5-FKBP11-CD81-the prostaglandin F2 receptor negative regulator (FPRP) complex [28] . When the complex is formed, the expressions of 5 interferon-induced genes are induced, including bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferoninduced protein with tetratricopeptide repeats 3 (Ifit3), b(2)microglobulin (B2m), and MHC class I antigen gene. Consequently, these results indicate that IFITM5 is involved not only in the bone formation but also in the immune system activity. In this study, we investigated the S-palmitoylation of IFITM5 and its role in the interaction with FKBP11 in mouse osteoblast cells. Cells transfected by a plasmid DNA encoding mouse IFITM5 were grown in the presence of an established chemical reporter, 17-octadecynoic acid (17-ODYA) [29, 30] , or an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP) [31] . The biochemical assays using these compounds revealed that the wild-type IFITM5 is S-palmitoylated. To identify the Spalmitoylation site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C86A, -C52A/C53A, and -C52A/53A/86A (Cys-less). The chemical reporter assay suggested that at least two out of three cysteines in IFITM5 are S-palmitoylated. The interaction of IFITM5 with FKBP11 was examined by immunoprecipitation assay, resulting in the loss of the interaction in the presence of 2BP. The same result was obtained in the two mutants, C52A/C53A and Cys-less. These results suggested that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain of IFITM5 is necessary for the interaction with FKBP11. On the other hand, Cys86 in the CP loop of IFITM5 was S-palmitoylated but not involved in the interaction. Because this interaction is important for the immunologically relevant gene expression, it was indicated that the role of the S-palmitoylation is to promote the interaction of IFITM5 with FKBP11 and to regulate the immune activity in the osteoblast cells. The possible interaction mechanism and the effect of the S-palmitoylation on the bone nodule formation will be discussed. For mammalian cell expression, plasmid vectors of wild-type IFITM5 (IFITM5-WT) and FLAG-fused FKBP11 (FKBP11-FLAG) were constructed by inserting the cloned genes into a pBApo-CMV Neo expression vector (Takara Bio, Shiga, Japan). The details of the recombinant DNA constructs were the same as described previously [19] . The genes of IFITM5 mutants (IFITM5-C86A, -C52A/53A, and -C52A/C53A/C86A (Cys-less)) were prepared using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The plasmid vectors of FLAG-fused IFITM5-WT, -C52A/53A, and Cys-less were constructed by inserting the cloned genes into the pBApo-CMV Neo expression vector. For E. coli cell expression, the plasmid vector of IFITM5-WT was constructed by inserting the cloned gene into a pET22b (Novagen, Madison, WI) expression vector. The forward primer 5'-GGAATTCCATATGGACACTTCATATCCCCGTG-3' and the reverse primer 5'-CCGCTCGAGGTTATAGTCCTCCTCATCAAACTTGG-3' were used to amplify the gene encoding the entire IFITM5 from the plasmid vector for mammalian cell expression described above. The underlined letters denote an NdeI and an XhoI cleavage site, respectively. The plasmids of IFITM5 mutants were prepared using a QuikChange site-directed mutagenesis kit. The sense and anti-sense primers used were 5'-GGCAGTATGGCTCCAAAGCCAAGGCGTACAACATCCTGG CTGC-3' and 5'-GCAGCCAGGATGTTGTACGCCTTGGCTTTGGAGCCATACT GCC-3' for IFITM5-C86A; and 5'-GCACGATGTACCTGAATCTGGCGGCGCTTGGATTCCTGG CGC-3' and 5'-GCGCCAGGAATCCAAGCGCCGCCAGATTCAGGTACATCG TGC-3' for IFITM5-C52A/C53A, respectively (Sigma-Aldrich, St. Louis, MO). Osteoblast-like MC3T3 cells were provided by the RIKEN, Cell Bank (RCB 1126). The procedures for cell culture, transfection, and protein expression were the same as reported previously. When necessary, 2-bromopalmitic acid (2BP; Wako, Osaka, Japan) and 17-octadecynoic acid (17-ODYA; Sigma-Aldrich) were dissolved in 99.5% dimethyl sulfoxide (DMSO; Wako) and added to differentiation medium at concentrations of 100 μM and 50 μM in less than 0.1% DMSO, respectively [30, 31] . Wild-type and mutant IFITM5 proteins were also produced using an E. coli recombinant expression system. E. coli BL21(DE3) cells transformed by the expression plasmid were grown at 37°C in LB medium containing 50 μg/mL ampicillin. After four-hour induction by 1 mM isopropyl β-Dthiogalactopyranoside (IPTG), cells were harvested by centrifugation (6,400 × g for 10 min at 4°C). The cells were suspended in 50 mM Tris-HCl buffer (pH 8) and disrupted by a French press (Ohtake, Tokyo, Japan) (100 MPa × 4 times). The crude membrane fraction was collected by ultracentrifugation (178,000 × g for 90 min at 4°C). The collected fraction was solubilized with 1.5% n-dodecyl-β-Dmaltopyranoside (DDM) (Dojindo Lab, Kumamoto, Japan) in 50 mM Tris-HCl, pH 8, containing 0.3 M NaCl and 5 mM imidazole. After the ultracentrifugation, the supernatant was incubated with Ni 2+ -NTA agarose resin (Qiagen, Hilden, Germany). The resin was applied to a chromatography column and washed with 50 mM imidazole containing 50 mM Tris-HCl (pH 8), 0.3 M NaCl and 0.1% DDM. The DDM-solubilized IFITM5 was collected by elution with the same buffer containing 0.3 M imidazole. The sample media were replaced by the appropriate buffer solution by two passages over a PD-10 column (GE Healthcare UK, Ltd., Amersham Place, England). The experimental details are described in previous reports [19, 28] . Briefly, total proteins were extracted from the osteoblast cells which co-expressed IFITM5 and FKBP11-FLAG using a total protein extraction kit (BioChain Institute Inc., Newark, CA). Then, the cell lysate was incubated with anti-FLAG M2 agarose gel (Sigma-Aldrich) at 4°C for 2 h. To recover FKBP11-FLAG, 500 ng/μL 3 × FLAG peptide (Sigma-Aldrich) dissolved in Tris-buffered saline was added to the collected gel at 4°C for 1 h. The recovered proteins and the cell lysate containing total proteins were analyzed by SDS-PAGE (15% ePAGEL; ATTO, Tokyo, Japan) and western blot. The anti-IFITM5 polyclonal antibody, which was prepared from the amino-terminal peptide sequence (TSYPREDPRAPSSRC), and anti-FLAG monoclonal antibody (Sigma-Aldrich) were used as primary antibodies. The HRP-conjugated goat anti-rabbit IgG (H+L) (Zymed Laboratories, San Francisco, CA) and goat anti-mouse IgG (H+L) (Sigma-Aldrich) antibodies were used as secondary antibodies for the anti-IFITM5 and anti-FLAG primary antibodies, respectively. The proteins were detected by chemiluminescent reaction (MercK-Millipore, Billerica, MA). The cell lysate extracted from the osteoblast cells metabolically labeled by 17-ODYA was incubated with anti-FLAG M2 agarose gel to obtain purified FLAG-fused IFITM5 proteins. The 17-ODYA-labeled proteins were chemically labeled with azide-PEG 3 -5(6)-carboxytetramethylrhodamine (TAMRA-azide; Click Chemistry Tools, Scottsdale, AZ) with reference to previous studies [10, 29, 30, 32] and the manufacturer's guide. The proteins separated by SDS-PAGE were visualized using a 532-nm laser for excitation and the fluorescence by TAMRA (565 nm) was detected using a 575nm long-path filter (Typhoon FLA 9000; GE Healthcare). The subcultured osteoblast MC3T3 cells were seeded at a density of 5,000 cells/cm 2 in 40 mm dishes and cultured in α-Modified Eagle's Medium (α-MEM; Sigma-Aldrich) containing 10% (v/v) fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan). On the next day, this was replaced with differentiation medium, containing 2 mM glycerophosphate and 50 μg/mL sodium ascorbate at final concentrations, to induce osteoblast differentiation. When necessary, 100 μM 2BP in less than 0.1% DMSO, or 0.1% DMSO alone was added to the differentiation medium at final concentrations. All cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 27 days. Mineralized nodules were stained with Alizarin Red S (Sigma-Aldrich). The standard staining procedure was used. The mineralized nodules were checked every three days. To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2BP, which inhibits the S-palmitoylation (Figure 2-A) [31] . Then, the cell lysate containing total protein was extracted for use in the SDS-PAGE and western blot analyses. For purposes of comparison, E. coli cells were also cultured in the absence of 2BP and the cell lysate was extracted. Figure 2 -B shows the results of the western blot assay for IFITM5-WT expressed in the osteoblast and the E. coli cells. In the osteoblast cells, IFITM5-WT exhibited a single band near the 17.4 kDa molecular-mass marker (see lane 1) in the absence of 2BP. However, in the presence of 2BP (see lane 4), the band appeared at a lower position than that in the absence of 2BP (lane 1). These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively. The S-palmitoylation is a reversible reaction, and therefore is depalmitoylated by a strong reductant such as hydroxylamine [10] . Following hydroxylamine treatment (see lane 2), the band appeared at the same position as in the presence of 2BP (lane 4). In prokaryote E. coli cells, the post-translational modification S-Palmitoylation on IFITM5 PLOS ONE | www.plosone.org does not occur. Hence, the band was also observed at the same lower position (see lane 3). In the case of IFITM3, the palmitoylation was also reported to induce a change in mobility on electrophoresis, just as in our present results [10] . For direct observation of the S-palmitoylation, an established chemical reporter, 17-ODYA (Figure 2-C) , was used. The osteoblast cells harboring the plasmid encoding IFITM5-WT were cultured in the presence of 17-ODYA to label the protein metabolically. Following the extraction and the purification of the cell lysate, the labeled IFITM5-WT was ligated with TAMRA-azide according to the Cu(I)-catalyzed [3+2] azidealkyne cycloaddition method [10, 29, 30, 32 ]. An in-gel fluorescence image of the 17-ODYA-TAMRA-labeled IFITM5-WT (see lane 2 in Figure 2 -D) showed that IFITM5 was Spalmitoylated in the osteoblast cells. The FLAG-tag attached to IFITM5 has no influence on the modification and chemical labeling (lanes 1 and 5). In addition, after the hydroxylamine treatment (see lane 6), the fluorescence became weak because of the dissociation of 17-ODYA from IFITM5, which was the same mechanism as the dissociation of the palmitic acid from IFITM5 by reduction as described above (lane 2 of Figure 2-B) . Therefore, we concluded that the IFITM5 expressed in the native osteoblast cells is S-palmitoylated. In addition, the bands corresponding to the high and the low molecular-mass forms shown in western blot analysis were tentatively assigned to the S-palmitoylated and the depalmitoylated forms, respectively. As described above in the Introduction, cysteine residues are the substrate for S-palmitoylation. IFITM5 possesses three cysteines, Cys52 and Cys53 in the TM1 domain, and Cys86 in the CP loop (Figure 1-A) . All of these cysteines are highly conserved among the mammalian IFITM family proteins (Figure 3-A) . To identify the modification site in IFITM5, we prepared cysteine-substituted mutants, IFITM5-C52A/C53A, -C86A, and -C52A/C53A/C86A (Cys-less). The osteoblast cells harboring each plasmid were cultured in the absence of 2BP, and then the cell lysate was extracted. Figure 3 -B shows the results of the western blot detecting the expression of all the mutants in the osteoblast cells. In the C52A/C53A and Cys-less mutants (see lanes 2 and 4), the low molecular-mass form was detected. This result indicates that either Cys52 or Cys53 is involved in the S-palmitoylation. In addition, as shown in Figure 2 -D, strong and weak fluorescence were detected in the C52A/ C53A mutant in the absence and presence of hydroxylamine (lanes 3 and 7) , respectively, but not in the Cys-less mutant (lanes 4 and 8) . These results suggested that the rest of the cysteine in the C52A/C53A mutant, Cys86, is S-palmitoylated and the Cys-less mutant completely lost the S-palmitoylation because all the cysteines were substituted. Therefore, we concluded that Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated. In addition, it was found that the S-palmitoylation on the TM1 domain has a major effect on the mobility in the gel (lower panel of Figure 2 -D and Figure 3-B) . Therefore, we hereafter refer to the high and low molecular-mass forms as the TM1palmitoylated and the TM1-depalmitoylated forms, respectively. Finally, we reassigned the bands shown in the western blot analysis as follows: IFITM5-WT is fully palmitoylated, the C86A mutant is partially palmitoylated at Cys52 and/or Cys53, the C52A/C53A mutant is partially palmitoylated at Cys86, and the Cys-less mutant is completely depalmitoylated. Previous studies have revealed that IFITM5 interacts with FKBP11 [19] . FKBP11 belongs to the FK506-binding protein family and has a transmembrane domain. The interaction between IFITM5 and FKBP11 is important for the immune activity because formation of the IFITM5-FKBP11-CD81-FPRP complex induces the expression of interferon-induced genesnamely, the Bst2, Irgm, Ifit3, B2m, and MHC class I antigen gene [28] . To investigate the effect of the S-palmitoylation on the interaction of IFITM5 with FKBP11, we carried out an immunoprecipitation assay. The osteoblast cells co-transfected by the plasmids encoding IFITM5-WT and FKBP11-FLAG were cultured in the absence and the presence of 2BP. Then, the extracted cell-lysate was incubated with anti-FLAG agarose gel. The gel was washed several times. Finally, the proteins were competitively eluted by the addition of FLAG peptide. If IFITM5 interacted with FKBP11, it was expected that IFITM5 The conserved cysteines are highlighted in orange and numbered. In the lower panel, the numbers given in parenthesis correspond to the residual number for IFITM2. For the calculation of probability, a total of 23 IFITM2, 23 IFITM3, and 17 IFITM5 sequences derived from mammalian species in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used. Sequence alignment was carried out using CLUSTALW. Sequence logos were generated using WEBLOGO 3. B) Western blot for the wild-type and cysteine-substituted mutants of IFITM5 expressed in the osteoblast cells. For detection, the anti-IFITM5 antibody was used as a primary antibody. The upper arrow indicates that C52 and/or C53 in the TM1 domain is Spalmitoylated (lanes 1 and 3) . The C52A/C53A (lane 2) and Cys-less (lane 4) mutants are partially and completely depalmitoylated. The experiment was carried out 2 times. doi: 10.1371/journal.pone.0075831.g003 would be obtained during this step and detected by immunoblotting. Figure 4 -A shows the results of the western blot for the co-immunoprecipitation of IFITM5-WT with FKBP-FLAG. The band corresponding to FKBP11 appeared in all the lanes (upper panel). Lanes 1 and 2 are controls to ensure that IFITM5 and FKBP11 are both contained in the cell lysate before the immunoprecipitation. The controls also ensured that IFITM5 was S-palmitoylated in the absence of 2BP (see lane 1), whereas IFITM5 was not S-palmitoylated in the presence of 2BP (see lane 2). After the immunoprecipitation, a single band corresponding to the S-palmitoylated IFITM5 appeared in the absence of 2BP (see lane 3), indicating the interaction of the Spalmitoylated IFITM5 with FKBP11. However, in the presence of 2BP, no band corresponding to IFITM5 appeared (see lane 4) , indicating that the two molecules do not interact with each other. These results suggest that the S-palmitoylation on IFITM5 contributes to the interaction with FKBP11. Next, we further investigated the relationship between the Spalmitoylation and the interaction with FKBP11 by using the IFITM5 mutants described above. The osteoblast cells cotransfected by the plasmids encoding IFITM5 mutants (C52A/ C53A, C86A, and Cys-less) and FKBP11-FLAG were cultured. The immunoprecipitation assay was carried out in the same way as described above. Figure 4 -B shows the results of the western blot for the co-immunoprecipitation of the wild-type and the IFITM5 mutants with FKBP11. Figure 4 -C shows the results of the control experiment using the cell lysate before the immunoprecipitation. As described in the previous section 3-3, the band corresponding to FKBP11 appeared in all the lanes (upper panels) because the immunoprecipitation was carried out using the anti-FLAG agarose gel. In the lower panel of Figure 4 -B, single bands were observed for the IFITM5-WT and -C86A mutant (lanes 1 and 3) but not for the -C52A/C53A and Cys-less mutants (lanes 2 and 4) . This result indicates that the wild-type and the C86A mutant interact with FKBP11, whereas the other two mutants do not. Interestingly, this tendency mirrored the trend for the S-palmitoylation profiles, which means that Cys52 and/or Cys53 in the TM1 domain of the IFITM5-WT and -C86A mutants is S-palmitoylated, whereas these residues are not S-palmitoylated in the C52A/C53A and Cys-less mutants (see Figures 2-D, 3 -B and the lower panel of Figure 4 -C). Because the S-palmitoylation contributes to the IFITM5-FKBP11 interaction, as described in the previous section 3-3 (also in Figure 4-A) , the results of Figure 4 -B suggest that the mutants which lost the S-palmitoylation site(s), Cys52 and/or Cys53, are not able to interact with FKBP11. In other words, the S-palmitoylation on these cysteines is necessary for the interaction of IFITM5 with FKBP11. As described above in the Introduction, previous studies have revealed that IFITM5 also contributes to bone formation [18] [19] [20] [21] . Therefore, we investigated the influence of Spalmitoylation on the bone nodule formation in osteoblast cells, in which native IFITM5 is expressed. Figure 5 shows the time-dependent nodule formation in the absence and the presence of 2BP ( Figure 5-A and -B) . Figure 5 -C shows the results of the control trial to verify the effect of DMSO, which was used as the solvent for 2BP, on the nodule formation. The mineralized nodule was stained with Alizarin Red, which reacts with deposited calcium. In Figure 5 -D, the area of the mineralized nodule was plotted against experimental time. In the absence of 2BP (Figure 5-A, -C, and -D) , the mineralization was started 15 days after the initiation of the cell differentiation (Day 0). On the other hand, in the presence of 2BP ( Figure 5-B and -D) , the nodule was formed on Day 12. The halftime for the maximum mineralization in the presence of 2BP was estimated to be 7 days earlier than that in the absence of 2BP (Figure 5-D) . In addition, differences in the form of the mineralized nodules were observed. Figure 5 -E shows an enlarged view of each nodule on Day 21. The stained nodules were diffused in the presence of 2BP (panel b), whereas in the absence of 2BP the nodules formed a large cluster (panels a and c). Therefore, our observations in this study suggested that the S-palmitoylation affects the bone nodule formation in the osteoblast cells. In this study, we confirmed the S-palmitoylation on IFITM5 in the osteoblast cells, which was the same as that previously reported for IFITM3 and IFITM2. As reported previously, in IFITM3 and IFITM2, which share 85% sequence similarity (Figure 1-C) , two cysteines in the TM1 domain (Cys71 and Cys72 for IFITM3, Cys70 and Cys71 for IFITM2) and one cysteine in the CP loop (Cys105 for IFITM3, Cys104 for IFITM2) are all S-palmitoylated in cells [10, 24] . On the other hand, although IFITM5 shares 68% and 66% sequence similarity to IFITM3 and IFITM2, respectively, more than one cysteine in the TM1 domain (Cys52 or Cys53) and one cysteine in the CP loop (Cys86) are S-palmitoylated. Taking into account the high conservation of three cysteines in the IFITM proteins (Figures 1-A and 3-A) , all the cysteines in IFITM5 may be involved in the S-palmitoylation just as in the case of IFITM3 and IFITM2 [10, 24] . The roles of the S-palmitoylation on IFITM3 have been studied intensively, and the S-palmitoylation has been shown to be crucial for the correct positioning in the membrane and the resistance to viral infection and internalization [10] (the roles are summarized in Figure 6 -A and discussed in detail below). A recent study has revealed that the S-palmitoylation on IFITM2 is also important for the protein clustering in the membrane [24] . However, we do not know the role of the Spalmitoylation of IFITM5 for the clustering in the membrane at present because we have not yet succeeded in obtaining a proper antibody for immunohistochemistry, despite our allocating much time to the search and considering a considerable number of antibodies. Dr. Hanagata and co-workers previously reported that IFITM5 lacking the TM1 domain and the CP loop, which and IFITM5 (lower panels), the anti-FLAG and the anti-IFITM5 antibodies were used as primary antibodies, respectively. Arrows indicate the existence of each protein and the S-palmitoylation on IFITM5. A) Western blot for the co-immunoprecipitation of the wild-type IFITM5 with the FLAG-fused FKBP11 (FKBP11-FLAG) in the osteoblast cells in the absence and the presence of 2BP (denoted as "-" and "+", respectively). Lanes 1 and 2 are the results for the control trials used to verify the existence of IFITM5 and FKBP11 before the immunoprecipitation, and Lanes 3 and 4 show the results after the immunoprecipitation. The experiment was repeated 3 times. B) Western blot for the co-immunoprecipitation of the wild-type and the cysteine-substituted mutants of IFITM5 with FKBP11-FLAG in the osteoblast cells. The band corresponding to FLAG peptide is not shown because of the smaller molecular-mass of FLAG peptide relative to FKBP11-FLAG. C) The control experiment of Figure 4 -B used to verify that IFITM5 and FKBP11 were both present in the cell lysate before the immunoprecipitation. The experiment was repeated 2 times. A) The functional mechanism of IFITM3 is summarized from previous studies. (i) IFITM3 is S-palmitoylated at Cys71, Cys72, and Cys105, (ii) which induces clustering and correct positioning in the membrane, (iii) resulting in the antiviral activity against influenza virus. B) The functional mechanism of IFITM5 is summarized by combining the results from the present and the previous studies. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylation allows IFITM5 to interact with FKBP11 in the osteoblast cells (iii). The dissociation of CD9 from the FKBP11-CD81-FPRP/CD9 complex is induced by formation of the IFITM5-FKBP11-CD81-FPRP complex and leads to the immunologically relevant gene expression. IFITM5 also contributes to the bone formation, but it is unknown which states as described in (i)-(iii) are important for the bone formation at present.At present, no interactive protein has been identified in IFITM3 and IFITM2. On the other hand, IFITM5 interacts with the partner protein, FKBP11, and the S-palmitoylation clearly makes a significant contribution to the interaction. Therefore, IFITM5 forms a hetero-oligomer in the cell membrane for its physiological function. contain the relevant modification sites, lost the ability to interact with FKBP11 [19] . In the present study, we determined that the S-palmitoylation on Cys52 and/or Cys53 in the TM1 domain is necessary for the interaction. From these results, we speculate that Cys52 and Cys53 face toward the interaction surface with FKBP11, and therefore IFITM5 and FKBP11 interact with each other through the palmitic acid(s) attached to the cysteine(s) (summarized in Figure 6 -B, discussed in detail later). Our investigation revealed that Cys86 is involved in the Spalmitoylation but does not contribute to the interaction with FKBP11. We speculate that some other residues in the CP loop located near the TM1 domain make some contribution to the interaction. Previous investigations also revealed that IFITM5 expressed in the heterologous fibroblast NIH3T3 cells exhibited direct interactions with CD81, the B cell receptor-associated protein 31 (BCAP31), and the hydroxysteroid (17-beta) dehydrogenase 7 (HSD17b7). These three proteins bind to the IFITM5 without the S-palmitoylation (low molecular-mass form; see Figure 3 -b in ref [19] . and Figure 1 -B in ref [28] .). In the fibroblast cells, the S-palmitoylation on IFITM5 is insufficient [19] . These interactions are not observed in the native osteoblast cells, and therefore are nonspecific. Taking these facts into consideration, we speculate that the S-palmitoylation on IFITM5 promotes the specific interaction with FKBP11 in the osteoblast cells. The role played by the S-palmitoylation of IFITM5 in immune activity of the osteoblast cells will be discussed by combining the results from the present and the previous studies. A specific interaction between IFITM5 and FKBP11 should be necessary to form the IFITM5-FKBP11-CD81-FPRP complex. CD81, also known as TAPA-1, is a member of the tetraspanin membrane protein family and a component of the B-cell coreceptor complex which mediates the B-cell signaling for immune responses. When forming this complex, CD9, a partner protein with CD81, dissociates from the FKBP11-CD81-FPRP/CD9 complex and consequently induces the osteoblastspecific expression of the interferon-induced genes, Bst2, Irgm, Ifit3, B2m, and the MHC class I antigen gene [28] . If the Spalmitoylation-mediated specific interaction of IFITM5 with FKBP11 were lost, the IFITM5-FKBP11-CD81-FPRP complex would not be formed, and consequently the interferon-induced gene expression would be inhibited because CD9 would remain associated with the FKBP11-CD81-FPRP/CD9 complex. In this respect, we speculate that IFITM5 is involved in the immune system activity in the osteoblast cells and the interaction of the S-palmitoylated IFITM5 with FKBP11 regulates the immune activity. In addition, it was suggested that the S-palmitoylation on IFITM5 contributes to the bone nodule formation, including morphology and time for mineralization, in the osteoblast cells ( Figure 5 ). It is difficult to conclude at present that the lack of the S-palmitoylation on IFITM5 causes the diffusion of the bone nodules (panel b of Figure 5 -E); we can say, however, that IFITM5 will probably not be S-palmitoylated in the cells in the presence of 2BP. While 2BP is commonly used as an inhibitor of palmitoylation, it also targets many metabolic enzymes [33, 34] . Thus, it is also difficult to interpret the results of the long-term incubation of the osteoblast cells in the presence of 2BP. In any case, these are interesting and key observations in terms of clarifying the role played by the S-palmitoylation of IFITM5 in bone formation, and further studies are required. Figure 6 describes a possible mechanism of the interaction of IFITM5 with FKBP11 and the role of IFITM5 in the osteoblast cell function by means of a comparison with IFITM3. In the case of IFITM3, as shown in Figure 6 -A, the following are observed. (i) The three cysteines are all S-palmitoylated (ii). The S-palmitoylation leads to the clustering and the correct positioning of IFITM3 molecules in the membrane (iii). The Spalmitoylation and the following clustering are crucial for the resistance to the influenza virus. When IFITM3 lacks the Spalmitoylation, the IFITM3 molecules do not cluster, which leads to the significant decrease in the antiviral activity. On the other hand, Figure 6 -B shows that the following observations are made in the case of IFITM5. (i) Cys86, plus one or two other cysteine residues in IFITM5, i.e., Cys52 and/or Cys53, are S-palmitoylated (ii). The S-palmitoylated IFITM5 is able to interact specifically with FKBP11. The interaction is presumed to be mediated by the palmitic acid(s) attached to the cysteine(s) facing toward the interaction surface on FKBP11. Cys86 is involved in the S-palmitoylation but not in the interaction of IFITM5 with FKBP11. At present, however, little is known about the role of the S-palmitoylation of IFITM5 for the localization in the membrane. When the S-palmitoylation affects the localization of IFITM5 as in the case of IFITM3 [10] , the S-palmitoylated IFITM5 molecules should be localized in the membrane or the depalmitoylated molecules should be delocalized. The loss of the interaction between IFITM5 and FKBP11 could be due to a relocalization of the depalmitoylated IFITM5 that prevents its association with FKBP11 (iii). The Spalmitoylated IFITM5 interacts with the FKBP11-CD81-FPRP/CD9 complex through FKBP11, which induces the dissociation of CD9 from the complex and the expression of 5 immunologically relevant genes. Finally, IFITM5 forms the IFITM5-FKBP11-CD81-FPRP complex. It is unknown at present which of the three states (i)~(iii) illustrated in Figure 6 -B is important for the bone mineralization of the osteoblast cells. The lack of the S-palmitoylation influences the interaction with FKBP11, which could account for the following complex formation and gene expression. In addition, the bone nodule formation is also affected. Note that the role of the Spalmitoylation has been involved in the bone formation [35] . It is indicated that the S-palmitoylation on IFITM5 plays roles not only for the regulation of the immune activity but also for the bone formation. In conclusion, we have revealed the S-palmitoylation on IFITM5 and its role in the interaction with FKBP11. Not only the immune activity but also the bone mineralization in the osteoblast cells is affected by the S-palmitoylation. In general, the functional role of the S-palmitoylation is different for each protein [36] . For many proteins, the palmitoylation and depalmitoylation cycle is constitutive and regulated by enzymes. Based on the present results, it is difficult to address (i) whether the S-palmitoylation on IFITM5 is constitutive or regulated, or (ii) when and where IFITM5 is S-palmitoylated in the osteoblast cells. Further studies are required and are currently underway.
What interaction is inhibited by the presence of 2-bromopalmitic acid (2BP)?
false
571
{ "text": [ "IFITM5 with FKBP11" ], "answer_start": [ 1807 ] }
1,602
High Burden of Non-Influenza Viruses in Influenza-Like Illness in the Early Weeks of H1N1v Epidemic in France https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157400/ SHA: f4c1afe385e9e31eb5678e15a3c280ba97326554 Authors: Schnepf, Nathalie; Resche-Rigon, Matthieu; Chaillon, Antoine; Scemla, Anne; Gras, Guillaume; Semoun, Oren; Taboulet, Pierre; Molina, Jean-Michel; Simon, François; Goudeau, Alain; LeGoff, Jérôme Date: 2011-08-17 DOI: 10.1371/journal.pone.0023514 License: cc-by Abstract: BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were positive for H1N1v. In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. The aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v. Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . Total nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample. Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP), Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA). Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] . Data from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests By using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] . All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown). Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ). Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05). Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS. Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) . As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%). Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] . Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . Viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients. Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections. In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.
What virus was the most common among the H1N1v negative patients?
false
5,265
{ "text": [ "rhinovirus" ], "answer_start": [ 11928 ] }
2,461
Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/ SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8 Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian Date: 2019-03-15 DOI: 10.1042/bsr20182028 License: cc-by Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets. Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] . As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] . It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics. As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system. In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection. The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China. Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days. Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde. The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] . The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] . The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] . Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately. PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect. Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant. PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine. Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system. The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01). CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets. SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection. A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets. Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection. The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection. Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] . It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells. Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection. As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10. In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets.
How can Bacilius subtilis be used as an oral vaccine?
false
602
{ "text": [ "recombinant vaccine carrier" ], "answer_start": [ 756 ] }
1,582
Exhaled breath condensate sampling is not a new method for detection of respiratory viruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059288/ SHA: f3b46e7e8f58799207cc44515f859c1daf5e4dfc Authors: Houspie, Lieselot; De Coster, Sarah; Keyaerts, Els; Narongsack, Phouthalack; De Roy, Rikka; Talboom, Ive; Sisk, Maura; Maes, Piet; Verbeeck, Jannick; Van Ranst, Marc Date: 2011-03-04 DOI: 10.1186/1743-422x-8-98 License: cc-by Abstract: BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. METHODS: In our study, 102 volunteers experiencing upper airway infection were recruited over the winter and early spring of 2008/2009 and the first half of the winter of 2009/2010. Ninety-nine EBCs were successfully obtained and screened for 14 commonly circulating respiratory viruses. To investigate the efficiency of virus isolation from EBC, a nasal swab was taken in parallel from a subset of volunteers. The combined use of the ECoVent device with the RTube™ allowed the registration of the exhaled volume and breathing frequency during collection. In this way, the number of exhaled viral particles per liter air or per minute can theoretically be estimated. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. Rhinovirus was detected in 6 EBCs and 1 EBC was Influenza B positive. We report a viral detection rate of 7% for the EBCs, which is much lower than the detection rate of 46.8% observed using nasal swabs. CONCLUSION: Although very promising, EBC collection using the RTube™ is not reliable for diagnosis of respiratory infections. Text: Human respiratory tract infections represent the most commonly encountered infections worldwide. In the majority of cases, the etiology of these infections remains undetermined due to rapid convalescence after infection. Respiratory tract infections in healthy adults can be caused by a variety of pathogens and the detection of these agents is currently based on their isolation from nasal swabs (NS), bronchoalveolar lavages (BAL), nasopharyngeal aspirates and sputum samples. The acquisition of these specimens by semi-invasive and invasive techniques is often unpleasant for the patient. Therefore, exhaled breath condensate (EBC) analysis has recently been explored as a new and non-invasive method to monitor lung inflammation and pulmonary disease such as chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer etc. EBCs mainly consist of water vapour but a small fraction contains respiratory droplets derived from the airway lining fluid [1, 2] . This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. At first, investigators suspected that turbulence of the inhaled air was responsible for the aerosolisation of the respiratory fluid. However, the effect of the turbulent airflow is limited to the upper airways since the turbulent airflow becomes laminar as it reaches the smaller bronchial airways and alveoli. Recently, the bronchiole fluid film burst model has been described [3] . This model suggests that aerosols are produced during inhalation by the bursting of fluid bubbles present in the bronchioles. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. Therefore we screened the EBC samples with virus specific PCR assays targeting 14 In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). The patient was instructed to breath orally at tidal volumes into a mouthpiece attached to a condenser for 10 minutes. No nose clips were used during collection and saliva contamination was avoided by the presence of a one-way valve and the T-shaped section of the mouthpiece. In a first part of the study that started during the winter and spring of 2008/2009, 70 EBC samples were collected from patients who voluntary presented themselves to our laboratory. The majority of these volunteers were students that responded to the information leaflet, distributed in the university buildings of the Catholic University of Leuven. The samples were collected with the aluminium cooler sleeve chilled at -80°C. In the fall and first half of the winter of 2009/2010, 32 condensates were collected from patients who presented themselves to their general practitioner. Due to practical circumstances, the condensates were collected with the cooler chilled at -20°C. For 13 out of 32 collections, the RTube™ was connected by a custom made connectingpiece to the ECoVent (Jaeger, Germany). This device registers ventilatory parameters such as the exhaled volume, breathing frequency and tidal volume. Additionally, a NS was obtained in parallel with the condensate collection from each patient. All EBCs were immediately stored at -20°C. Nasal swabs (NS) were refrigerated. After viral DNA and RNA extraction, EBC samples and nasal swabs were stored at -80°C. Three specimens were excluded from the study due to incorrect condensate collection. A short questionnaire was used to document the date of birth, the severity of respiratory complaints and to record the days of symptomatic illness from all volunteers. This study was approved by the Medical Ethics Committee of the University Hospital of Leuven and informed consents were received from all participants. Viral DNA and RNA were isolated with the QIAamp MinElute Virus kit (Qiagen, Westburg, The Netherlands) according to the instruction manual. EBC extracts were eluted in 60 μl elution buffer and NS extracts in 110 μl elution buffer. The breath condensates were screened for 11 respiratory RNA viruses (CoV NL63, E229 and OC43, RV, HMPV, InfA&B and PIV1-4) [4] [5] [6] [7] using a OneStep RT-PCR Kit (Qiagen, Westburg, The Netherlands) in a 50 μl reaction containing 10 μl of the extracted RNA, 0.6 μM of forward and reverse primers (Table 2), 1.5 μl One Step Enzyme Mix, 10 μl 5 × One Step RT-PCR Buffer and 400 μM of each dNTP. For adenovirus screening, a DNA PCR was carried out for which the amplification reaction mix contained 0.5 μM forward primer (AdFW) and reverse primer (AdRV), 0.4 mM dNTPs, 10 μl Buffer C and 1 U Taq polymerase in a final volume of 50 μl. The PCR primers used were located in conserved regions of the genomes of the respiratory pathogens ( Table 2 ). The reactions were carried out in a T3000 Thermocycler 48 (Westburg, Leusden, The Netherlands) with an initial reverse transcription step for RNA viruses at 50°C for 30 min, followed by PCR activation at 95°C for 30 s, 45 cycles of amplification followed by a final extension step for 10 min at 72°C. The DNA amplification program was initiated with a denaturation step at 94°C for 3 min, followed by 45 cycles of 94°C for 30 s, 55°C for 30 s and a final extension step at 72°C for 1 min. The amplicons were subjected to a 6% polyacrylamide gel and visualised under UV light by staining with ethidium bromide. PCR products were purified using the Invitek MSB Spin PCRapace Kit and cycle sequenced in forward and reverse direction using the ABI PRISM Big-Dye Termination Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed with the ABI3130 Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Consensus sequences were obtained using the SeqMan II software (DNASTAR, Madison, Wis.). For samples from HRSV was detected using a RT-PCR assay as previously described [8, 9] . In brief, a multiplex mix was prepared in a final volume of 25 μl using 5 μl extracted RNA, 12.5 μl of Eurogentec One-Step Reverse Transcriptase qPCR Master Mix containing ROX as a passive reference, 0.125 μl Euroscript + RT & RNase inhibitor (Eurogentec, Seraing, Belgium) 200 nM of HRSV-A and -B specific forward and reverse primers and 100 nM of HRSV-A and -B MGB probes. cRNA standards were constructed using the MEGAshortscript T7 kit (Ambion, Austin, TX, USA) and spectrophotometrically quantified. The viral load of RV positive samples were quantified by qRT-PCR as described in the manuscript published by Lu and coworkers [10] . The Eurogentec One-Step Reverse Transcriptase qPCR kit was used for preparation of the master mix as described above. The primerset HRSV-AF F 669-695 ctgtgatagarttccaacaaaagaaca [8, 9] HRSV-AF F 718-745 agttacacctgcattaacactaaattcc [8, 9] HRSV-BN N 435-458 ggctccagaatataggcatgattc [8, 9] HRSV-BN N 480-508 tggttattacaagaagagcagctatacacagt [8, 9] MGB probes and probe, located in 5'UTR, were added to a final concentration of 1 μM and 0.1 μM, respectively. cRNA standards were constructed based on the PCR product of sample 1 using the MegaScript kit (Ambion, Austin, TX, USA). Quantification was performed with a spectrophotometer at 260 nm and converted to the molecule number [11] . Tenfold serial dilutions, allowing detection in a range of 8.6 × 10 6 to 8.6 × 10 2 RNA copies were used. The RT-PCR assays were carried out on a ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). An initial reverse transcription step was performed at 48°C for 30 min, followed by a denaturation step at 95°C for 10 min. Finally, an amplification step of 45 cycli at 95°C for 15 sec and 1 min at 60°C was completed. (37.5%) men, with a median age of 29 (range 9 -46 years). Age and gender was missing for 2 participants of the second group. In total, 52% of the participants were between 20-30 years old. Only 6% were younger than 20 years old and 3% were older than 70 years. In totality, 80 patients (78.4%) were already feeling ill for 1 to 7 days at the day the sample was obtained. Seven volunteers (6.8%) were symptomatic for 8 to 14 days and 9 participants (8.8%) were already ill for more than 14 days at the day of sample collection. Data on the duration of symptoms was lacking for 6 patients. Almost all volunteers experienced at least 2 symptoms except for two patients (Table 1) . Forty-seven (46.1%) volunteers complained about a constant runny or stuffy nose, 43 (42.2%) had frequent sneezing events and 38 (37.3%) participants had a serious sore throat (Table 1) . In a first part of the study, we collected 70 EBCs. Screening of the EBCs for 14 respiratory viruses (Table 2) , showed 5 RV (7.1%) positive samples (Table 3 ). In a second part, we collected 32 EBCs from patients that presented themselves to their general practitioner. Two of these EBCs were positive for one of the 14 investigated respiratory viruses, 1 for RV and 1 for InfB. To inspect the detection rate of respiratory viruses in the condensate, a NS was taken from this second group of volunteers for comparison. In 15 out of 32 NS (46.8%), one or more viral pathogens were isolated. Viral screening of the NS resulted in the detection of RV, InfA (subtype H1N1) and HRSV-B. Quantification of the HRSV-B viral load demonstrated for samples 72 and 101 viral titers of 8.0 × 10 4 RNA copies/ml and 6.8 × 10 7 RNA copies/ml respectively. The RV RT-PCR assay did not allow the quantification of all samples that tested positive for RV by PCR ( Table 3) . Presence of the same pathogen in both the EBC and the NS was confirmed for only 1 sample: sample 71, which tested positive for RV in both the EBC and the NS. For sample 81, RV was detected in the NS and analysis of the EBC demonstrated an InfB infection. For EBC samples that were collected in the fall and winter of 2009/2010, measurements with the ECoVent in (Table 3 , sample 81) was positive for InfB when using the RTube™ in combination with the EcoVent. In theory, the viral generation rate (number of viral RNA copies exhaled per minute) can be predicted by quantification of the exhaled viral load. Then, an estimation of the RNA copies per litre exhaled air or per minute can be calculated. Quantification of the exhaled InfB would allow us to predict the generation rate for this virus. Due to insufficient sample volume, we could not determine the number of RNA copies in the sample. Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract. The collection of EBC is easy to perform and can be conducted in a home environment. This method is much more agreeable for the patient when compared to the unpleasant and invasive collection of nasal swabs, BAL, aspirates, etc. This aspect renders the method very attractive for routine laboratory diagnostics of viral infections. Most studies that perform breath analyses for viral detection use modified face masks, with a removable central region in electret or a removable Teflon filter on which exhaled particles impact [12] [13] [14] . With the RTube™ collection device, aerosolized particles of the airway lining fluid are precipitated into a condensate when the breath is cooled which serves as an immediate starting point for molecular testing. Until now, this is the study with the largest subset of volunteers that investigated EBC as a specimen for the detection of respiratory viruses. Previous studies reported the inclusion of a limited subset of participants and investigated the presence of a limited number of viruses in the breath samples. The study performed by Fabian and colleagues, included 12 volunteers [12] . Huynh and co-workers recruited 9 volunteers for exhaled breath sampling [13] . In the study by Stelzer-Braid et al., 50 EBCs were analysed [14] and St-George et al. report the participation of 12 adults [15] . These studies have focused on the detection of InfA and -B, PIV1-3, HRSV and HMPV, while we have screened the samples for a panel of 14 commonly circulating respiratory viruses. Based on the analysis of 99 EBCs (3 EBCs were excluded), our results support the exhalation of RV and InfB in 7% of our samples. Since many of the volunteers had already been experiencing symptoms for 1 to 7 days, we initially presumed that they were already recovering from the infection and were no longer exhaling the virus. For common cold infections it is suggested that a person may already be infectious for 1 or 2 days before experiencing any symptoms. However, in a second part of our study we started collecting EBCs in parallel with nasal swabs from patients presenting themselves to their medical doctor, 1 to 3 days after onset of symptoms. Only for 1 condensate the same pathogen was detected in both the EBC and the NS. The detection rate for respiratory viral pathogens in the NS was 46.8% which is much higher than the 7% detection rate in the EBCs. The low detection of virus positive condensates can therefore not be attributed to the fact that volunteers were no longer infectious. The discrepant detection rate between samples may also be explained by different severity of respiratory infection, since comparator samples were of different parts of the respiratory tract. Patients that delivered a positive NS may have possibly suffered from an upper airway infection whereas EBC positive volunteers may have experienced a more advanced, lower respiratory tract infection. However, the effect of nasal inhalation on EBC collection, guiding formed particles in the upper respiratory tract to the lower compartments, in stead of oral inhalation was not investigated. Patients with positive EBC samples were experiencing symptoms for maximum two days at the time of collection. However, this was not different for 7 patients with positive NS. Six patients that provided positive NS were experiencing symptoms for a longer period at the time of collection (Table 3 ). In the group of volunteers that provided an EBC negative or EBC and NS negative sample, the manifestation of symptoms were reported ranging from 1 day to more than two weeks. When reported symptoms were compared between EBC positive patients (7) and NS positive patients (15) , 27% and 33% in the positive NS group experienced shivering and muscle pain whereas this symptom was not indicated by any patient of the EBC positive group. In all groups fever, headache, watering eyes, stuffed nose, frequent sneezing, sore throat and coughing were reported. Volunteers were not diagnosed with other pathogens before participation in the study. Since we did not test these samples for other than viral pathogens, we can not exclude the possibility that some of the negative NS are positive for bacteria or other pathogens causing respiratory illness. Recently, one study reported a detection rate of 5% for influenza in EBC [15] . This is in the same range of the detection rate that we report for respiratory viruses in general. Other studies with a limited number of patients, describe a markedly higher sensitivity of 33 to 36% [12] [13] [14] but the higher percentage may be due to the low number of participants subjects were included [12] . Remarkably, the studies reporting this higher detection rate used collections masks, while the study using the RTube™ reported comparable findings. Face masks consist of electret which trap viruses based on permanently charged fibres [13] . In addition, the Teflon filter has 2 μm pores which will retain all larger particles. Possibly, the lower detection rate can partly be explained by the fact that the RTube™ is manufactured in polypropylene and does not possess a virus attracting and filtering feature like the aforementioned materials. The qRT-PCR developed by Lu and coworkers for the detection of RV, did not allow the assessment of the viral load present in the EBC samples [10] . Also for 4 NS, the viral titer remained undetermined, probably due to the limited sensitivity of the assay. For diagnosis, more sensitive methods might be necessary to detect respiratory viruses present in EBC since it is unpredictable how diluted the viral particles in the specimen are. Recently, nested qRT-PCR assays have been developed to allow a more sensitive detection of viruses in aerosols [16] . Also person-dependent factors, such as the number of particles produced, the exhaled volume and the age of the patient, have been suggested to play an important role for exhalation of viral particles. The participants that were recruited in the study of Fabian and coworkers were 12 years of age and older [12] . For hospitalized children a much higher rate of virus positive samples is reported [14] . In our study, the majority of volunteers were between 20 and 30 years old. Only two children less than 10 years and 3 elderly people (> 70 years) were included. One of the children tested positive for InfA in the NS, but the infection was not confirmed in the EBC. For influenza, an exhaled generation rate of <3.2 to 20 influenza RNA copies per minute was predicted by quantifying the virus aerosols that impacted on a removable Teflon filter of a collection mask [12] . We used the RTube™ in combination with the ECoVent, that allowed the registration of additional ventilation parameters such as breathing frequency and exhaled volume. In this way, when the number of RNA copies in the EBC is quantified, the amount of viral particles that are exhaled per litre or per minute can be estimated. Unfortunately, we were not able to predict a virus generation rate for InfB since viral load remained undetermined. Although an inventive, new and promising method, EBC collected by the RTube™ does not appear to be appropriate for diagnosis of respiratory infections. Nonetheless, this method may provide an alternative for current sample procurement for epidemiological studies of circulating viruses. This technique also confirms the observation that viruses are able to disseminate through normal breathing, particularly RV. In addition, EBC collection from patients during respiratory infections may be further investigated for biomarker patterns. In calves that were experimentally infected with bovine RSV, an increase in leukotriene B 4 , indicating oxidative stress, was observed. This increased level was also associated with the development of bronchial hyperresponsiveness [17] . In humans, a transiently elevated H 2 O 2 level was observed during common cold infection. This marker returned to baseline values when volunteers recovered from infection. H 2 O 2 has also been recognized as an interesting marker in asthma, where it is associated with chronic lower airway inflammation [18] . In InfA infected volunteers, an increased CO level was observed during upper respiratory infection. This observation might imply that CO is an indicator of airway inflammation or represents one of the host defence mechanisms against viral infection [19] . Therefore, a better identification of the biomarker signature in condensates of individuals experiencing a viral infection might imply interesting findings towards the identification of markers reflecting inflammation or antiviral protection. This may contribute to the biomarker profiles established for diseases like asthma and COPD, for which viral infections are suggested to trigger or exacerbate symptoms [20] .
How many patients were i this study?
false
5,194
{ "text": [ "102" ], "answer_start": [ 609 ] }
2,669
Frontiers in antiviral therapy and immunotherapy https://doi.org/10.1002/cti2.1115 SHA: facbfdfa7189ca9ff83dc30e5d241ab22e962dbf Authors: Heaton, Steven M Date: 2020 DOI: 10.1002/cti2.1115 License: cc-by Abstract: nan Text: Globally, recent decades have witnessed a growing disjunction, a 'Valley of Death' 1,2 no less, between broadening strides in fundamental biomedical research and their incommensurate reach into the clinic. Plumbing work on research funding and development pipelines through recent changes in the structure of government funding, 2 new public and private joint ventures and specialist undergraduate and postgraduate courses now aim to incorporate pathways to translation at the earliest stages. Reflecting this shift, the number of biomedical research publications targeting 'translational' concepts has increased exponentially, up 1800% between 2003 and 2014 3 and continuing to rise rapidly up to the present day. Fuelled by the availability of new research technologies, as well as changing disease, cost and other pressing issues of our time, further growth in this exciting space will undoubtedly continue. Despite recent advances in the therapeutic control of immune function and viral infection, current therapies are often challenging to develop, expensive to deploy and readily select for resistance-conferring mutants. Shaped by the hostvirus immunological 'arms race' and tempered in the forge of deep time, the biodiversity of our world is increasingly being harnessed for new biotechnologies and therapeutics. Simultaneously, a shift towards host-oriented antiviral therapies is currently underway. In this Clinical & Translational Immunology Special Feature, I illustrate a strategic vision integrating these themes to create new, effective, economical and robust antiviral therapies and immunotherapies, with both the realities and the opportunities afforded to researchers working in our changing world squarely in mind. Opening this CTI Special Feature, I outline ways these issues may be solved by creatively leveraging the so-called 'strengths' of viruses. Viral RNA polymerisation and reverse transcription enable resistance to treatment by conferring extraordinary genetic diversity. However, these exact processes ultimately restrict viral infectivity by strongly limiting virus genome sizes and their incorporation of new information. I coin this evolutionary dilemma the 'information economy paradox'. Many viruses attempt to resolve this by manipulating multifunctional or multitasking host cell proteins (MMHPs), thereby maximising host subversion and viral infectivity at minimal informational cost. 4 I argue this exposes an 'Achilles Heel' that may be safely targeted via host-oriented therapies to impose devastating informational and fitness barriers on escape mutant selection. Furthermore, since MMHPs are often conserved targets within and between virus families, MMHP-targeting therapies may exhibit both robust and broadspectrum antiviral efficacy. Achieving this through drug repurposing will break the vicious cycle of escalating therapeutic development costs and trivial escape mutant selection, both quickly and in multiple places. I also discuss alternative posttranslational and RNA-based antiviral approaches, designer vaccines, immunotherapy and the emerging field of neo-virology. 4 I anticipate international efforts in these areas over the coming decade will enable the tapping of useful new biological functions and processes, methods for controlling infection, and the deployment of symbiotic or subclinical viruses in new therapies and biotechnologies that are so crucially needed. Upon infection, pathogens stimulate expression of numerous host inflammatory factors that support recruitment and activation of immune cells. On the flip side, this same process also causes immunopathology when prolonged or deregulated. 5 In their contribution to this Special Feature, Yoshinaga and Takeuchi review endogenous RNA-binding proteins (RBPs) that post-transcriptionally control expression of crucial inflammatory factors in various tissues and their potential therapeutic applications. 6 These RBPs include tristetraprolin and AUF1, which promote degradation of AU-rich element (ARE)-containing mRNA; members of the Roquin and Regnase families, which respectively promote or effect degradation of mRNAs harbouring stem-loop structures; and the increasingly apparent role of the RNA methylation machinery in controlling inflammatory mRNA stability. These activities take place in various subcellular compartments and are differentially regulated during infection. In this way, mRNA-destabilising RBPs constitute a 'brake' on the immune system, which may ultimately be toggled therapeutically. I anticipate continued efforts in this area will lead to new methods of regaining control over inflammation in autoimmunity, selectively enhancing immunity in immunotherapy, and modulating RNA synthesis and virus replication during infection. Another mRNA under post-transcriptional regulation by Regnase-1 and Roquin is Furin, which encodes a conserved proprotein convertase crucial in human health and disease. Furin, along with other PCSK family members, is widely implicated in immune regulation, cancer and the entry, maturation or release of a broad array of evolutionarily diverse viruses including human papillomavirus (HPV), influenza (IAV), Ebola (EboV), dengue (DenV) and human immunodeficiency virus (HIV). Here, Braun and Sauter review the roles of furin in these processes, as well as the history and future of furin-targeting therapeutics. 7 They also discuss their recent work revealing how two IFN-cinducible factors exhibit broad-spectrum inhibition of IAV, measles (MV), zika (ZikV) and HIV by suppressing furin activity. 8 Over the coming decade, I expect to see an ever-finer spatiotemporal resolution of host-oriented therapies to achieve safe, effective and broad-spectrum yet costeffective therapies for clinical use. The increasing abundance of affordable, sensitive, high-throughput genome sequencing technologies has led to a recent boom in metagenomics and the cataloguing of the microbiome of our world. The MinION nanopore sequencer is one of the latest innovations in this space, enabling direct sequencing in a miniature form factor with only minimal sample preparation and a consumer-grade laptop computer. Nakagawa and colleagues here report on their latest experiments using this system, further improving its performance for use in resource-poor contexts for meningitis diagnoses. 9 While direct sequencing of viral genomic RNA is challenging, this system was recently used to directly sequence an RNA virus genome (IAV) for the first time. 10 I anticipate further improvements in the performance of such devices over the coming decade will transform virus surveillance efforts, the importance of which was underscored by the recent EboV and novel coronavirus (nCoV / COVID-19) outbreaks, enabling rapid deployment of antiviral treatments that take resistance-conferring mutations into account. Decades of basic immunology research have provided a near-complete picture of the main armaments in the human antiviral arsenal. Nevertheless, this focus on mammalian defences and pathologies has sidelined examination of the types and roles of viruses and antiviral defences that exist throughout our biosphere. One case in point is the CRISPR/Cas antiviral immune system of prokaryotes, which is now repurposed as a revolutionary gene-editing biotechnology in plants and animals. 11 Another is the ancient lineage of nucleocytosolic large DNA viruses (NCLDVs), which are emerging human pathogens that possess enormous genomes of up to several megabases in size encoding hundreds of proteins with unique and unknown functions. 12 Moreover, hundreds of human-and avian-infective viruses such as IAV strain H5N1 are known, but recent efforts indicate the true number may be in the millions and many harbour zoonotic potential. 13 It is increasingly clear that host-virus interactions have generated truly vast yet poorly understood and untapped biodiversity. Closing this Special Feature, Watanabe and Kawaoka elaborate on neo-virology, an emerging field engaged in cataloguing and characterising this biodiversity through a global consortium. 14 I predict these efforts will unlock a vast wealth of currently unexplored biodiversity, leading to biotechnologies and treatments that leverage the host-virus interactions developed throughout evolution. When biomedical innovations fall into the 'Valley of Death', patients who are therefore not reached all too often fall with them. Being entrusted with the resources and expectation to conceive, deliver and communicate dividends to society is both cherished and eagerly pursued at every stage of our careers. Nevertheless, the road to research translation is winding and is built on a foundation of basic research. Supporting industry-academia collaboration and nurturing talent and skills in the Indo-Pacific region are two of the four pillars of the National Innovation and Science Agenda. 2 These frame Australia's Medical Research and Innovation Priorities, which include antimicrobial resistance, global health and health security, drug repurposing and translational research infrastructure, 15 capturing many of the key elements of this CTI Special Feature. Establishing durable international relationships that integrate diverse expertise is essential to delivering these outcomes. To this end, NHMRC has recently taken steps under the International Engagement Strategy 16 to increase cooperation with its counterparts overseas. These include the Japan Agency for Medical Research and Development (AMED), tasked with translating the biomedical research output of that country. Given the reciprocal efforts at accelerating bilateral engagement currently underway, 17 the prospects for new areas of international cooperation and mobility have never been more exciting nor urgent. With the above in mind, all contributions to this CTI Special Feature I have selected from research presented by fellow invitees to the 2018 Awaji International Forum on Infection and Immunity (AIFII) and 2017 Consortium of Biological Sciences (ConBio) conferences in Japan. Both Australia and Japan have strong traditions in immunology and related disciplines, and I predict that the quantity, quality and importance of our bilateral cooperation will accelerate rapidly over the short to medium term. By expanding and cooperatively leveraging our respective research strengths, our efforts may yet solve the many pressing disease, cost and other sustainability issues of our time.
Why may MMHP-targeting therapies exhibit both robust and broadspectrum antiviral efficacy?
false
4,131
{ "text": [ "since MMHPs are often conserved targets within and between virus families," ], "answer_start": [ 2852 ] }
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?
false
1,169
{ "text": [ "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." ], "answer_start": [ 5150 ] }
2,440
Optimization Method for Forecasting Confirmed Cases of COVID-19 in China https://doi.org/10.3390/jcm9030674 SHA: 1d7f8850c5244fdc9b387038e7eeae9bcbbde6d2 Authors: Al-Qaness, Mohammed A. A.; Ewees, Ahmed A.; Fan, Hong; Abd El Aziz, Mohamed Date: 2020 DOI: 10.3390/jcm9030674 License: cc-by Abstract: In December 2019, a novel coronavirus, called COVID-19, was discovered in Wuhan, China, and has spread to different cities in China as well as to 24 other countries. The number of confirmed cases is increasing daily and reached 34,598 on 8 February 2020. In the current study, we present a new forecasting model to estimate and forecast the number of confirmed cases of COVID-19 in the upcoming ten days based on the previously confirmed cases recorded in China. The proposed model is an improved adaptive neuro-fuzzy inference system (ANFIS) using an enhanced flower pollination algorithm (FPA) by using the salp swarm algorithm (SSA). In general, SSA is employed to improve FPA to avoid its drawbacks (i.e., getting trapped at the local optima). The main idea of the proposed model, called FPASSA-ANFIS, is to improve the performance of ANFIS by determining the parameters of ANFIS using FPASSA. The FPASSA-ANFIS model is evaluated using the World Health Organization (WHO) official data of the outbreak of the COVID-19 to forecast the confirmed cases of the upcoming ten days. More so, the FPASSA-ANFIS model is compared to several existing models, and it showed better performance in terms of Mean Absolute Percentage Error (MAPE), Root Mean Squared Relative Error (RMSRE), Root Mean Squared Relative Error (RMSRE), coefficient of determination ( R 2 ), and computing time. Furthermore, we tested the proposed model using two different datasets of weekly influenza confirmed cases in two countries, namely the USA and China. The outcomes also showed good performances. Text: A large family of viruses, called coronaviruses, are severe pathogens for human beings, which infect respiratory, hepatic, gastrointestinal, and neurologic diseases. They are distributed among humans, birds, livestock, mice, bats, and other wild animals [1] [2] [3] . The outbreaks of two previous coronaviruses, SARS-CoV and MERS-CoV in 2003 and 2012, respectively, have approved the transmission from animal to animal, and human to human [4] . In December 2019, the World Health Organization (WHO) received notifications from China for many cases of respiratory illness that were linked to some people who had visited a seafood market in Wuhan [5] . Currently, Wuhan city suffers from the spreading of a novel coronavirus, called COVID-19 (previously, it was called 2019-nCoV). In [6] , the authors concluded that COVID-19 likely originated in bats, because it is more similar to two bat-derived coronavirus strains. However, the source of the COVID-19 is not confirmed yet, and it communities, Hong Kong and Toronto, were 1.2 and 1.32, respectively. Ong et al. [20] proposed a monitoring and forecasting model for influenza A (H1N1-2009). Furthermore, Nah et al. [21] proposed a probability-based model to predict the spread of the MERS. The Adaptive Neuro-Fuzzy Inference System (ANFIS) [22] is widely applied in time series prediction and forecasting problems, and it showed good performance in many existing applications. It offers flexibility in determining nonlinearity in the time series data, as well as combining the properties of both artificial neural networks (ANN) and fuzzy logic systems. It has been applied in various forecasting applications, for example, in [23] , a stock price forecasting model was proposed using ANFIS and empirical mode decomposition. Chen et al. [24] proposed a TAIEX time series forecasting model based on a hybrid of ANFIS and ordered weighted averaging (OWA). In [25] , another time series forecasting method was presented for electricity prices based on ANFIS. Svalina et al. [26] proposed an ANFIS based forecasting model for close price indices for a stock market for five days. Ekici and Aksoy [27] presented an ANFIS based building energy consumption forecasting model. More so, ANFIS is also applied to forecast electricity loads [28] . Kumar et al. [29] proposed an ANFIS based model to forecast return products. Ho and Tsai [30] applied ANFIS to forecast product development performance. However, estimating ANFIS parameters is a challenge that needs to be improved. Therefore, in previous studies, some individual swarm intelligence (SI) methods have been applied to the ANFIS parameters to enhance time series forecasting because these parameters have a significant effect on the performance of ANFIS. The SI methods include the particle swarm optimization (PSO) [31, 32] , social-spider optimization [33] , sine-cosine algorithm (SCA) [34] , and multi-verse optimizer (MVO) [35] . For example, in [34] SCA algorithm was applied to improve the ANFIS model to forecast oil consumption in three countries, namely, Canada, Germany, and Japan. In the same context, in [35] , The MVO algorithm was used to enhance the ANFIS model to forecast oil consumption in two countries. In addition, in [36] the PSO was used with ANFIS to predict biochar yield. However, individual SI algorithms may stock at local optima. Therefore, one solution is to apply hybrid SI algorithms to avoid this problem. In [37] , a hybrid of two SI algorithms, namely GA and SSA, was presented to improve the ANFIS model. The proposed new model called GA-SSA-ANFIS was applied to forecast crude oil prices for long-term time series data. However, the previously mentioned methods suffer from some limitations that can affect the performance of the forecasting output such as slow convergence and the ability to balance between exploration and exploitation phases can influence the quality of the final output. This motivated us to propose an alternative forecasting method dependent on the hybridization concept. This concept avoids the limitations of traditional SI techniques by combining the strengths of different techniques, and this produces new SI techniques that are better than traditional ones. In the current study, we propose an improved ANFIS model based on a modified flower pollination algorithm (FPA) using the salp swarm algorithm (SSA). The FPA is an optimization algorithm proposed by Yang [38] , which was inspired by the flow pollination process of the flowering plants. The FPA was employed in various optimization applications, for example to estimate solar PV parameter [39, 40] , solving sudoku puzzles [41] , feature selection [42] , antenna design [43] , and other applications [44] [45] [46] [47] . Moreover, SSA is also an optimization algorithm proposed by Mirjalili et al. [48] inspired by the behavior of salp chains. In recent years, the SSA was utilized to solve different optimization problems, such as feature selection [49, 50] , data classification [51] , image segmentation [52] , and others [53, 54] . The proposed method called FPASSA is a hybrid of FPA and SSA, in which the SSA is applied as a local search method for FPA. The proposed FPASSA starts by receiving the historical COVID-19 dataset. Then a set of solutions is generated where each of them represents the value for the parameters of the ANFIS model. Then the quality of each solution is calculated using the fitness value, and the solution that has the best fitness value is chosen to represent the best solution. Then the probability of each solution is computed. Then the current solution will be updated, either using global or local strategy in FPA. However, in the case of local strategy, the operators of SSA or FPA will be used according to the probability of the fitness value for each solution. The process of updating the solutions is repeated until reaching the stop condition, and the best parameter configurations are used to forecast the number of confirmed cases of COVID-19. The main contribution points of the current study are as follows: 1. We propose an efficient forecasting model to forecast the confirmed cases of the COVID-19 in China for the upcoming ten days based on previously confirmed cases. An improved ANFIS model is proposed using a modified FPA algorithm, using SSA. We compare the proposed model with the original ANFIS and existing modified ANFIS models, such as PSO, GA, ABC, and FPA. The rest of this study is organized as follows. The preliminaries of ANFIS, FPA, and SSA are described in Section 2. Section 3 presents the proposed FPASSA, and Section 4 presents the experimental setup and results. We conclude this study in Section 5. The principles of the ANFIS are given in this section. The ANFIS model links the fuzzy logic and neural networks [22] . It generates a mapping between the input and output by applying IF-THEN rules (it is also called Takagi-Sugeno inference model). Figure 1 illustrates the ANFIS model where, y and x define the inputs to Layer 1 whereas, O 1i is its output of node i that is computed as follows: where µ denotes the generalized Gaussian membership functions. A i and B i define the membership values of µ. α i and ρ i denote the premise parameters set. The output of Layer 2 (it is also known as the firing strength of a rule) is calculated as follows: Meanwhile, the output of Layer 3 (it is also known as the normalized firing strength) is calculated as follows: The output of Layer 4 (it is also known as an adaptive node) is calculated as follows: where r i , q i , and p i define the consequent parameters of the node i. Layer 5 contains only one node; its output is computed as: Flower Pollination Algorithm is an optimization method proposed by Yang [38] . It simulates the transfer of flowers' pollen by pollinators in nature. This algorithm utilizes the two types of pollination (i.e., self-pollination and cross-pollination). In self-pollination, the pollination occurs with no pollinators, whereas, in cross-pollination, the pollens are moved between different plants. In more detail, the self-pollination can be represented as a local pollination while the cross-pollination can be called global pollination. The global pollination or cross-pollination can be mathematically formed as follows: where x t i defines the pollen i at iteration t. L denotes the pollination's strength or the step size. F * is the target position or best solution. In some cases, insects can fly with different distance steps for a long space; therefore, Levy fly distribution is applied to simulate this movement. where λ = 1.5. Γ(λ) denotes the gamma function. This distribution is available for large steps s > 0. The self-pollination or local pollination can be mathematically formed as follows: where x t i and x k i represent pollens from different flower in the same plant. in the range [0,1] The process of pollination can be done using cross-pollination or self-pollination. Therefore, the random variable p, in the range [0, 1], is used to determine this process. SSA is an optimization technique introduced by [48] . It simulates the Salps' behavior in nature. This behavior is called salp chain. The mathematical model of SSA begins by splinting its population into a leader group and followers group. The leader is the front salp, whereas, the followers are the other salps. The search space is determined in n-dimensions with n variables. Equation (10) works to update the salps' positions. where x 1 j denotes the leader's position in j-th dimension. F j is the target position. ub j and lb j represent the max and min bounds, respectively. c 2 and c 3 denote random numbers in [0, 1]. c 1 is an important parameter; it balances between the exploration and exploitation phases. It is computed as follows: where the current loop number is t and the max loop' number is t max . Then, the followers' position is updated as follows: where x i j defines the i-th position of the follower in j-th dimension. i > 1. This section explains the proposed FPASSA-ANFIS method. It is a time series method for forecasting the confirmed cases of the COVID-19, as given in Figure 2 . The FPASSA-ANFIS utilizes the improved FPA to train the ANFIS model by optimizing its parameters. The FPASSA-ANFIS contains five layers as the classic ANFIS model. Layer 1 contains the input variables (the historical COVID-19 confirmed cases). Whereas Layer 5 produces the forecasted values. In the learning phase, the FPASSA is used to select the best weights between Layer 4 and Layer 5. The FPASSA-ANFIS starts by formatting the input data in a time series form. In our case, the autocorrelation function (ACF) was considered. ACF is one of the methods applied to find patterns in the data; it presents information about the correlation between points separated by various time lags. Therefore, in this paper, the variables with ACF greater than 0.2 are considered i.e., 5-lags. Besides, the training data contains 75% of the dataset, whereas the testing data contains 25% of them. The number of clusters is defined by the fuzzy c-mean (FCM) method to construct the ANFIS model. The parameters of the ANFIS model are prepared by the FPASSA algorithm. In the training phase, the calculation error (as in Equation (13)) between the real data and the predicted data is used to evaluate the parameters' quality. where T is the real data, and P is the predicted data. N s is the sample length. The smaller values of the objective function indicate good ANFIS's parameter. On the other hand, the updating phase of the followers' positions in the SSA algorithm is applied to improve the global pollination phase in the FPA algorithm. In this improvement, there is a random variable (r) used to switch between both phases. If r > 0.5, then the operators of the SSA is used; otherwise, the operators of the FPA are used. In general, The FPASSA starts by constructing the population (X); afterward, the objective function is calculated for each solution. The solution with the lowest error value is saved to the next iteration. This sequence is repeated until meeting the stop condition, which in this paper, is the maximum number of iterations. Then the best solution is passed to train the parameters of the ANFIS model. After finishing the training phase, the testing phase is started with the best solution to compute the final output. The performance of the proposed method is evaluated by comparing the real data with the predicted data using the performance measures. Finally, the FPASSA produces a foretasted value for confirmed cases of COVID-19 in China in the next day. The steps of the proposed FPASSA are presented in Algorithm 1. Input: Historical COVID-19 dataset, size of population N, total number of iterations t max . Divide the data into training and testing sets. Using Fuzzy c-mean method to determine the number of membership functions. Constructing the ANFIS network. Set the initial value for N solutions (X). Return the best solution that represents the best configuration for ANFIS. Apply the testing set to the best ANFIS model. Forecasting the COVID-19 for the next ten days. This section presents the description of the used dataset, the performance measures, the parameter setting for all methods, the experiment results, and discussions. The main dataset of this study is COVID-19 dataset. It was collected from the WHO website (https: //www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/). It contains the daily confirmed cases in China from 21 January 2020 to 18 February 2020, as shown in Table 1 . We used 75% from the dataset to train the model while the rest is used to test it. Moreover, we evaluated the performance of the proposed method using two datasets of weekly influenza confirmed cases. The first one is called DS1; it was collected from the Centers for Disease Control and Prevention (CDC) (https://www.cdc.gov/flu/weekly/). It starts from week number 40 in 2015 and continues until week number 6 in 2020. Whereas, the second one is called DS2. It was collected from the WHO website (https://www.who.int/influenza). It contains the data of weekly influenza confirmed cases in China from week number 1 in 2016 to week number 8 in 2020. The quality of the proposed method is evaluated using a set of performance metrics as follows: • Root Mean Square Error (RMSE): where Yp and Y are the predicted and original values, respectively. • Mean Absolute Error (MAE): • Mean Absolute Percentage Error (MAPE): • Root Mean Squared Relative Error (RMSRE): N s represents the sample size of the data. • Coefficient of Determination (R 2 ): where Y represents the average of Y. The lowest value of RMSE, MAE, MAPE, and RMSRE refers to the best method. The higher value of R 2 indicates better correlation for the method. This paper aims to assess the ability of the FPASSA to forecast the COVID-19 by comparing its performance with other methods, namely the ANFIS and the trained ANFIS models using PSO, GA, ABC, FPA, and FPASSA. The parameters' setting for these models is listed in Table 2 . The common parameters, such as population size, are set to 25 and 100 iterations are applied. Besides, each algorithm is performed for 30 independent runs to fair comparisons. The selected parameters are chosen because they produced good behavior in previous experiments, such as [34, 35, 55, 56] . Table 2 . Parameters' setting. Parameters Setting Max. epochs = 100, Error goal = 0, Initial step = 0.01, Decrease rate = 0.9, Increase rate = 1. In this section, the performance of the proposed FPASSA to predict the DS1 and DS2 is discussed. It can be concluded from Table 3 that the performance of FPASSA outperformed the compared methods in all measures, whereas the FPA is ranked second. The results of DS2 indicate that the FPASSA is ranked first in terms of RMSE, MAPE, R 2 , and the CPU time. Whereas, the PSO is ranked second, followed by the FPA, GA, then ABC. These results denote that the proposed method can optimize the parameters of the ANFIS model effectively and produce good results in terms of the performance measures. Comparison results between the proposed FPASSA and other models to forecast COVID-19 are given in Table 4 . It can be concluded that the FPASSA outperforms other models. For example, by analyzing the results of RMSE, MAE, MAPE, RMSRE, and CPU time(s) it can be observed that the FPASSA achieves the smallest value among the comparison algorithms, and this indicates the high quality of the FPASSA. Meanwhile, the FPA allocates the second rank, which provides better results than the rest of the methods. Moreover, the value of R 2 refers to the high correlation between the prediction obtained by the proposed FPASSA method and the original COVID-19, which has nearly 0.97. This can also be noticed from Figure 3 , which depicts the training of the algorithms using the historical data of the COVID-19 as well as their forecasting values for ten days. Table 5 depicts the forecasting value for the confirmed cases of the COVID-19 in China from 19/2/2020 to 28/2/2020. From these results, it can be noticed that the outbreak will reach its highest level on the day 28/2/2020. The average percentage of the increase over the forecasted period is 10%, the highest percentage is 12% on 28/2/2020, and the lowest percentage is 8.7% on 19/2/2020. From the previous results, it can be concluded that the proposed FPASSA-ANFIS has a high ability to forecast the COVID-19 dataset. These results avoid the limitations of traditional ANFIS because of the combination with the modified FPA method. Moreover, the operators of SSA are combined with the local strategy of FPA to enhance their exploitation ability. However, the time computational of the proposed FPASSA method still requires more improvements. This paper proposed a modified version for the flower pollination algorithm (FPA) using the salp swarm algorithm (SSA). This modified version, called FPASSA, is applied to improve the performance of the ANFIS through determining the optimal value for its parameters. The developed FPASSA-ANFIS model is applied as a forecasting technique for a novel coronavirus, called COVID-19, that was discovered in Wuhan, China at the end of last year and January of the current year. The proposed FPASSA-ANFIS model has a high ability to predict the number of confirmed cases within ten days. Besides, FPASSA-ANFIS outperforms other forecasting models in terms of RMSE, MAE, MAPE, RMSRE, and R 2 . Furthermore, two datasets of weekly influenza confirmed cases in the USA and China were used to evaluate the proposed method, and the evaluation outcomes showed its good performance. According to the promising results obtained by the proposed FPASSA-ANFIS, it can be applied in different forecasting applications.
What are the large family of viruses, called coronaviruses?
false
4,404
{ "text": [ "pathogens for human beings, which infect respiratory, hepatic, gastrointestinal, and neurologic diseases." ], "answer_start": [ 1942 ] }
1,560
Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
What element does hepcidin play a roles in regulating during metabolism?
false
2,134
{ "text": [ "Fe" ], "answer_start": [ 2569 ] }
1,580
Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285652/ SHA: ef7110a9022bac2e50c995b0f6b826ff071e48f8 Authors: Curtis, Kelly A.; Rudolph, Donna L.; Nejad, Irene; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard; LaBarre, Paul; Owen, S. Michele Date: 2012-02-23 DOI: 10.1371/journal.pone.0031432 License: cc0 Abstract: BACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC. Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed [1] . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required [2] . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes [3] , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus [4] . Rapid tests are currently a key component of HIV screening at the point-of-care (POC), significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings. Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks [3, 5] . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth [6, 7] . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests (NAAT) are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection [5] . In their current form, however, NAAT's are not feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay (Gen-Probe, Inc., http://www.fda.gov/ BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466) is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing. To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time (,1 hour)is desirable [8] . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs [8] . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed [9] . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification (LAMP) [10] , has been optimized for the detection of DNA and/or RNA (RT-LAMP) from a wide range of bacterial and viral pathogens [11, 12, 13, 14, 15, 16, 17, 18, 19] , including HIV [20, 21] . LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC [22] . LAMP has also proven to be less sensitive to biological inhibitors than PCR [23, 24] , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 [20, 21, 25] . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP assay for real-time or immediate naked-eye detection [15, 17, 21, 22, 26] . The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification (NINA) devices that generate heat from the exothermic reaction of calcium oxide and water [27, 28] . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals. Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health (PATH, Seattle, WA), as described [27, 28] . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide (CaO; Sigma-Aldrich, St. Louis, MO) and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids (Fig. 1) . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material (PCM) that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer (DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT). DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 [29] , using a QIAamp DNA blood mini kit (QIAGEN, Valencia, CA). Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell [29] . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially (PRD801; SeraCare Life Sciences, Mil- ford, MA) and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit (QIAGEN). Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC [30] and RNA extracted from HIV-2 NIH-Z purified virus (Advanced Biotechnologies Inc., Columbia, MD). Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study [31] , or obtained commercially (SeraCare Life Sciences). All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA (Bio-Rad Laboratories, Redmond, WA), GS HIV-1 Western blot (Bio-Rad Laboratories), Aptima HIV-1 RNA assay (Gen-Probe, Inc., San Diego, CA), and Amplicor HIV-1 DNA assay (Roche Diagnostics, Branchburg, NJ ). Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples (SeraCare Life Sciences) were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL (Advanced Biotechnologies Inc.). HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase (RT) gene. The six primers required for the RT-LAMP reaction, forward outer (F3), backward outer (B3), forward inner (FIP), backward inner (BIP), and the loop primers (LoopF and LoopB), were designed using the PrimerExplorer V4 software (Eiken Chemical Co. Ltd.; http:// primerexplorer.jp/e/). The LAMP primers and amplification cycle have been described in detail by Nagamine et al. [32] . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described [20] , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products [21] . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 (BHQ-1) added to the 39 end. The HIV-1 HXB2 sequence (GenBank accession number AF033819) was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 . The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM (final concentration) of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine (Sigma-Aldrich), 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer (New England Biolabs, Ipswich, MA), 16 U Bst DNA polymerase (New England Biolabs) and 2 U AMV reverse transcriptase (Invitrogen, Carlsbad, CA). The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer (2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA), as previously described [21] . The reaction mixture was incubated at 60uC for 60 minutes, using a GeneAmpH PCR System (Applied Biosystems, Foster City, CA) or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction. The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described [21] . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain (Invitrogen), which was subsequently visualized using the ChemiDoc XRS system. To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300. The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC. To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates (60%). For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler ( Table 2) . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 (Table 3 ). The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree. The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory (28uC; Table 2 ). The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC ( Table 3 ). The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction (data not shown). The temperature profiles at the ambient temperature of 37uC, however, were similar to those at 28uC. Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices (Table 4 ). Amplification consistency was most evident with two of the patient samples (patient #4 and #5) that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative (data not shown). A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes. In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid (NINA) heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated [27, 28] . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler. Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option. We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory. For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product. The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis [33] . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC.
What percentage of patients do not return for followup after HIV testing?
false
4,438
{ "text": [ "35 to 50%" ], "answer_start": [ 2553 ] }
2,669
Frontiers in antiviral therapy and immunotherapy https://doi.org/10.1002/cti2.1115 SHA: facbfdfa7189ca9ff83dc30e5d241ab22e962dbf Authors: Heaton, Steven M Date: 2020 DOI: 10.1002/cti2.1115 License: cc-by Abstract: nan Text: Globally, recent decades have witnessed a growing disjunction, a 'Valley of Death' 1,2 no less, between broadening strides in fundamental biomedical research and their incommensurate reach into the clinic. Plumbing work on research funding and development pipelines through recent changes in the structure of government funding, 2 new public and private joint ventures and specialist undergraduate and postgraduate courses now aim to incorporate pathways to translation at the earliest stages. Reflecting this shift, the number of biomedical research publications targeting 'translational' concepts has increased exponentially, up 1800% between 2003 and 2014 3 and continuing to rise rapidly up to the present day. Fuelled by the availability of new research technologies, as well as changing disease, cost and other pressing issues of our time, further growth in this exciting space will undoubtedly continue. Despite recent advances in the therapeutic control of immune function and viral infection, current therapies are often challenging to develop, expensive to deploy and readily select for resistance-conferring mutants. Shaped by the hostvirus immunological 'arms race' and tempered in the forge of deep time, the biodiversity of our world is increasingly being harnessed for new biotechnologies and therapeutics. Simultaneously, a shift towards host-oriented antiviral therapies is currently underway. In this Clinical & Translational Immunology Special Feature, I illustrate a strategic vision integrating these themes to create new, effective, economical and robust antiviral therapies and immunotherapies, with both the realities and the opportunities afforded to researchers working in our changing world squarely in mind. Opening this CTI Special Feature, I outline ways these issues may be solved by creatively leveraging the so-called 'strengths' of viruses. Viral RNA polymerisation and reverse transcription enable resistance to treatment by conferring extraordinary genetic diversity. However, these exact processes ultimately restrict viral infectivity by strongly limiting virus genome sizes and their incorporation of new information. I coin this evolutionary dilemma the 'information economy paradox'. Many viruses attempt to resolve this by manipulating multifunctional or multitasking host cell proteins (MMHPs), thereby maximising host subversion and viral infectivity at minimal informational cost. 4 I argue this exposes an 'Achilles Heel' that may be safely targeted via host-oriented therapies to impose devastating informational and fitness barriers on escape mutant selection. Furthermore, since MMHPs are often conserved targets within and between virus families, MMHP-targeting therapies may exhibit both robust and broadspectrum antiviral efficacy. Achieving this through drug repurposing will break the vicious cycle of escalating therapeutic development costs and trivial escape mutant selection, both quickly and in multiple places. I also discuss alternative posttranslational and RNA-based antiviral approaches, designer vaccines, immunotherapy and the emerging field of neo-virology. 4 I anticipate international efforts in these areas over the coming decade will enable the tapping of useful new biological functions and processes, methods for controlling infection, and the deployment of symbiotic or subclinical viruses in new therapies and biotechnologies that are so crucially needed. Upon infection, pathogens stimulate expression of numerous host inflammatory factors that support recruitment and activation of immune cells. On the flip side, this same process also causes immunopathology when prolonged or deregulated. 5 In their contribution to this Special Feature, Yoshinaga and Takeuchi review endogenous RNA-binding proteins (RBPs) that post-transcriptionally control expression of crucial inflammatory factors in various tissues and their potential therapeutic applications. 6 These RBPs include tristetraprolin and AUF1, which promote degradation of AU-rich element (ARE)-containing mRNA; members of the Roquin and Regnase families, which respectively promote or effect degradation of mRNAs harbouring stem-loop structures; and the increasingly apparent role of the RNA methylation machinery in controlling inflammatory mRNA stability. These activities take place in various subcellular compartments and are differentially regulated during infection. In this way, mRNA-destabilising RBPs constitute a 'brake' on the immune system, which may ultimately be toggled therapeutically. I anticipate continued efforts in this area will lead to new methods of regaining control over inflammation in autoimmunity, selectively enhancing immunity in immunotherapy, and modulating RNA synthesis and virus replication during infection. Another mRNA under post-transcriptional regulation by Regnase-1 and Roquin is Furin, which encodes a conserved proprotein convertase crucial in human health and disease. Furin, along with other PCSK family members, is widely implicated in immune regulation, cancer and the entry, maturation or release of a broad array of evolutionarily diverse viruses including human papillomavirus (HPV), influenza (IAV), Ebola (EboV), dengue (DenV) and human immunodeficiency virus (HIV). Here, Braun and Sauter review the roles of furin in these processes, as well as the history and future of furin-targeting therapeutics. 7 They also discuss their recent work revealing how two IFN-cinducible factors exhibit broad-spectrum inhibition of IAV, measles (MV), zika (ZikV) and HIV by suppressing furin activity. 8 Over the coming decade, I expect to see an ever-finer spatiotemporal resolution of host-oriented therapies to achieve safe, effective and broad-spectrum yet costeffective therapies for clinical use. The increasing abundance of affordable, sensitive, high-throughput genome sequencing technologies has led to a recent boom in metagenomics and the cataloguing of the microbiome of our world. The MinION nanopore sequencer is one of the latest innovations in this space, enabling direct sequencing in a miniature form factor with only minimal sample preparation and a consumer-grade laptop computer. Nakagawa and colleagues here report on their latest experiments using this system, further improving its performance for use in resource-poor contexts for meningitis diagnoses. 9 While direct sequencing of viral genomic RNA is challenging, this system was recently used to directly sequence an RNA virus genome (IAV) for the first time. 10 I anticipate further improvements in the performance of such devices over the coming decade will transform virus surveillance efforts, the importance of which was underscored by the recent EboV and novel coronavirus (nCoV / COVID-19) outbreaks, enabling rapid deployment of antiviral treatments that take resistance-conferring mutations into account. Decades of basic immunology research have provided a near-complete picture of the main armaments in the human antiviral arsenal. Nevertheless, this focus on mammalian defences and pathologies has sidelined examination of the types and roles of viruses and antiviral defences that exist throughout our biosphere. One case in point is the CRISPR/Cas antiviral immune system of prokaryotes, which is now repurposed as a revolutionary gene-editing biotechnology in plants and animals. 11 Another is the ancient lineage of nucleocytosolic large DNA viruses (NCLDVs), which are emerging human pathogens that possess enormous genomes of up to several megabases in size encoding hundreds of proteins with unique and unknown functions. 12 Moreover, hundreds of human-and avian-infective viruses such as IAV strain H5N1 are known, but recent efforts indicate the true number may be in the millions and many harbour zoonotic potential. 13 It is increasingly clear that host-virus interactions have generated truly vast yet poorly understood and untapped biodiversity. Closing this Special Feature, Watanabe and Kawaoka elaborate on neo-virology, an emerging field engaged in cataloguing and characterising this biodiversity through a global consortium. 14 I predict these efforts will unlock a vast wealth of currently unexplored biodiversity, leading to biotechnologies and treatments that leverage the host-virus interactions developed throughout evolution. When biomedical innovations fall into the 'Valley of Death', patients who are therefore not reached all too often fall with them. Being entrusted with the resources and expectation to conceive, deliver and communicate dividends to society is both cherished and eagerly pursued at every stage of our careers. Nevertheless, the road to research translation is winding and is built on a foundation of basic research. Supporting industry-academia collaboration and nurturing talent and skills in the Indo-Pacific region are two of the four pillars of the National Innovation and Science Agenda. 2 These frame Australia's Medical Research and Innovation Priorities, which include antimicrobial resistance, global health and health security, drug repurposing and translational research infrastructure, 15 capturing many of the key elements of this CTI Special Feature. Establishing durable international relationships that integrate diverse expertise is essential to delivering these outcomes. To this end, NHMRC has recently taken steps under the International Engagement Strategy 16 to increase cooperation with its counterparts overseas. These include the Japan Agency for Medical Research and Development (AMED), tasked with translating the biomedical research output of that country. Given the reciprocal efforts at accelerating bilateral engagement currently underway, 17 the prospects for new areas of international cooperation and mobility have never been more exciting nor urgent. With the above in mind, all contributions to this CTI Special Feature I have selected from research presented by fellow invitees to the 2018 Awaji International Forum on Infection and Immunity (AIFII) and 2017 Consortium of Biological Sciences (ConBio) conferences in Japan. Both Australia and Japan have strong traditions in immunology and related disciplines, and I predict that the quantity, quality and importance of our bilateral cooperation will accelerate rapidly over the short to medium term. By expanding and cooperatively leveraging our respective research strengths, our efforts may yet solve the many pressing disease, cost and other sustainability issues of our time.
What happens to these activities during infection?
false
4,148
{ "text": [ "are differentially regulated" ], "answer_start": [ 4591 ] }
1,674
Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523942/ SHA: f00f183d0bce0091a02349ec1eab44a76dad9bc4 Authors: Henry, Kevin A.; Arbabi-Ghahroudi, Mehdi; Scott, Jamie K. Date: 2015-08-04 DOI: 10.3389/fmicb.2015.00755 License: cc-by Abstract: For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage (genera Inovirus and Plectrovirus) are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort (1915) and d 'Hérelle (1917) , the first filamentous phage, f1, was isolated in Loeb (1960) and later characterized as a member of a larger group of phage (Ff, including f1, M13, and fd phage) specific for the E. coli conjugative F pilus (Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964) . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry (If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972) , and over time the list of known filamentous phage has expanded to over 60 members (Fauquet et al., 2005) , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012) . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues (Smith, 1985; Parmley and Smith, 1988) . Based on the ideas described in Parmley and Smith (1988) , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991) . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications (Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010) . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( Table 1) . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pVIII, type 3 display on pIII, etc.), resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith (1988), McConnell et al. (1994) , Rondot et al. (2001) Hybrid (type 33 and 3+3 systems) Type 3+3 system <1 2 Smith and Scott (1993) , Smith and Petrenko (1997) pVI Hybrid (type 6+6 system) Yes <1 2 >25 kDa Hufton et al. (1999) pVII Fully recombinant (type 7 system) No ∼5 >25 kDa Kwasnikowski et al. (2005) Hybrid (type 7+7 system) Yes <1 2 Gao et al. (1999) pVIII Fully recombinant (landscape phage; type 8 system) No 2700 3 ∼5-8 residues Kishchenko et al. (1994) , Petrenko et al. (1996) Hybrid (type 88 and 8+8 systems) Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith (1990) , Greenwood et al. (1991) , Smith and Fernandez (2004) pIX Fully recombinant (type 9+9 * system) Yes ∼5 >25 kDa Gao et al. (2002) Hybrid (type 9+9 system) No <1 2 Gao et al. (1999) , Shi et al. (2010) , Tornetta et al. (2010) 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type 3 * +3 display). By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display (up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number (1-5 per phage particle). While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion (Sidhu et al., 2000) . For the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (Meynell and Lawn, 1968 ) and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies (Pratt et al., 1969; Woolford et al., 1977) . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. (1988) . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pVIII display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII (de la Cruz et al., 1988) . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; Table 2) , including malaria and human immunodeficiency virus type 1 (HIV-1). When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits (de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996) , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants (or mimics thereof) of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di Marzo Veronese et al., 1994; Scala et al., 1999) . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( Table 2) . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (Irving et al., 2010 ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (Van Regenmortel, 2012); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (Frenkel et al., 2000 (Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011) , possibly reduced amyloid plaque formation in mice (Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008) , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease (Lavie et al., 2004) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. (2001) found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules (Rudolf et al., 1998) , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes (Abdennebi et al., 1999) . Phage displaying or chemically Rubinchik and Chow (2000) conjugated to sperm antigen peptides or peptide mimics (Samoylova et al., 2012a,b) and gonadotropin-releasing hormone (Samoylov et al., 2012) are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals ( Table 2) , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions (Greenwood et al., 1991) possibly due to copy number differences (pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996) . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH; Melzer et al., 2003; Su et al., 2007) , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target (Henry et al., 2011) . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. (2010) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (Henry et al., 2011) . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see Immunological Mechanisms of Vaccination with Filamentous Phage below). The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII ( Table 3) . Early work, showing that immunization with phage elicited T-cell help (Kölsch et al., 1971; Willis et al., 1993) , was confirmed by several subsequent studies (De Berardinis et al., 1999; Ulivieri et al., 2008) . From the perspective of vaccination against infectious disease, De Berardinis et al. (2000) showed that a cytotoxic T-cell (CTL) epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins (Mascolo et al., 2007) . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus (Wan et al., 2001) and Candida albicans (Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial (Manoutcharian et al., 2004; Morales et al., 2008) . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (Plotkin, 2010) , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (Gram et al., 1993) . E. coli F17a-G adhesin (Van Gerven et al., 2008) , hepatitis B core antigen (Bahadir et al., 2011) , and hepatitis B surface antigen (Balcioglu et al., 2014) all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (Rao et al., 2003) . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv (mimicking a Vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from Vibrio anguillarum challenge (Xia et al., 2005) . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model (Roehnisch et al., 2013) , and was welltolerated and immunogenic in patients with multiple myeloma (Roehnisch et al., 2014) . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone (Cuesta et al., 2006) , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (Figure 1) . The phage particle is immunogenic without adjuvant in all species tested to date, including mice (Willis et al., 1993) , rats (Dente et al., 1994) , rabbits (de la Cruz et al., 1988) , guinea pigs (Frenkel et al., 2000; Kim et al., 2004) , fish (Coull et al., 1996; Xia et al., 2005) , non-human primates (Chen et al., 2001) , and humans (Roehnisch et al., 2014) . Various routes of immunization have been employed, including oral administration (Delmastro et al., 1997) as well as subcutaneous (Grabowska et al., 2000) , intraperitoneal (van Houten et al., 2006) , intramuscular (Samoylova et al., 2012a) , intravenous (Vaks and Benhar, 2011) , and intradermal injection (Roehnisch et al., 2013) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: (i) the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice (Terry et al., 1997; Kneissel et al., 1999) ; (ii) the N-terminal N1 and N2 domains of pIII (van Houten et al., 2010) ; and (iii) bacterial lipopolysaccharide (LPS) embedded in the phage coat (Henry et al., 2011) . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization (Frenkel et al., 2000) . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes (Hashiguchi et al., 2010) . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van Houten et al., 2010) . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | www.frontiersin.org Although serum anti-phage antibody titers appear to be at least partially T-cell dependent (Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010) , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses (Murira, 2014) . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules (Gaubin et al., 2003; Ulivieri et al., 2008) and can activate T H 1, T H 2, and T H 17 helper T cells (Yang et al., 2005a; Wang et al., 2014d) . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII (Kajihara et al., 2000) . Phage proteins can also be cross-presented on MHC class I molecules (Wan et al., 2005) and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help (Del Pozzo et al., 2010) . The latter CTLs mediate a delayed-type hypersensitivity reaction (Fang et al., 2005; Del Pozzo et al., 2010) . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins (Grabowska et al., 2000) . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors (Hashiguchi et al., 2010) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (Molenaar et al., 2002) , particularly of the liver and spleen, where it is retained for days (Zou et al., 2004) , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe (reviewed in Sulakvelidze et al., 2001) . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "Trojan horse" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease (Hagens and Blasi, 2003; Hagens et al., 2004) , lambda phage S holin (Hagens and Blasi, 2003) or a lethal catabolite gene activator protein (Moradpour et al., 2009) effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS (Hagens and Blasi, 2003; Hagens et al., 2004) . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (Hagens et al., 2006) or phage engineered to repress the cellular SOS response (Lu and Collins, 2009) . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli (May et al., 2011) . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (Figure 2) . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression (Poul and Marks, 1999) . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol (Yacoby et al., 2006; Vaks and Benhar, 2011) . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro (Ghosh et al., 2012a) . Tumorspecific peptide:pVIII fusion proteins selected from "landscape" phage (Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a) were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models (Jayanna et al., 2010b; Wang et al., 2014b,c) . Using the B16-OVA tumor model, Eriksson et al. (2007) showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (Eriksson et al., 2009) . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic (Frenkel and Solomon, 2002) and therapeutic (Solomon, 2008) reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (Ksendzovsky et al., 2012) . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (Rakover et al., 2010) . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units (EU)/mL (Boratynski et al., 2004; Branston et al., 2015) , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (Smith and Gingrich, 2005; Branston et al., 2015) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (Boratynski et al., 2004; Zakharova et al., 2005) , polymyxin B chromatography (Grabowska et al., 2000) , and treatment with detergents such as Triton X-100 or Triton X-114 (Roehnisch et al., 2014; Branston et al., 2015) . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate (LAL) assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in Dalmasso et al., 2014) or for detection of foodborne pathogens post-production (reviewed in Schmelcher and Loessner, 2014) . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance (Nanduri et al., 2007) , piezoelectric transducers (Olsen et al., 2006) , linear dichroism (Pacheco-Gomez et al., 2012) , and magnetoelastic sensor technology (Lakshmanan et al., 2007; Huang et al., 2009) were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (Li et al., 2010b) and eggs (Chai et al., 2012) . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (Henry et al., 2011) . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (Figure 3) . The most commonly used approach is functionalization of amine groups with NHS esters (van Houten et al., 2006 (van Houten et al., , 2010 Yacoby et al., 2006) , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (Li et al., 2010a) . Carrico et al. (2012) developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (Ng et al., 2012) or enzymes (Chen et al., 2007; Hess et al., 2012) , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (Welsh et al., 1996) . Lee et al. (2002) engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012) , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (Mao et al., 2003 (Mao et al., , 2004 , nanoparticles , and nanocomposites (Oh et al., 2012; Chen et al., 2014) . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. (2006) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (Nam et al., 2008) . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides (Lee et al., 2009) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging (Ghosh et al., 2012b; Yi et al., 2012) , photocatalytic water splitting (Nam et al., 2010a; Neltner et al., 2010) , light harvesting (Nam et al., 2010b; Chen et al., 2013) , photoresponsive technologies (Murugesan et al., 2013) , neural electrodes (Kim et al., 2014) , and piezoelectric energy generation (Murugesan et al., 2013) . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions (Hansen et al., 1998 (Hansen et al., , 2000 Dahlke Ojennus et al., 1999) in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013) . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (Marks et al., 1992; Bradbury et al., 2011) . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; Koide and Koide, 2012) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations (Lippow et al., 2007) and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. (2011) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (PACE), which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. (2011) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. (2014) later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis (Meyer and Ellington, 2011) . This approach represents a promising avenue for both basic research in molecular evolution (Dickinson et al., 2013) and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil (Murugaiyan et al., 2011) , coastal fresh water (Xue et al., 2012) , alpine lakes (Hofer and Sommaruga, 2001) and deep sea bacteria (Jian et al., 2012) , but not, perhaps surprisingly, the human gut (Kim et al., 2011) . The environmental "phageome" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles (Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005) . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from 0 to 100% of all viral particles (Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001) . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (Hofer and Sommaruga, 2001) . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (Roux et al., 2012) and reclaimed and potable water (Rosario et al., 2009) but have much higher frequencies in wastewater and sewage (Cantalupo et al., 2011; Alhamlan et al., 2013) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution (reviewed in Canchaya et al., 2003) . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage (Waldor and Mekalanos, 1996) . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 (Hassan et al., 2010) . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis (Derbise et al., 2007) , Neisseria meningitidis (Bille et al., 2005 (Bille et al., , 2008 , Vibrio parahaemolyticus (Iida et al., 2001) , E. coli 018:K1:H7 (Gonzalez et al., 2002) , Xanthomonas campestris (Kamiunten and Wakimoto, 1982) , and P. aeruginosa (Webb et al., 2004) , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum (Yamada, 2013) . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (Lin et al., 2011) . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (Petrenko and Makowski, 1993) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (Curtis et al., 2011 (Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (Curtis et al., 2009 ). The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text.
What application is a natural extension of the ability to display recombinant exogenous sequences on its surface?
false
1,739
{ "text": [ "as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide" ], "answer_start": [ 9597 ] }
1,585
Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3789439/ SHA: efba2008a6ccf1ad2614aebd79a6a741ea6538b9 Authors: Jin, Yi; Zhang, Yuewei; Wan, Chunyan; Wang, Hongjun; Hou, Lingyu; Chang, Jianyu; Fan, Kai; Xie, Xiangming Date: 2013-09-18 DOI: 10.1155/2013/194976 License: cc-by Abstract: The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products. Text: Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2] . The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4] . The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/ influenza/human animal interface/H5N1 cumulative table archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6] . Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important. The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8] . Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China's middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7] . Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating 2 Evidence-Based Complementary and Alternative Medicine properties [9] . In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10] . However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP). The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11] . Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [12] [13] [14] [15] . In recent reports, polysaccharidebased adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17] . However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza. In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products. Virus. The H5N1 influenza virus (A/bar-headed goose/ Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37 ∘ C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80 ∘ C. Viral titers were determined by plaque assay as described previously [18] . Animal and Cells. 8-10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University. The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier's recommendations. Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4 ∘ C. The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80 ∘ C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40 ∘ C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H 2 SO 4 method [19] . Vitro. 2.5 mL A549 and Raw 264.7 cells (4 × 10 5 /mL) per well were plated in 6-well plates and cultured at 37 ∘ C under 5% CO 2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 g/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR). Assay. Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaqueforming units (PFU) of H5N1 influenza virus in 50 L. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded. 2.6. Plaque Assay. MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 g/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 g/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37 ∘ C, 5% CO 2 for 60-72 h and stained with 1% crystal violet. Total RNA from 1 × 10 6 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer's instructions. DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3 5 -CTC CTT AAT GTC ACG CAC GAT TTC-3 h IL-6 5 -CCT TCG GTC CAG TTG CCT TCT-3 5 -CCA GTG CCT CTT TGC TGC TTT C-3 h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8 Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.
What factors make H5N1 a worldwide threat to public health?
false
5,217
{ "text": [ "rapid spread and high pathogenicity" ], "answer_start": [ 1731 ] }
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?
false
2,181
{ "text": [ "Personal Protective Equipment" ], "answer_start": [ 6407 ] }
2,652
The Long Noncoding RNA NEAT1 Exerts Antihantaviral Effects by Acting as Positive Feedback for RIG-I Signaling https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391460/ SHA: d18636f47e3c7dd93da309d556ba464d964fd24f Authors: Ma, Hongwei; Han, Peijun; Ye, Wei; Chen, Hesong; Zheng, Xuyang; Cheng, Linfeng; Zhang, Liang; Yu, Lan; Wu, Xing'an; Xu, Zhikai; Lei, Yingfeng; Zhang, Fanglin Date: 2017-04-13 DOI: 10.1128/jvi.02250-16 License: cc-by Abstract: Hantavirus infection, which causes zoonotic diseases with a high mortality rate in humans, has long been a global public health concern. Over the past decades, accumulating evidence suggests that long noncoding RNAs (lncRNAs) play key regulatory roles in innate immunity. However, the involvement of host lncRNAs in hantaviral control remains uncharacterized. In this study, we identified the lncRNA NEAT1 as a vital antiviral modulator. NEAT1 was dramatically upregulated after Hantaan virus (HTNV) infection, whereas its downregulation in vitro or in vivo delayed host innate immune responses and aggravated HTNV replication. Ectopic expression of NEAT1 enhanced beta interferon (IFN-β) production and suppressed HTNV infection. Further investigation suggested that NEAT1 served as positive feedback for RIG-I signaling. HTNV infection activated NEAT1 transcription through the RIG-I–IRF7 pathway, whereas NEAT1 removed the transcriptional inhibitory effects of the splicing factor proline- and glutamine-rich protein (SFPQ) by relocating SFPQ to paraspeckles, thus promoting the expression of RIG-I and DDX60. RIG-I and DDX60 had synergic effects on IFN production. Taken together, our findings demonstrate that NEAT1 modulates the innate immune response against HTNV infection, providing another layer of information about the role of lncRNAs in controlling viral infections. IMPORTANCE Hantaviruses have attracted worldwide attention as archetypal emerging pathogens. Recently, increasing evidence has highlighted long noncoding RNAs (lncRNAs) as key regulators of innate immunity; however, their roles in hantavirus infection remain unknown. In the present work, a new unexplored function of lncRNA NEAT1 in controlling HTNV replication was found. NEAT1 promoted interferon (IFN) responses by acting as positive feedback for RIG-I signaling. This lncRNA was induced by HTNV through the RIG-I–IRF7 pathway in a time- and dose-dependent manner and promoted HTNV-induced IFN production by facilitating RIG-I and DDX60 expression. Intriguingly, NEAT1 relocated SFPQ and formed paraspeckles after HTNV infection, which might reverse inhibitive effects of SFPQ on the transcription of RIG-I and DDX60. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA NEAT1 in host innate immunity after HTNV infection. In summary, our findings provide additional insights regarding the role of lncRNAs in controlling viral infections. Text: glycoprotein (GP), and viral RNA-dependent polymerase protein (RdRp), respectively. Humans become infected by inhaling contaminated aerosols or by coming into contact with rodent excreta, and they develop two severe acute diseases, namely, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (2) . Hantavirus infection affects up to 100,000 to 200,000 humans annually, with fulminant HFRS cases most represented in China (3) . Chinese HFRS cases, mainly caused by Hantaan virus (HTNV) infection, account for approximately 90% of all global cases, with a mortality rate ranging from 0.1 to 15% (4) . Since there is neither an effective therapeutic nor FDA-licensed vaccine, further understanding of host immune responses against hantaviral infection is of great significance for global public health and safety. The innate immune system, characterized by interferon (IFN) responses and immunocyte activation, provides the initial defense against viral invasions. Cellular pathogen recognition receptors (PRRs), including Toll-like receptors (TLRs) and RIG-I like receptors (RLRs), can detect distinct pathogen-associated molecular patterns (PAMPs) and trigger the expression of IFNs and cytokines. RIG-I has been shown to recognize hantaviral invasion, but its regulatory process remains unclear (5) . Long noncoding RNAs (lncRNAs) have emerged as important modulators of gene expression. lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is an essential architectural constituent of paraspeckles in the mammalian nucleus, interacting with Drosophila DBHS RNA-binding proteins such as the splicing factor proline-and glutamine-rich protein (SFPQ) and the non-POU domain-containing, octamer-binding protein (NONO/p54) (6) . To date, two isoform transcripts of the NEAT1 gene have been identified, namely, the 3.7-kb NEAT1-1 (MEN) and the 23-kb NEAT1-2 (MEN␤) (Fig. 1A) . A large amount of research has shown that NEAT1 is associated with oncogenesis and tumor progression (7) (8) (9) , promoting cancer formation in mice by dampening oncogene-dependent activation of p53 (10) . Nevertheless, studies assessing the function of NEAT1 in viral infections are scarce. Here, the human umbilical vein endothelial cell (HUVEC) transcriptome was analyzed after HTNV infection by digital gene expression (DGE) profiling, and lncRNA NEAT1 was found to be remarkably upregulated by viral infection. Silencing NEAT1 in vitro or in vivo suppressed host immune responses and aggravated HTNV infection, whereas NEAT1 overexpression in vitro enhanced beta interferon (IFN-␤) production and inhibited HTNV replication. Further investigation showed that NEAT1 promoted RIG-I and DDX60 expression by relocating SFPQ and removing the transcriptional inhibitory effects of SFPQ, which are critical for IFN responses against HTNV infection. We also found that RIG-I signaling, rather than TLR3 and TLR4, accounted for the elevation of HTNV-induced NEAT1. Taken together, our findings provide novel insights into the lncRNA-mediated regulatory mechanism of host innate defense against HTNV infection. To explore the potential role of long noncoding RNAs in host innate immune responses, DGE analysis of HUVECs for whole-genome profiling was performed at 24 h post-HTNV infection. As shown in Fig. 1B , the NEAT1 level in the HTNV group was higher than that in the mock group (P ϭ 6.86 ϫ 10 Ϫ13 , false discovery rate [FDR] ϭ 9.75 ϫ 10 Ϫ12 ) or the 60 Co-inactivated HTNV group (P ϭ 1.75 ϫ 10 Ϫ14 , FDR ϭ 3.10 ϫ 10 Ϫ13 ); however, the difference between the 60 Co-inactivated HTNV group and the mock group was not significant (P ϭ 0.21034, FDR ϭ 0.58211). To confirm the profiling results, two primer pairs from the published literature (11) , one recognizing both NEAT1-1 and NEAT1-2 and the other specific for NEAT1-2 (Fig. 1A) , were applied to quantify NEAT1 RNA isoforms by quantitative real-time PCR (qRT-PCR). It has been reported that NEAT1-2 rather than NEAT1-1 plays a key regulatory role in paraspeckle formation (11) , and we also found that elevated NEAT1 levels depend on live HTNV infection rather than 60 Co-inactivated HTNV stimulation (Fig. 1C) . Fluorescence in situ hybridization (FISH) with probes specific for NEAT1-2 was performed with HUVECs, and the results confirmed increased NEAT1-2 expression and the aggregation of NEAT1-2 in the nucleus at 24 and 48 h postinfection (hpi) (Fig. 1D) . To further investigate whether NEAT1 expression was altered in other cell lines, HEK293, HeLa, and A549 cells were used. All these cells could be infected by HTNV ( Fig. 1E and F) and generated hantavirus progeny (Fig. 1G ). Similar to the data obtained from HUVECs, NEAT1 was indeed upregulated by HTNV at a multiplicity of infection (MOI) of 1 beginning at 24 hpi in HUVECs and A549, HEK293, and HeLa cells, and the increasing tendency occurred in a time-dependent manner (Fig. 1H ). Of note, the NEAT1 elevation at 2 hpi might have been unrelated to the virus but resulted in cellular stress responses. Besides, NEAT1 expression increased from an MOI of 0.1 to 1, indicating that the elevation occurred in a viral dose-dependent manner (Fig. 1I) . NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. The abovedescribed data showed that HTNV infection increased NEAT1, and we wondered how NEAT1 could reciprocally influence HTNV replication. The small interfering RNA (siRNA) transfection efficiency in HUVECs was confirmed by flow cytometry, and NEAT1 expression was significantly decreased, as assessed by qRT-PCR after RNA interference (RNAi) ( Fig. 2A) . Of note, si-NEAT1 targets both NEAT1-1 and NEAT1-2, whereas the stealth siRNA NEAT1-2 (st-NEAT1-2) is specific for NEAT1-2. Compared with the cells transfected with control siRNA (negative control [NC]), HUVECs with si-NEAT1 could dramatically promote HTNV NP production, and NP expression seemed to be related to the amount of applied si-NEAT1 (Fig. 2B) . Intriguingly, depletion of NEAT1-2 alone could mimic the antiviral effects of simultaneous NEAT1-1 and NEAT1-2 silencing (Fig. 2C) , indicating that NEAT1-2 was critical for the antiviral responses. Consistent with those data, the expressions of HTNV mRNA of S segment (Fig. 2D ) and HTNV titers (Fig. 2E ) were increased after NEAT1 silencing. On the other hand, plasmids, of which pCMV-NEAT1-1 is transcribed into the 3.7-kb NEAT1-1 (MEN) and pCMV-NEAT1-2 is transcribed into the 2-to 3-kb NEAT1-2 (MEN␤), were applied to directly investigate the role of NEAT1 in HTNV infection (Fig. 2F) . Surprisingly, we found NEAT1-1 overexpression restricted NEAT1-2 transcription (Fig. 2F ). Overexpression of NEAT1 with both pCMV-NEAT1-1 and pCMV-NEAT1-2 could conspicuously repress HTNV NP expression, and NP expression seemed to be associated with the transfected plasmids (Fig. 2G) . Furthermore, overexpression of NEAT1-2 instead of NEAT1-1 could efficiently suppress HTNV replication (Fig. 2H ). NEAT1-1 upregulation even aggravated HTNV infection (Fig. 2H ), which may be the result of downregulation of NEAT1-2. Consistently, through analysis of viral load detected by qRT-PCR and the 50% tissue culture infective dose (TCID 50 ) test by ELISA, we found that expression of HTNV-specific mRNA (Fig. 2I ) and HTNV titers (Fig. 2J ) were limited in HUVECs in which NEAT1-2 was ectopically expressed in comparison to those transfected with control vector or pCMV-NEAT1-1. These data further showed that NEAT1-2 and not NEAT1-1 suppresses HTNV replication in HUVECs. Alteration of NEAT1-2 affects HTNV-induced IFN expression in HUVECs. IFN-␤ production or pretreatment at an early infection stage plays an important role in limiting HTNV infection, while IFN-␤ treatment after 24 hpi exerts little antiviral effect (12, 13) . It has been reported that the GnT of hantaviruses suppressed IFN-␤ expression of host cells at an early stage of infection (14) . Here, we also found that HUVECs could not efficiently produce IFN-␤ until 12 hpi at an MOI of 0.1 or until 24 hpi at an MOI of 1 (Fig. 3A) , which indicated that high doses of HTNV could hamper prompt IFN responses. Notably, enhanced NEAT1-2 transcription appeared at 8 hpi at an MOI of 0.1 or at 20 hpi at an MOI of 1 (Fig. 3B) , suggesting that NEAT1-2 expression increased just before IFN production. We found that expression of endogenous IFN-␤ mRNA was much lower in cells transfected with si-NEAT1-2 at MOIs of both 0.1 (Fig. 3C ) and 1 (Fig. 3D ) than in those transfected with control siRNA (NC). In contrast, overexpression of NEAT1 in HUVECs increased IFN-␤ expression after HTNV infection (MOI ϭ 1) at 24 and 48 hpi (Fig. 3E ). More importantly, HUVECs transfected with pCMV-NEAT1-2 conspicuously increased IFN-␤ gene expression compared with those cells with vector plasmids at 12 hpi (MOI ϭ 1), demonstrating that NEAT1-2 overexpression accelerated robust IFN responses in host cells against HTNV infection. With a dual luciferase reporter system Twenty-four hours after transfection, the cells expressing FAM were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with NC sequences, si-NEAT1, or the stealth siRNA NEAT1-2 (st-NEAT1-2) (3 g). Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD (n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the NC group). NS, nonsignificant. (B) HUVECs in six-well plates were transfected with NC sequences (the amount of Si-NEAT1-2 is considered 0 g) or increasing amounts of si-NEAT1 (0.1, 0.5, 1, and 3 g). Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 0.1 for 48 h. The expression of HTNV NP was measured by Western blotting. (C) HUVECs were treated as described for panel A, right, but at an MOI of 0.1. In-cell Western (ICW) analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence (NP/␤-actin) was analyzed using Student's t test. (n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NC group). (D) HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD (n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group). (E) HUVECs were treated as described for panel A, right, but at an MOI of 0.1. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD (n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the NC group). (F) Left, HUVECs in six-well plates were transfected with vectors or pGFP (3 g). Twenty-four hours after transfection, the cells expressing green fluorescent protein (GFP) were calculated by flow cytometry. Right, HUVECs in six-well plates were transfected with control plasmids (vector), pCMV-NEAT1-1, or pCMV-NEAT1-2 (3 g). Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1. At (Continued on next page) NEAT1 Promotes Innate Antiviral Responses Journal of Virology maintaining IFN-␤ promoters, we found NEAT1-2 silencing or overexpression could inhibit or increase the promoter activity of the IFN-␤ gene after HTNV infection, respectively, whereas silencing NEAT1-2 or ectopically expressing NEAT1-2 without HTNV infection could not inhibit or enhance IFN-␤ expression (Fig. 3F) . These results showed that NEAT1-2 regulated HTNV-induced IFN-␤ expression. To explore whether the antihantavirus effects of NEAT1 were caused by IFN-␤ alteration, a series of compensatory experiments was designed. In NEAT1-2 knockdown HUVECs, the addition of IFN-␤ at 12 hpi could efficiently block HTNV NP production (MOI ϭ 0.1), and such phenomena were also determined by the amount of applied IFN-␤ (Fig. 3G ). In addition, in cells with high NEAT1-2 expression, treatment with neutralizing antibodies (NAbs) of IFN-␣ and IFN-␤ could counteract the antiviral effects of NEAT1-2 (MOI ϭ 1), and the compensatory effects were dependent on the magnitude of the NAbs. Together these results demonstrated that NEAT1-2 especially enhanced the host antihantaviral innate immune responses by regulating IFN-␤ signaling. RIG-I and DDX60 regulated by NEAT1-2 facilitate HTNV-induced IFN-␤ production. PRRs maintain a vital role in the promotion of IFN responses, and we conjectured that NEAT1 might amplify IFN responses by modulating these molecules. TLR3, TLR4, and RIG-I have been shown to recognize HTNV infection (5, 15, 16) . DDX60 was recently reported as an important activator of RIG-I, but the antiviral effects of DDX60 remain a subject of debate (17, 18) , Here, we found that multiple Toll-like receptors like TLR1, TLR2, TLR3, and TLR4, as well as MDA5, were increased after HTNV infection, but none of them were influenced by silencing NEAT1-2 ( Fig. 4A ). The upregulated RIG-I and DDX60 were blocked in the cells with low NEAT1-2 expression after HTNV infection (Fig. 4A ). HUVECs with declining NEAT1-2 expression showed gradually decreasing expression of RIG-I and DDX60 (Fig. 4B) , and increasing NEAT1-2 transcription was found to activate RIG-I and DDX60 production accordingly (Fig. 4C ). These data indicated that NEAT1-2 could positively modulate RIG-I and DDX60 expression, while the role of RIG-I and DDX60 upon HTNV infection is obscure. We then found that RIG-I and DDX60 colocalized after HTNV infection (Fig. 4D ), implying that RIG-I and DDX60 might collaborate with each other to exert antiviral effects. To verify the antiviral role of RIG-I and DDX60, we designed a series of siRNAs targeting RIG-I and DDX60, and we selected the si-RIG-I-2 and siRNA-DDX60-1 with the highest knockdown efficiency by qRT-PCR in HUVECs (data not shown). Simultaneously knocking down RIG-I and DDX60 significantly promoted HTNV NP expression (Fig. 4E ), and knockdown of both of them could greatly affect IFN-␤ expression ( Fig. 4F and G). Ectopic expression of either RIG-I or DDX60 inhibited viral replication, whereas overexpression of both resulted in superior antiviral effects (Fig. 4H ), indicating that efficient anti-HTNV responses might depend on the interactive effects of DDX60 and RIG-I. More importantly, RIG-I or/and DDX60 overexpression enhanced HTNV-induced IFN-␤ expression, and they had synergistic effects on IFN-␤ production ( Fig. 4I and J). Consequently, NEAT1 might regulate IFN-␤ production by upregulating RIG-I and DDX60, and thus we were interested in how NEAT1 regulated RIG-I and DDX60 expression. SFPQ, which is relocated by NEAT1 HTNV infection, regulates the expression of RIG-I and DDX60. NEAT1 was found to interact with SFPQ by RNA immunoprecipitation (RIP) after HTNV infection (Fig. 5A) , indicating that modulatory effects of NEAT1 48 hpi, the NEAT1 expression levels were measured by qRT-PCR. Values are means Ϯ SD (n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; ***, P Ͻ 0.0001; Student's t test, compared with the vector group). (G) HUVECs in six-well plates were transfected with control plasmids (vector, the amount of pCMV-NEAT1-1 plus pCMV-NEAT1-2 is considered 0 g) or increasing amounts of pCMV-NEAT1-1 plus pCMV-NEAT1-2 (0.05 ϩ 0.05 g, 0.25 ϩ 0.25 g, 0.5 ϩ 0.5 g, 1.5 ϩ 1.5 g, respectively). Twenty-four hours after transfection, the cells were infected with HTNV at an MOI of 1 for 48 h. The expression of HTNV NP was measured by Western blotting. (H) HUVECs were treated as described for panel F, right. In-Cell Western (ICW) analysis for HTNV NP was performed at 48 hpi. The ICW for HTNV NP staining is shown on the left, while the relative intensity of fluorescence (NP/␤-actin) was analyzed using Student's t test. (n ϭ 4; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group). (H) HUVECs were treated as described for panel F, right. The expression of HTNV S segment was measured by qRT-PCR. Values are means Ϯ SD (n ϭ 3; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the vector group). (I) HUVECs were treated as described for panel F, right. The propagated HTNV was acquired at 72 hpi, and viral titers were detected by TCID 50 with ELISA in Vero E6 cells. Values are means Ϯ SD (n ϭ 3; *, P Ͻ 0.01; Student's t test, compared with the vector group). might be involved in SFPQ. Interestingly, the protein level of SFPQ, as well as another paraspeckle-forming constituent, NONO, remained unchanged after HTNV infection (Fig. 5B ) or after NEAT1 overexpression and knockdown (Fig. 5C ). However, SFPQ became centralized rather than diffuse in the nucleus after HTNV infection (Fig. 5D) . The enhanced interaction of SFPQ and NONO indicated excess formation of paraspeckles in the nucleus (Fig. 5E ) and relocalization of SFPQ. SFPQ knockdown could inhibit HTNV replication ( Fig. 5F and G), which might have been related to the increase in RIG-I (Fig. 5H ) and DDX60 (Fig. 5I ). SFPQ has been suggested to bind to the promoter region of RIG-I and DDX60 (11), thus preventing the expression of RIG-I and DDX60. Taken together, the above results suggested that NEAT1 might relocate SFPQ from the RIG-I signaling is crucial for NEAT1 expression after HTNV infection. Elevated NEAT1 exerts antiviral effects by modulating the innate immune response, yet it is unclear how HTNV triggers NEAT1 transcription. Interestingly, overexpression of the S or M segment of HTNV in HEK293 cells failed to induce NEAT1 expression, suggesting that NEAT1 transcription was closely related to live viral replication (Fig. 6A) . Of note, the upregulation of NEAT1 by HTNV could not be reversed by applying IFN-I neutralizing antibodies (Fig. 6B) . Meanwhile, NEAT1 expression could not be induced by stimulation with different types of IFNs (Fig. 6C, D, and E) or cytokines (Fig. 6F and G) . We conjectured that NEAT1 expression was related to the activation of PRRs. By knocking down several PRRs, we found that the RIG-I and TLR4 pathways played important roles in HTNV-induced NEAT1 upregulation (Fig. 7A) . Using RIG-I-and TLR4-deficient cell lines which could be well infected by HTNV (Fig. 7B) , RIG-I was confirmed to be indispensable for NEAT1 induction after HTNV infection (Fig. 7C) . Moreover, using STAT1 as a positive control, we found that the transcription factor IRF7, rather than IRF3 and p65, translocated into the nucleus in HTNV-infected HUVECs at 2 dpi (Fig. 7D) . Furthermore, IRF7 knockdown blocked HTNV-induced NEAT1 upregulation (Fig. 7E) . Therefore, HTNV caused transcriptional activation of the NEAT1 gene, probably via the RIG-I-IRF7 pathway. NEAT1 silencing has profound effects on innate immune responses after HTNV infection in mice. Although cell-based experiments revealed that NEAT1-2 is a crucial regulator of innate antihantaviral responses, its function in vivo has remained unclear. To address this question, we intravenously injected siRNAs targeting mouse NEAT1-2 at 1 day before HTNV infection. NEAT1-2 expression levels in the liver, kidney, and spleen were reduced at 2 dpi (Fig. 8A) . Previous studies have shown that NEAT1 knockout does not affect physiological processes except potentia generandi in mice; hence, we assessed its role under pathological conditions. Body weight loss in NEAT1-2-depleted mice was observed from 2 dpi to 5 dpi, and the IFN production in serum was remarkably decreased in the NEAT1-2 silenced group than those in the NC group at 3 dpi (Fig. 8B) . As expected, NEAT1-2 knockdown mice showed considerably higher HTNV NP levels in the liver, spleen, and kidney at 3 dpi (Fig. 8C) . Moreover, the virus titers in related organs were higher in the NEAT1-2 silenced group than in the NC group (Fig. 8D ). In addition, reduced inflammatory cell filtration but increased tissue injury was found in NEAT1-2 knockdown mice during the early stage of infection (Fig. 8E) . Infiltration of macrophages in the spleen was attenuated (Fig. 8F) , and the activation of macrophages was also suppressed (by flow cytometry; data not shown). Moreover, CD8 ϩ IFN-␥ ϩ T cells were reduced in the spleens of NEAT1-2 knockdown mice in comparison to those in the NC group at 3 dpi (Fig. 8G) . Nevertheless, NEAT1-2 silencing had no effect on the production of neutralizing antibodies at 7 dpi (data not shown). The above-described findings indicated that NEAT1-2 depletion might influence multiple aspects of the innate immune response in HTNV-infected mice. Innate immunity is a phylogenetically ancient and conserved system that counteracts invading microbes, the regulatory mechanism of which is sophisticated and complex. Long noncoding RNAs, which were once considered dark materials in the mammalian genome, have been shown to exert vital modulatory effects on host innate immunity (19) . In this report, we first demonstrated that NEAT1 was induced by HTNV through the RIG-I-IRF7 pathway and served as positive feedback for RIG-I signaling. Using DGE analysis, we observed upregulated NEAT1 and confirmed its alteration in To further determine the function of NEAT1 after HTNV infection in vivo, mice were injected intravenously with si-NEAT1-2 (1 g/g) or nontarget control siRNA (NC) (1 g/g); 1 day later, they were infected with HTNV (100 LD 50 ) by intramuscular injection. (A) To maintain high knockdown efficiency, siRNAs were injected intravenously every other day. (A) The knockdown efficiency was assessed by qRT-PCR in kidney, liver, and spleen samples at 2 dpi (n ϭ 6 in each group). (B) The effects of NEAT1 on HTNV virulence in mice were determined by body weight loss from 0 to 10 dpi (left panel, n ϭ 10 in each group). The IFN-␤ in sera of different groups was measured by ELISA at 3dpi (right panel, n ϭ 8 in each group). Values are means Ϯ SD (*, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group). (C) Mice were sacrificed at 3 dpi, and livers, spleens, and kidneys were collected for ELISA detection of HTNV NP titers (upper panels, n ϭ 8 in each group) and qRT-PCR to assess HTNV S segment levels (bottom panels, n ϭ 8 in each group) at 3 dpi. Values are means Ϯ SD (*, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group). (D) NEAT1 effects on HTNV infection kinetics at 3 dpi were determined by testing the HTNV titers in livers, spleens, and kidneys. Values are means Ϯ SD (n ϭ 8; *, P Ͻ 0.01; **, P Ͻ 0.001; Student's t test, compared with the NCϩHTNV group). (E) Hematoxylin and eosin (H&E) staining for mouse liver, spleen, or kidney specimens was performed (3 dpi, n ϭ 8 in each group). After HTNV infection, livers in the NC group showed inflammatory cell infiltration in certain regions, while those in the si-NEAT1-2 group showed slight acute viral hepatitis. Spleens in the NC group showed lymph node hyperplasia, while those in the si-NEAT1-2 group were severely congestive. Kidneys in the NC group also showed inflammatory cell infiltration, while those in the si-NEAT1-2 group had moderate interstitial congestion. (F) Macrophage infiltration in spleens was analyzed by detecting CD11b and F4/80 by flow cytometry at 3 dpi, and the results obtained for four mice in each group are presented. (G) CD3 ϩ CD8 ϩ IFN-␥ ϩ T cells were analyzed by flow cytometry at 3 dpi, and the results obtained for three mice in each group are presented. different cell lines. To assess its effects on HTNV replication, NEAT1 was silenced both in vitro and in vivo, which resulted in increased HTNV infection and suppressed innate immune responses. Further analysis indicated that NEAT1 might interact with SFPQ and regulate DDX60 and RIG-I expression. By virtue of RNAi, the RIG-I-IRF7 pathway was confirmed to be necessary for HTNV-triggered NEAT1 elevation. Recently, large-scale transcriptomic studies identified numerous noncoding transcripts in the mammalian genome, which were speculated to influence diverse biological processes. Among these noncoding RNAs (ncRNAs), long noncoding RNAs (lncRNAs) emerged as important regulators of gene expression and are closely related to the activation of the host innate immune system. TLR2 controls lncRNA-COX2 expression in a MyD88-and NF-B-dependent manner, whereas lncRNA-COX either promotes interleukin 6 (IL-6) secretion or represses ISG15 and CCL5 expression (20) . TLR2 activation or tumor necrosis factor alpha (TNF-␣) stimulation induces transcription of the lncRNA THRIL, the downregulation of which impairs TNF-␣ and IL-6 secretion (21) . TLR4 signaling in response to lipopolysaccharide (LPS) induces lncRNA IL-1␤-eRNA and IL-1␤-RBT46, the knockdown of which attenuates IL-1␤ and CXCL8 release (22) . The lncRNA Lethe, triggered by TNF-␣ and IL-1␤, acts as a negative feedback regulator of NF-B signaling (23) . The roles of lncRNAs in host-virus interactions have been progressively unveiled. Various viruses, such as influenza virus (IAV), coronavirus, enterovirus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Japanese encephalitis virus (JEV), and rabies virus, have been reported to activate the transcription of different lncRNAs in host cells (11, (24) (25) (26) . Importantly, multiple lncRNAs have been shown to affect the IFN response in recent years and have gradually become hot spots in the field of antiviral research. NeST was shown to enhance IFN-␥ production, controlling the susceptibility of mice to persistent Theiler's virus infection as well as resistance to Salmonella enterica serovar Typhimurium infection (27) . Both CMPK2 and NRAV were identified as negative regulators of IFN immune reactions. CMPK2, induced by IFN-␣ or HCV infection, suppresses various ISGs, the knockdown of which dramatically blocks HCV replication (26) . NRAV inhibits some critical ISGs, such as IFITM3 and Mx1, the depletion of which suppresses IAV replication both in vitro and in vivo (25) . Numerous lncRNAs, including lnc-ISG15 and ISR2, respond to IFNs such as ISGs, although their actual function requires further investigation (28) . Considering the poor evolutionary conservation but rapid divergence of lncRNAs, their functions may be highly species and virus specific. Though considerable progress has been achieved to demonstrate the antiviral effects of lncRNAs on model viruses, there are no published reports assessing the role of lncRNAs in hantaviral infection. NEAT1 has been reported to interact with Drosophila DBHS RNA-binding proteins (e.g., SFPQ, NONO-p54nrb, and PSPC1), recruiting them to paraspeckles, a nuclear substructure found in all cultured and primary cells except embryonic stem cells (24) . The versatile function of NEAT1 is rapidly progressing in multiple areas of biology. NEAT1 has been reported to be involved in the pathogenesis of multiple types of cancer (7) (8) (9) . NEAT1 also participates in neurodegenerative diseases such as Huntington's disease (29) and seems to potentially contribute to the elevated production of a number of cytokines and chemokines in patients with systemic lupus erythematosus (SLE) (30) . Furthermore, poly I·C can activate NEAT1 transcription through the TLR3 pathway, whereas NEAT1 positively regulates IL-8 transcription and potentially affects the expression of multiple ISGs after poly I·C stimulation (11) . In addition, NEAT1 has been reported to suppress the export of Rev-dependent instability element (INS)containing HIV-1 mRNAs from the nucleus to the cytoplasm, thus inhibiting HIV replication (24) . However, the role of NEAT1 in hantaviral infection remains unclear. In this report, NEAT1 has been identified as an important regulator of the host innate immune system against HTNV infection. Elevated NEAT1 promotes IFN secretion, most likely by enhancing RIG-I and DDX60 expression. DDX60, a DEXD/H box RNA helicase similar to Saccharomyces cerevisiae Ski2, is induced after viral infection (31) . DDX60 recognizes viral RNA and activates endogenous RIG-I, thereby promoting the RIG-I signaling-related IFN response. However, the antiviral effects of DDX60 seem to vary among viruses (17) . We found that NEAT1-regulated DDX60 was involved in IFN production in response to HTNV infection. In HTNV-infected cells, double-stranded RNA (dsRNA) could not be detected, and it is unclear how host PRRs, especially RIG-I, recognize HTNV invasion (5) . Here, considering the interaction of RIG-I and DDX60 and the effect of DDX60 on IFN-␤ production, we hypothesize that DDX60 might mediate RIG-I signaling activation upon HTNV infection, which requires further investigation. Of note, we applied multiple cell lines to explore the role of NEAT1 during HTNV infection. HTNV primarily targets vascular endothelial cells in vivo and contributes to the increased vascular permeability and coagulation disorders in HFRS; hence, HUVECs are the most common in vitro cell model to study host innate immunity against HTNV infection or viral pathogenesis (32) . EVC-304 cells are also endovascular cells, whereas EVC-304 TLR4 Ϫ cells are TLR4-deleted cells, both of which have been used for HTNV infection related studies (15, 33) . A549 cells were once used to isolate HTNV, and they were confirmed to be a mature model of infection (34) (35) (36) (37) . Additionally, Huh 7.0 and Huh 7.5 (RIG-I Ϫ ) cells used in our study have been reported to be infected by HTNV by Lee et al. (5) and can be used as a cell model to study immune responses against HTNV replication (38, 39) . Additionally, HEK293 (40) and HeLa (41) cells have been reported to be infected by HTNV. Using qRT-PCR, Western blotting, and immunofluorescence assays, we have also shown that both HEK293 and HeLa cells can be infected by HTNV. To study the molecular mechanism underlying the effect of NEAT1 on IFN expression and HTNV infection, it may be suitable to use HEK293 and HeLa cells as a cell model, especially under conditions in which HTNV NP can be detected using Western blot or immunofluorescence analyses. In experiments to assess the effect of NEAT1 on the control of hantaviruses, In-Cell Western (ICW) analysis was applied to qualify HTNV NP production. Alterations in the relative fluorescence intensity of NP after silencing or overexpressing NEAT1-2 did not seem to be as remarkable as qRT-PCR or Western blot analysis results. The NP spotted and exhibited in the ICW results forms obvious stains that mimic PFU. However, the specific values scanned and analyzed by the ICW assay reflect only the fluorescence intensity of the integral well instead of the number of spots. As a consequence, the intensity represented the quantity of NP production but could not directly indicate the virulence, which was better shown by plaque-forming assays. The RNAi studies in vivo are encouraging (Fig. 8) , but the NC used by our group was not mutated si-NEAT1-2 (i.e., same sense strand, but with a point mutation in the targeting strand). The results would be more compelling if the control mice had been treated with the mutated si-NEAT1-2. One major finding of our study is that the lncRNA NEAT1 serves as positive feedback for RIG-I signaling. After observing that NEAT1 can regulate IFN expression by HTNV infection, we were interested in the function of NEAT1. We noticed that silencing NEAT1-2 or ectopically expressing NEAT1-2 could not inhibit or enhance IFN expression without HTNV infection (Fig. 3F) , which indicated that NEAT1-2 could not directly affect IFN-␤ expression. This finding excludes the possibility that NEAT1-2 directly promoted IFN-␤ and that IFN-␤ promoted the expression of PRRs such as RIG-I. Thereafter, NEAT1 was found to modulate HTNV-induced RIG-I and DDX60 expression. Recent findings have shown that RIG-I signaling is essential for an efficient polyfunctional T cell response during IAV infection (42) . Indeed, we found that the function of T cells was suppressed after NEAT1-2 depletion in our animal experiments (Fig. 8G) , which might be due to the disrupted RIG-I signaling in NEAT1-2 silenced T cells. In conclusion, this is the first study to describe the role of NEAT1 in HTNV infection. HTNV infection induced NEAT1 expression through the RIG-I-IRF7 pathway, while NEAT1 displayed positive feedback for RIG-I signaling. NEAT1 relocated SFPQ from the potential promoter region of several antiviral genes to the paraspeckles, removing the transcriptional inhibitory effects of SFPQ. This phenomenon would facilitate the expression of DDX60 and RIG-I, thus promoting IFN responses and suppressing HTNV infection (Fig. 9) . nontarget control (i.e., negative control [NC]) or targeted RIG-I and DDX60 were designed by Gene-Pharma as follows: NC, 5=- UUCUUCGAACGUGUCACGUTT-3=; si-RIG-I-1, 5=-GCCCAUUUAAACCAAGAAATT-3=, si-RIG-I-2, 5=-GGUGGAGGAUAUUUGAACUTT-3=, and si-RIG-I-3, 5=-CCCAACGAUAUCAUUUCUTT-3=; si-DDX60-1, 5=-GUCCAGGUGUCAGUUUGAUTT-3=, si-DDX60-2, 5=-CCGAAGUGAAGAAGGUAAATT-3=, and si-DDX60-3, 5=-GAUGGAUGCUAGGAAAUAUTT-3=. The pCMV-NEAT1-1 and pCMV-NEAT1-2 plasmids, which transcribe NEAT1-1 and NEAT1-2, respectively, were provided by Nakagawa Shinichi (10) . The Flag-RIG-I and pUNO-DDX60 plasmids were purchased from Invitrogen. Reagents. Mouse monoclonal antibody (MAb) 1A8 for the HTNV nucleocapsid protein (NP) was produced as previously described (43) . Abs against RIG-I, IRF3, IRF7, p65, and STAT1, as well as the neutralizing antibodies against IFN-␣ and IFN-␤, were purchased from Abcam (Cambridge, MA, USA). Phorbol myristate acetate (PMA) and Ab against DDX60 were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Abs against SFPQ, NONO, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and ␤-actin were purchased from Protein Tech, Inc. (Wuhan, China). The Abs targeting CD11b, F4/80, CD3, CD8, IFN-␥, inducible nitric oxide synthase (iNOS), and CD206 for flow cytometry were purchased from BD Biosciences (San Jose, CA, USA). IFN-␣, -␤, and -␥, TNF-␣, and IL-1␤ were from PeperoTech (Rocky Hill, NJ). ELISA kits for IFN-␤ detection were manufactured by R&D Systems, Inc. (Minneapolis, MN, USA) . RNA extraction and quantitative real-time PCR (qRT-PCR) analysis. Total cellular RNAs were extracted with RNAiso (TaKaRa, Dalian, China), the concentration of which was measured using a NanoDrop 1000 spectrophotometer. Reverse transcription (RT) was then performed with PrimeScript RT master mix (TaKaRa) according to the instructions provided by the manufacturer. Each cDNA was denatured at 95°C for 5 min and amplified for 40 cycles of 15 s at 98°C, 30 s at 58°C, and 30 s at 72°C using a LightCycler 96 (Roche, Basel, Switzerland). The mRNA expression level of each target gene was normalized to the respective ␤-actin and analyzed. The qRT-PCR primer sequences for NEAT1, NEAT1-2, IFN-␤, HTNV S segment, RIG-I, DDX60, ␤-actin, and GAPDH were obtained from previous reports (24, 45) . The methods used to quantify HTNV RNA load have been described by our group previously (46) . DGE analysis and lncRNA sequencing. HUVECs with a confluence of 80% in 6 wells were mock infected or infected with live or 60 Co-inactivated HTNV at an MOI of 1. RNAs were extracted as previously described at 24 hpi, and the quality was analyzed using FastQC software by the Beijing Genomics Institute (BGI, Shenzhen, China). Digital gene expression (DGE) tags were annotated to the human transcriptome (Ensembl version 58) by mapping the reads to the sequence flanking NlaIII restriction sites on both coding and noncoding strands. Tags matching more than one gene region were discarded. Tag counts were normalized to TPM (transcripts per million) by dividing the raw tag count by the total number of tags from each library and multiplying by 1 million. To avoid the possible noise signal from high-throughput sequencing, the genes with average TPM of less than 1 in these three states were excluded. In this study, an absolute fold change of no less than 1.5 and a false discovery rate (FDR) of less than 0.001 were used to define the differentially expressed genes. Genes were selected as differentially expressed using a P value threshold of 0.01. Genes were selected as differentially expressed using a P value threshold of 0.01. FISH and immunofluorescence assays (IFA). Fluorescence in situ hybridization (FISH) was performed with a FISH kit (Ribobio Co.) according to the manufacturer's instructions. In brief, cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Prehybridization was performed with lncRNA FISH probe mix at 37°C for 30 min, and then hybridization was performed by adding NEAT1-2 FISH probe mix and incubating the mixture at 37°C overnight. After washing with 4ϫ, 2ϫ, and 1ϫ SSC, the cell nuclei were stained with DAPI (4=,6-diamidino-2-phenylindole). Finally, the samples were observed using a BX60 fluorescence microscope (Olympus, Tokyo, Japan). IFA was performed after FISH or independently. The cells were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 15 min. Primary Abs were added and incubated at 37°C for 2 h. After five washes with DPBS, secondary Cy3-or fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit or goat anti-mouse IgG (Sangon, Shanghai, China) was added and incubated at 37°C for 2 h. Cell nuclei were stained with DAPI. Finally, the samples were observed using a BX60 fluorescence microscope (Olympus). Cells were washed twice with ice-cold DPBS and lysed with 1ϫ SDS protein loading buffer (50 mM Tris, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol, and 0.1% bromophenol blue). The samples were then boiled at 95°C for 10 min. The lysates were resolved by 10%, 12%, or 15% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were incubated with the primary antibodies, followed by secondary antibodies labeled with infrared dyes (Li-Cor Biosciences, Lincoln, NE, USA). The signals on the PVDF membrane were visualized using an Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE, USA). ICW assay. The In-Cell Western (ICW) assay was performed using an Odyssey imaging system (Li-Cor) according to the manufacturer's instructions. HUVECs were either mock transfected or transfected with NC sequences, si-NEAT1, st-NEAT1-2, vector plasmids, pCMV-NEAT1-1, or pCMV-NEAT1-2 and grown in 96-well plates (2 ϫ 10 4 cells/well). Twenty-four hours posttransfection, the cells were either infected or mock infected with HTNV at an MOI of 1. At 48 hpi, HUVECs were washed twice with ice-cold DPBS, fixed with 4% PFA for 10 min, and permeabilized with 1.0% Triton X-100 for 15 min. Cells were added with Li-Cor Odyssey blocking solution at room temperature for 30 min and incubated at 4°C overnight with mouse IgG MAb 1A8 against HTNV NP together with rabbit IgG antibody against ␤-actin, both of which were diluted in PBS containing 3% bovine serum albumin (BSA; HyClone). Subsequently, the cells were washed and stained with goat anti-mouse IgG IRDye 800 antibody (1:5,000; Li-Cor) and goat anti-rabbit
What evidence suggests that RIG-I and DDX60 collaborate to exert antiviral effects?
false
5,312
{ "text": [ "colocalized after HTNV infection" ], "answer_start": [ 16456 ] }
2,504
Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.
What is the effect of viral components remaining in the airway?
false
3,974
{ "text": [ "antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation" ], "answer_start": [ 17828 ] }
1,595
Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3 Inflammasome https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361828/ SHA: f02d0c1e8b0109648e578662dc250abe349a033c Authors: Chen, I-Yin; Moriyama, Miyu; Chang, Ming-Fu; Ichinohe, Takeshi Date: 2019-01-29 DOI: 10.3389/fmicb.2019.00050 License: cc-by Abstract: Nod-like receptor family, pyrin domain-containing 3 (NLRP3) regulates the secretion of proinflammatory cytokines interleukin 1 beta (IL-1β) and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus (EMCV) 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus (SARS-CoV) activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K(+) efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus (SARS-CoV), a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame (ORF) 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins (Tan et al., 2005) . The 3 one-third of the genome encodes four structural proteins, spike (S), envelope (E), matrix (M) and nucleocapsid (N), and non-structural proteins, along with a set of accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b) (Perlman and Dandekar, 2005; Tan et al., 2005) . SARS-CoV is the etiological agent of SARS (Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003) . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, were detected in autopsy tissues from SARS patients (He et al., 2006) . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (Medzhitov, 2001; Kawai and Akira, 2010) . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 (NLRP3) is activated by a wide variety of stimuli, including virus infection (Bauernfeind et al., 2011) . Four models describing activation of the NLRP3 inflammasome have been proposed thus far (Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010) . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role (Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013) . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals (Duewell et al., 2010) , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species (ROS) or mitochondrial DNA from damaged mitochondria (Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012) . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 (Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015) . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling (MAVS) or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013) ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion (Kayagaki et al., 2015; Shi et al., 2015) . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins (Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015) , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels (Tan et al., 2005; Chen and Ichinohe, 2015) . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome (Nieto-Torres et al., 2015) . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains (Zeng et al., 2004; Lu et al., 2006) , are thought to act as Na + /K + and K + channels, respectively (Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011) , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science (The University of Tokyo). Bone marrow-derived macrophages (BMMs) were prepared as described previously (Ichinohe et al., 2009) . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque). Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum (FBS), and L-glutamine (2 mM) at 37 • C/5% CO 2 . HEK293FT cells (a human embryonic kidney cell line) and HeLa cells (a human epithelial carcinoma cell line) were maintained in DMEM supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 µg/ml) (Nacalai Tesque). MDCK cells (Madin-Darby canine kidney cells) and HT-1080 cells (a human fibrosarcoma cell line) were grown in Eagle's minimal essential medium (E-MEM; Nacalai Tesque) supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 µg/ml) (Nacalai Tesque). Influenza A virus strain A/PR8 (H1N1) was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs (Ichinohe et al., 2009) . The viral titer was quantified in a standard plaque assay using MDCK cells (Pang et al., 2013) . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain (Matsuyama et al., 2005) were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang (National Taiwan University College of Medicine, Taipei, Taiwan). To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors (Invitrogen). To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively (Ito et al., 2012) . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells (TOYOBO). To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP (green fluorescent protein), or pLenti-M2 using polyethylenimine (PEI) Max. At 24 h post-transfection, the cells were lysed with RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate (SDS), 150 mM NaCl and 1 mM EDTA). And the lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by electroblotting onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated over night with mouse anti-V5-tag (R960-25, Invitrogen), mouse anti-influenza A virus M2 (14C2, Abcam), mouse anti-GFP (GF200, Nacalai Tesque), or rabbit antitubulin (DM1A, Santa Cruz) antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories) or anti-rabbit IgG (Invitrogen). After washing 3 times with washing buffer (0.05% Tween-20/PBS), the membranes were exposed using Chemi-Lumi One Super (Nacalai Tesque), and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus (GE Healthcare). To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector (Invitrogen) using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish (10 cm in diameter) were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix (Invitrogen) using Lipofectamine 2000 (Invitrogen). The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter (Millipore) at 72-96 h post-transfection (Ito et al., 2012) . The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection (MOI) of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay (ELISA) using paired antibodies (eBioscience) (Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi (Ito et al., 2012) . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody (M2, Sigma) followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Life Technologies). To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA (561, MBL) and mouse anti-NLRP3 (Cryo-2; AdipoGen) antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) and Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) (Life Technologies). Fluorescent signals were observed by confocal microscopy (A1R + , Nikon). Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein (a proton-selective ion channel), its H37G mutant (which has lost its proton selectivity and enables the transport of other cations such as Na + and K + ), and the EMCV 2B protein (a Ca 2+ channel) stimulates NLRP3 inflammasome-mediated IL-1β secretion (Ichinohe et al., 2010; Ito et al., 2012) . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome (Nieto- Torres et al., 2015) . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis (Figures 1A-C) . We next transduced lipopolysaccharide (LPS)-primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports (Ichinohe et al., Figure 1D) . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs ( Figure 1D) . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells ( Figure 1E) . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F [located in the transmembrane domain (from amino acid residues 7-38)] prevent ion conductivity (Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012) . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain (amino acid residues 127-133) that is involved in the formation of a homodimer to generate the ion channel (Lu et al., 2006; Chan et al., 2009) . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein (Chan et al., 2009) . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS (Chan et al., 2009; Figure 2A) . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report (Nieto -Torres et al., 2015) , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs ( Figure 2B) . Notably, the 3a-CS mutant completely abrogated IL-1β secretion (Figure 2B) , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h (lentiviruses) or 6 h (ATP) post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports (Yu et al., 2004; Yuan et al., 2005) , the 3a protein localized to the Golgi apparatus ( Figure 3A ). In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum (ER) (Figure 3B ). By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures (Figures 3A,B) . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation (Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016) . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells (Figure 4) . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel (Lu et al., 2006) . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome (Mariathasan et al., 2006; Petrilli et al., 2007) . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result (Ichinohe et al., 2010) , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM ( Figure 5A) . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins ( Figure 5B ). Since mitochondrial ROS are important for NLRP3 inflammasome activation (Nakahira et al., 2011; Zhou et al., 2011) , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009) . As reported previously (Nakahira et al., 2011; Ito et al., 2012) , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP ( Figure 6A) . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO ( Figure 6B) . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 (Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015) . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome (Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015) . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome (Ichinohe et al., 2010) . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 (H37G), causing loss of proton selectivity, enables transport of other cations (i.e., Na + and K + ), thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 (EV71), and human rhinovirus (a member of the Picornaviridae family) triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments (Ito et al., 2012; Triantafilou et al., 2013) . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin (Negash et al., 2013; Farag et al., 2017) . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex (Wang et al., 2017) . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome (Nieto-Torres et al., 2015) . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation (Yue et al., 2018) . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation (Lu et al., 2006) , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 (Yue et al., 2018) , the ion channel activity-loss mutant 3a-CS (Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133) (Chan et al., 2009 ) completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane (Minakshi and Padhan, 2014) and acts as a K + channel (Lu et al., 2006) , thereby (presumably) stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV (Castano-Rodriguez et al., 2018) activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis.
How many laboratory-confirmed cases of SARS coronavirus infections were reported between November 2002 and July 2003?
false
288
{ "text": [ "At least 8,098" ], "answer_start": [ 2507 ] }
2,486
Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review https://doi.org/10.3390/jcm9030623 SHA: 9b0c87f808b1b66f2937d7a7acb524a756b6113b Authors: Pang, Junxiong; Wang, Min Xian; Ang, Ian Yi Han; Tan, Sharon Hui Xuan; Lewis, Ruth Frances; Chen, Jacinta I. Pei; Gutierrez, Ramona A.; Gwee, Sylvia Xiao Wei; Chua, Pearleen Ee Yong; Yang, Qian; Ng, Xian Yi; Yap, Rowena K. S.; Tan, Hao Yi; Teo, Yik Ying; Tan, Chorh Chuan; Cook, Alex R.; Yap, Jason Chin-Huat; Hsu, Li Yang Date: 2020 DOI: 10.3390/jcm9030623 License: cc-by Abstract: Rapid diagnostics, vaccines and therapeutics are important interventions for the management of the 2019 novel coronavirus (2019-nCoV) outbreak. It is timely to systematically review the potential of these interventions, including those for Middle East respiratory syndrome-Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS)-CoV, to guide policymakers globally on their prioritization of resources for research and development. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Supplementary strategies through Google Search and personal communications were used. A total of 27 studies fulfilled the criteria for review. Several laboratory protocols for confirmation of suspected 2019-nCoV cases using real-time reverse transcription polymerase chain reaction (RT-PCR) have been published. A commercial RT-PCR kit developed by the Beijing Genomic Institute is currently widely used in China and likely in Asia. However, serological assays as well as point-of-care testing kits have not been developed but are likely in the near future. Several vaccine candidates are in the pipeline. The likely earliest Phase 1 vaccine trial is a synthetic DNA-based candidate. A number of novel compounds as well as therapeutics licensed for other conditions appear to have in vitro efficacy against the 2019-nCoV. Some are being tested in clinical trials against MERS-CoV and SARS-CoV, while others have been listed for clinical trials against 2019-nCoV. However, there are currently no effective specific antivirals or drug combinations supported by high-level evidence. Text: Since mid-December 2019 and as of early February 2020, the 2019 novel coronavirus (2019-nCoV) originating from Wuhan (Hubei Province, China) has infected over 25,000 laboratory-confirmed cases across 28 countries with about 500 deaths (a case-fatality rate of about 2%). More than 90% of the cases and deaths were in China [1] . Based on the initial reported surge of cases in Wuhan, the majority were males with a median age of 55 years and linked to the Huanan Seafood Wholesale Market [2] . Most of the reported cases had similar symptoms at the onset of illness such as fever, cough, and myalgia or fatigue. Most cases developed pneumonia and some severe and even fatal respiratory diseases such as acute respiratory distress syndrome [3] . The 2019 novel coronavirus (2019-nCoV), a betacoronavirus, forms a clade within the subgenus sarbecovirus of the Orthocoronavirinae subfamily [4] . The severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also betacoronaviruses that are zoonotic in origin and have been linked to potential fatal illness during the outbreaks in 2003 and 2012, respectively [5, 6] . Based on current evidence, pathogenicity for 2019-nCoV is about 3%, which is significantly lower than SARS-CoV (10%) and MERS-CoV (40%) [7] . However, 2019-nCoV has potentially higher transmissibility (R0: 1.4-5.5) than both SARS-CoV (R0: [2] [3] [4] [5] and MERS-CoV (R0: <1) [7] . With the possible expansion of 2019-nCoV globally [8] and the declaration of the 2019-nCoV outbreak as a Public Health Emergency of International Concern by the World Health Organization, there is an urgent need for rapid diagnostics, vaccines and therapeutics to detect, prevent and contain 2019-nCoV promptly. There is however currently a lack of understanding of what is available in the early phase of 2019-nCoV outbreak. The systematic review describes and assesses the potential rapid diagnostics, vaccines and therapeutics for 2019-nCoV, based in part on the developments for MERS-CoV and SARS-CoV. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies examining the diagnosis, therapeutic drugs and vaccines for Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and the 2019 novel coronavirus (2019-nCoV), in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. There were two independent reviewers each focusing on SARS, MERS, and 2019-nCoV, respectively. A third independent reviewer was engaged to resolve any conflicting article of interest. We used the key words "SARS", "coronavirus", "MERS", "2019 Novel coronavirus", "Wuhan virus" to identify the diseases in the search strategy. The systematic searches for diagnosis, therapeutic drugs and vaccines were carried out independently and the key words "drug", "therapy", "vaccine", "diagnosis", "point of care testing" and "rapid diagnostic test" were used in conjunction with the disease key words for the respective searches. Examples of search strings can be found in Table S1 . We searched for randomized controlled trials (RCTs) and validation trials (for diagnostics test) published in English, that measured (a) the sensitivity and/or specificity of a rapid diagnostic test or a point-of-care testing kit, (b) the impact of drug therapy or (c) vaccine efficacy against either of these diseases with no date restriction applied. For the 2019-nCoV, we searched for all in vitro, animal, or human studies published in English between 1 December 2019 and 6 February 2020, on the same outcomes of interest. In addition, we reviewed the references of retrieved articles in order to identify additional studies or reports not retrieved by the initial searches. Studies that examined the mechanisms of diagnostic tests, drug therapy or vaccine efficacy against SARS, MERS and 2019-nCoV were excluded. A Google search for 2019-nCoV diagnostics (as of 6 February 2020; Table S2 ) yielded five webpage links from government and international bodies with official information and guidelines (WHO, Europe CDC, US CDC, US FDA), three webpage links on diagnostic protocols and scientific commentaries, and five webpage links on market news and press releases. Six protocols for diagnostics using reverse transcriptase polymerase chain reaction (RT-PCR) from six countries were published on WHO's website [9] . Google search for 2019-nCoV vaccines yielded 19 relevant articles. With the emergence of 2019-nCoV, real time RT-PCR remains the primary means for diagnosing the new virus strain among the many diagnostic platforms available ( [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ; Table S3 ). Among the 16 diagnostics studies selected, one study discussed the use of RT-PCR in diagnosing patients with 2019-nCoV [11] ( Table 1 ). The period and type of specimen collected for RT-PCR play an important role in the diagnosis of 2019-nCoV. It was found that the respiratory specimens were positive for the virus while serum was negative in the early period. It has also suggested that in the early days of illness, patients have high levels of virus despite the mild symptoms. Apart from the commonly used RT-PCR in diagnosing MERS-CoV, four studies identified various diagnostic methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), RT-insulated isothermal PCR (RT-iiPCR) and a one-step rRT-PCR assay based on specific TaqMan probes. RT-LAMP has similar sensitivity as real time RT-PCR. It is also highly specific and is used to detect MERS-CoV. It is comparable to the usual diagnostic tests and is rapid, simple and convenient. Likewise, RT-iiPCR and a one-step rRT-PCR assay have also shown similar sensitivity and high specificity for MER-CoV. Lastly, one study focused on the validation of the six commercial real RT-PCR kits, with high accuracy. Although real time RT-PCR is a primary method for diagnosing MERS-CoV, high levels of PCR inhibition may hinder PCR sensitivity (Table 1) . There are eleven studies that focus on SARS-CoV diagnostic testing (Table 1) . These papers described diagnostic methods to detect the virus with the majority of them using molecular testing for diagnosis. Comparison between the molecular test (i.e RT-PCR) and serological test (i.e., ELISA) showed that the molecular test has better sensitivity and specificity. Hence, enhancements to the current molecular test were conducted to improve the diagnosis. Studies looked at using nested PCR to include a pre-amplification step or incorporating N gene as an additional sensitive molecular marker to improve on the sensitivity (Table 1 ). In addition, there are seven potential rapid diagnostic kits (as of 24 January 2020; Table 2 ) available on the market for 2019-nCoV. Six of these are only for research purposes. Only one kit from Beijing Genome Institute (BGI) is approved for use in the clinical setting for rapid diagnosis. Most of the kits are for RT-PCR. There were two kits (BGI, China and Veredus, Singapore) with the capability to detect multiple pathogens using sequencing and microarray technologies, respectively. The limit of detection of the enhanced realtime PCR method was 10 2 -fold higher than the standard real-time PCR assay and 10 7fold higher than conventional PCR methods In the clinical aspect, the enhanced realtime PCR method was able to detect 6 cases of SARS-CoV positive samples that were not confirmed by any other assay [25] • The real time PCR has a threshold sensitivity of 10 genome equivalents per reaction and it has a good reproducibility with the inter-assay coefficients of variation of 1.73 to 2.72%. • 13 specimens from 6 patients were positive with viral load range from 362 to 36,240,000 genome equivalents/mL. The real-time RT-PCR reaction was more sensitive than the nested PCR reaction, as the detection limit for the nested PCR reaction was about 10 3 genome equivalents in the standard cDNA control. [34] Real-time reverse-transcription PCR (rRT-PCR); RNA-dependent RNA polymerase (RdRp); open reading frame 1a (ORF1a); Loop-mediated isothermal amplification (LAMP); enzyme-linked immunosorbent assay (ELISA); immunofluorescent assay (IFA); immunochromatographic test (ICT); nasopharyngeal aspirate (NPA). With the emergence of 2019-nCoV, there are about 15 potential vaccine candidates in the pipeline globally (Table 3 ), in which a wide range of technology (such as messenger RNA, DNA-based, nanoparticle, synthetic and modified virus-like particle) was applied. It will likely take about a year for most candidates to start phase 1 clinical trials except for those funded by Coalition for Epidemic Preparedness Innovations (CEPI). However, the kit developed by the BGI have passed emergency approval procedure of the National Medical Products Administration, and are currently used in clinical and surveillance centers of China [40] . Of the total of 570 unique studies on 2019-nCoV, SARS CoV or MERS-CoV vaccines screened, only four were eventually included in the review. Most studies on SARS and MERS vaccines were excluded as they were performed in cell or animal models ( Figure 1 ). The four studies included in this review were Phase I clinical trials on SARS or MERS vaccines (Table 4 ) [44] [45] [46] [47] . There were no studies of any population type (cell, animal, human) on the 2019-nCoV at the point of screening. The published clinical trials were mostly done in United States except for one on the SARS vaccine done in China [44] . All vaccine candidates for SARS and MERS were reported to be safe, well-tolerated and able to trigger the relevant and appropriate immune responses in the participants. In addition, we highlight six ongoing Phase I clinical trials identified in the ClinicalTrials.gov register ( [48, 49] ); Table S4 ) [50] [51] [52] . These trials are all testing the safety and immunogenicity of their respective MERS-CoV vaccine candidates but were excluded as there are no results published yet. The trials are projected to complete in December 2020 (two studies in Russia [50, 51] ) and December 2021 (in Germany [52] ). Existing literature search did not return any results on completed 2019-nCoV trials at the time of writing. Among 23 trials found from the systematic review (Table 5) , there are nine clinical trials registered under the clinical trials registry (ClinicalTrials.gov) for 2019-nCoV therapeutics [53] [54] [55] [56] [57] [58] [59] [60] [61] . Of which five studies on hydroxychloroquine, lopinavir plus ritonavir and arbidol, mesenchymal stem cells, traditional Chinese medicine and glucocorticoid therapy usage have commenced recruitment. The remaining four studies encompass investigation of antivirals, interferon atomization, darunavir and cobicistat, arbidol, and remdesivir usage for 2019-nCoV patients (Table 5) . Seroconversion measured by S1-ELISA occurred in 86% and 94% participants after 2 and 3 doses, respectively, and was maintained in 79% participants up to study end at week 60. Neutralising antibodies were detected in 50% participants at one or more time points during the study, but only 3% maintained neutralisation activity to end of study. T-cell responses were detected in 71% and 76% participants after 2 and 3 doses, respectively. There were no differences in immune responses between dose groups after 6 weeks and vaccine-induced humoral and cellular responses were respectively detected in 77% and 64% participants at week 60. [47] Molecules developed by the university scientists inhibit two coronavirus enzymes and prevent its replication. The discovered drug targets are said to be more than 95% similar to enzyme targets found on the SARS virus. Researchers note that identified drugs may not be available to address the ongoing outbreak but they hope to make it accessible for future outbreaks. [85] Besides the six completed randomized controlled trials (RCT) selected from the systematic review (Table 6) , there is only one ongoing randomized controlled trial targeted at SARS therapeutics [92] . The studies found from ClinicalTrials.gov have not been updated since 2013. While many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir or ribavirin only, there has yet to be well-designed clinical trials investigating their usage. Three completed randomized controlled trials were conducted during the SARS epidemic-3 in China, 1 in Taiwan and 2 in Hong Kong [93] [94] [95] [96] [97] . The studies respectively investigated antibiotic usage involving 190 participants, combination of western and Chinese treatment vs. Chinese treatment in 123 participants, integrative Chinese and Western treatment in 49 patients, usage of a specific Chinese medicine in four participants and early use of corticosteroid in 16 participants. Another notable study was an open non-randomized study investigating ribavirin/lopinavir/ritonavir usage in 152 participants [98] . One randomized controlled trial investigating integrative western and Chinese treatment during the SARS epidemic was excluded as it was a Chinese article [94] . There is only one ongoing randomized controlled trial targeted at MERS therapeutics [99] . It investigates the usage of Lopinavir/Ritonavir and Interferon Beta 1B. Likewise, many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir/ribavirin, interferon, and convalescent plasma usage. To date, only one trial has been completed. One phase 1 clinical trial investigating the safety and tolerability of a fully human polyclonal IgG immunoglobulin (SAB-301) was found in available literature [46] . The trial conducted in the United States in 2017 demonstrated SAB-301 to be safe and well-tolerated at single doses. Another trial on MERS therapeutics was found on ClinicalTrials.gov-a phase 2/3 trial in the United States evaluating the safety, tolerability, pharmacokinetics (PK), and immunogenicity on coadministered MERS-CoV antibodies REGN3048 & REGN3051 [100]. Rapid diagnostics plays an important role in disease and outbreak management. The fast and accurate diagnosis of a specific viral infection enables prompt and accurate public health surveillance, prevention and control measures. Local transmission and clusters can be prevented or delayed by isolation of laboratory-confirmed cases and their close contacts quarantined and monitored at home. Rapid diagnostic also facilitates other specific public health interventions such as closure of high-risk facilities and areas associated with the confirmed cases for prompt infection control and environmental decontamination [11, 101] . Laboratory diagnosis can be performed by: (a) detecting the genetic material of the virus, (b) detecting the antibodies that neutralize the viral particles of interest, (c) detecting the viral epitopes of interest with antibodies (serological testing), or (d) culture and isolation of viable virus particles. The key limitations of genetic material detection are the lack of knowledge of the presence of viable virus, the potential cross-reactivity with non-specific genetic regions and the short timeframe for accurate detection during the acute infection phase. The key limitations of serological testing is the need to collect paired serum samples (in the acute and convalescent phases) from cases under investigation for confirmation to eliminate potential cross-reactivity from non-specific antibodies from past exposure and/or infection by other coronaviruses. The limitation of virus culture and isolation is the long duration and the highly specialized skills required of the technicians to process the samples. All patients recovered. Significantly shorted time from the disease onset to the symptom improvement in treatment (5.10 ± 2.83 days) compared to control group (7.62 ± 2.27 days) (p < 0.05) No significant difference in blood routine improvement, pulmonary chest shadow in chest film improvement and corticosteroid usgae between the 2 groups. However, particularly in the respect of improving clinical symptoms, elevating quality of life, promoting immune function recovery, promoting absorption of pulmonary inflammation, reducing the dosage of cortisteroid and shortening the therapeutic course, treatment with integrative chinese and western medicine treatment had obvious superiority compared with using control treatment alone. Single infusions of SAB-301 up to 50 mg/kg appear to be safe and well-tolerated in healthy participants. [46] Where the biological samples are taken from also play a role in the sensitivity of these tests. For SARS-CoV and MERS-CoV, specimens collected from the lower respiratory tract such as sputum and tracheal aspirates have higher and more prolonged levels of viral RNA because of the tropism of the virus. MERS-CoV viral loads are also higher for severe cases and have longer viral shedding compared to mild cases. Although upper respiratory tract specimens such as nasopharyngeal or oropharyngeal swabs can be used, they have potentially lower viral loads and may have higher risk of false-negatives among the mild MERS and SARS cases [102, 103] , and likely among the 2019-nCoV cases. The existing practices in detecting genetic material of coronaviruses such as SARS-CoV and MERS-CoV include (a) reverse transcription-polymerase chain reaction (RT-PCR), (b) real-time RT-PCR (rRT-PCR), (c) reverse transcription loop-mediated isothermal amplification (RT-LAMP) and (d) real-time RT-LAMP [104] . Nucleic amplification tests (NAAT) are usually preferred as in the case of MERS-CoV diagnosis as it has the highest sensitivity at the earliest time point in the acute phase of infection [102] . Chinese health authorities have recently posted the full genome of 2019-nCoV in the GenBank and in GISAID portal to facilitate in the detection of the virus [11] . Several laboratory assays have been developed to detect the novel coronavirus in Wuhan, as highlighted in WHO's interim guidance on nCoV laboratory testing of suspected cases. These include protocols from other countries such as Thailand, Japan and China [105] . The first validated diagnostic test was designed in Germany. Corman et al. had initially designed a candidate diagnostic RT-PCR assay based on the SARS or SARS-related coronavirus as it was suggested that circulating virus was SARS-like. Upon the release of the sequence, assays were selected based on the match against 2019-nCoV upon inspection of the sequence alignment. Two assays were used for the RNA dependent RNA polymerase (RdRP) gene and E gene where E gene assay acts as the first-line screening tool and RdRp gene assay as the confirmatory testing. All assays were highly sensitive and specific in that they did not cross-react with other coronavirus and also human clinical samples that contained respiratory viruses [11] . The Hong Kong University used two monoplex assays which were reactive with coronaviruses under the subgenus Sarbecovirus (consisting of 2019-nCoV, SARS-CoV and SARS-like coronavirus). Viral RNA extracted from SARS-CoV can be used as the positive control for the suggested protocol assuming that SARS has been eradicated. It is proposed that the N gene RT-PCR can be used as a screening assay while the Orf1b assay acts as a confirmatory test. However, this protocol has only been evaluated with a panel of controls with the only positive control SARS-CoV RNA. Synthetic oligonucleotide positive control or 2019-nCoV have yet to be tested [106] . The US CDC shared the protocol on the real time RT-PCR assay for the detection of the 2019-nCoV with the primers and probes designed for the universal detection of SARS-like coronavirus and the specific detection of 2019-nCoV. However, the protocol has not been validated on other platforms or chemistries apart from the protocol described. There are some limitations for the assay. Analysts engaged have to be trained and familiar with the testing procedure and result interpretation. False negative results may occur due to insufficient organisms in the specimen resulting from improper collection, transportation or handling. Also, RNA viruses may show substantial genetic variability. This could result in mismatch between the primer and probes with the target sequence which can diminish the assay performance or result in false negative results [107] . Point-of-care test kit can potentially minimize these limitations, which should be highly prioritized for research and development in the next few months. Serological testing such as ELISA, IIFT and neutralization tests are effective in determining the extent of infection, including estimating asymptomatic and attack rate. Compared to the detection of viral genome through molecular methods, serological testing detects antibodies and antigens. There would be a lag period as antibodies specifically targeting the virus would normally appear between 14 and 28 days after the illness onset [108] . Furthermore, studies suggest that low antibody titers in the second week or delayed antibody production could be associated with mortality with a high viral load. Hence, serological diagnoses are likely used when nucleic amplification tests (NAAT) are not available or accessible [102] . Vaccines can prevent and protect against infection and disease occurrence when exposed to the specific pathogen of interest, especially in vulnerable populations who are more prone to severe outcomes. In the context of the current 2019-nCoV outbreak, vaccines will help control and reduce disease transmission by creating herd immunity in addition to protecting healthy individuals from infection. This decreases the effective R0 value of the disease. Nonetheless, there are social, clinical and economic hurdles for vaccine and vaccination programmes, including (a) the willingness of the public to undergo vaccination with a novel vaccine, (b) the side effects and severe adverse reactions of vaccination, (c) the potential difference and/or low efficacy of the vaccine in populations different from the clinical trials' populations and (d) the accessibility of the vaccines to a given population (including the cost and availability of the vaccine). Vaccines against the 2019-nCoV are currently in development and none are in testing (at the time of writing). On 23 January 2020, the Coalition for Epidemic Preparedness Innovations (CEPI) announced that they will fund vaccine development programmes with Inovio, The University of Queensland and Moderna, Inc respectively, with the aim to test the experimental vaccines clinically in 16 weeks (By June 2020). The vaccine candidates will be developed by the DNA, recombinant and mRNA vaccine platforms from these organizations [109] . Based on the most recent MERS-CoV outbreak, there are already a number of vaccine candidates being developed but most are still in the preclinical testing stage. The vaccines in development include viral vector-based vaccine, DNA vaccine, subunit vaccine, virus-like particles (VLPs)-based vaccine, inactivated whole-virus (IWV) vaccine and live attenuated vaccine. The latest findings for these vaccines arebased on the review by Yong et al. (2019) in August 2019 [110] . As of the date of reporting, there is only one published clinical study on the MERS-CoV vaccine by GeneOne Life Science & Inovio Pharmaceuticals [47] . There was one SARS vaccine trial conducted by the US National Institute of Allergy and Infectious Diseases. Both Phase I clinical trials reported positive results, but only one has announced plans to proceed to Phase 2 trial [111] . Due to the close genetic relatedness of SARS-CoV (79%) with 2019-nCoV [112] , there may be potential cross-protective effect of using a safe SARS-CoV vaccine while awaiting the 2019-nCoV vaccine. However, this would require small scale phase-by-phase implementation and close monitoring of vaccinees before any large scale implementation. Apart from the timely diagnosis of cases, the achievement of favorable clinical outcomes depends on the timely treatment administered. ACE2 has been reported to be the same cell entry receptor used by 2019-nCoV to infect humans as SARS-CoV [113] . Hence, clinical similarity between the two viruses is expected, particularly in severe cases. In addition, most of those who have died from MERS-CoV, SARS-CoV and 2019-nCoV were advance in age and had underlying health conditions such as hypertension, diabetes or cardiovascular disease that compromised their immune systems [114] . Coronaviruses have error-prone RNA-dependent RNA polymerases (RdRP), which result in frequent mutations and recombination events. This results in quasispecies diversity that is closely associated with adaptive evolution and the capacity to enhance viral-cell entry to cause disease over time in a specific population at-risk [115] . Since ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, coronaviruses are likely to infect the upper respiratory and gastrointestinal tract and this may influence the type of therapeutics against 2019-nCoV, similarly to SAR-CoV. However, in the years following two major coronavirus outbreaks SARS-CoV in 2003 and MERS-CoV in 2012, there remains no consensus on the optimal therapy for either disease [116, 117] . Well-designed clinical trials that provide the gold standard for assessing the therapeutic measures are scarce. No coronavirus protease inhibitors have successfully completed a preclinical development program despite large efforts exploring SARS-CoV inhibitors. The bulk of potential therapeutic strategies remain in the experimental phase, with only a handful crossing the in vitro hurdle. Stronger efforts are required in the research for treatment options for major coronaviruses given their pandemic potential. Effective treatment options are essential to maximize the restoration of affected populations to good health following infections. Clinical trials have commenced in China to identify effective treatments for 2019-nCoV based on the treatment evidence from SARS and MERS. There is currently no effective specific antiviral with high-level evidence; any specific antiviral therapy should be provided in the context of a clinical study/trial. Few treatments have shown real curative action against SARS and MERS and the literature generally describes isolated cases or small case series. Many interferons from the three classes have been tested for their antiviral activities against SARS-CoV both in vitro and in animal models. Interferon β has consistently been shown to be the most active, followed by interferon α. The use of corticosteroids with interferon alfacon-1 (synthetic interferon α) appeared to have improved oxygenation and faster resolution of chest radiograph abnormalities in observational studies with untreated controls. Interferon has been used in multiple observational studies to treat SARS-CoV and MERS-CoV patients [116, 117] . Interferons, with or without ribavirin, and lopinavir/ritonavir are most likely to be beneficial and are being trialed in China for 2019-nCoV. This drug treatment appears to be the most advanced. Timing of treatment is likely an important factor in effectiveness. A combination of ribavirin and lopinavir/ritonavir was used as a post-exposure prophylaxis in health care workers and may have reduced the risk of infection. Ribavirin alone is unlikely to have substantial antiviral activities at clinically used dosages. Hence, ribavirin with or without corticosteroids and with lopinavir and ritonavir are among the combinations employed. This was the most common agent reported in the available literature. Its efficacy has been assessed in observational studies, retrospective case series, retrospective cohort study, a prospective observational study, a prospective cohort study and randomized controlled trial ranging from seven to 229 participants [117] . Lopinavir/ritonavir (Kaletra) was the earliest protease inhibitor combination introduced for the treatment of SARS-CoV. Its efficacy was documented in several studies, causing notably lower incidence of adverse outcomes than with ribavirin alone. Combined usage with ribavirin was also associated with lower incidence of acute respiratory distress syndrome, nosocomial infection and death, amongst other favorable outcomes. Recent in vitro studies have shown another HIV protease inhibitor, nelfinavir, to have antiviral capacity against SARS-CoV, although it has yet to show favorable outcomes in animal studies [118] . Remdesivir (Gilead Sciences, GS-5734) nucleoside analogue in vitro and in vivo data support GS-5734 development as a potential pan-coronavirus antiviral based on results against several coronaviruses (CoVs), including highly pathogenic CoVs and potentially emergent BatCoVs. The use of remdesivir may be a good candidate as an investigational treatment. Improved mortality following receipt of convalescent plasma in various doses was consistently reported in several observational studies involving cases with severe acute respiratory infections (SARIs) of viral etiology. A significant reduction in the pooled odds of mortality following treatment of 0.25 compared to placebo or no therapy was observed [119] . Studies were however at moderate to high risk of bias given their small sample sizes, allocation of treatment based on the physician's discretion, and the availability of plasma. Factors like concomitant treatment may have also confounded the results. Associations between convalescent plasma and hospital length of stay, viral antibody levels, and viral load respectively were similarly inconsistent across available literature. Convalescent plasma, while promising, is likely not yet feasible, given the limited pool of potential donors and issues of scalability. Monoclonal antibody treatment is progressing. SARS-CoV enters host cells through the binding of their spike (S) protein to angiotensin converting enzyme 2 (ACE2) and CD209L [118] . Human monoclonal antibodies to the S protein have been shown to significantly reduce the severity of lung pathology in non-human primates following MERS-CoV infection [120] . Such neutralizing antibodies can be elicited by active or passive immunization using vaccines or convalescent plasma respectively. While such neutralizing antibodies can theoretically be harvested from individuals immunized with vaccines, there is uncertainty over the achievement of therapeutic levels of antibodies. Other therapeutic agents have also been reported. A known antimalarial agent, chloroquine, elicits antiviral effects against multiple viruses including HIV type 1, hepatitis B and HCoV-229E. Chloroquine is also immunomodulatory, capable of suppressing the production and release of factors which mediate the inflammatory complications of viral diseases (tumor necrosis factor and interleukin 6) [121] . It is postulated that chloroquine works by altering ACE2 glycosylation and endosomal pH. Its anti-inflammatory properties may be beneficial for the treatment of SARS. Niclosamide as a known drug used in antihelminthic treatment. The efficacy of niclosamide as an inhibitor of virus replication was proven in several assays. In both immunoblot analysis and immunofluorescence assays, niclosamide treatment was observed to completely inhibit viral antigen synthesis. Reduction of virus yield in infected cells was dose dependent. Niclosamide likely does not interfere in the early stages of virus attachment and entry into cells, nor does it function as a protease inhibitor. Mechanisms of niclosamide activity warrant further investigation [122] . Glycyrrhizin also reportedly inhibits virus adsorption and penetration in the early steps of virus replication. Glycyrrhizin was a significantly potent inhibitor with a low selectivity index when tested against several pathogenic flaviviruses. While preliminary results suggest production of nitrous oxide (which inhibits virus replication) through induction of nitrous oxide synthase, the mechanism of Glycyrrhizin against SARS-CoV remains unclear. The compound also has relatively lower toxicity compared to protease inhibitors like ribavirin [123] . Inhibitory activity was also detected in baicalin [124] , extracted from another herb used in the treatment of SARS in China and Hong Kong. Findings on these compounds are limited to in vitro studies [121] [122] [123] [124] . Due to the rapidly evolving situation of the 2019-nCoV, there will be potential limitations to the systematic review. The systematic review is likely to have publication bias as some developments have yet to be reported while for other developments there is no intention to report publicly (or in scientific platforms) due to confidentiality concerns. However, this may be limited to only a few developments for review as publicity does help in branding to some extent for the company and/or the funder. Furthermore, due to the rapid need to share the status of these developments, there may be reporting bias in some details provided by authors of the scientific articles or commentary articles in traditional media. Lastly, while it is not viable for any form of quality assessment and metaanalysis of the selected articles due to the limited data provided and the heterogeneous style of reporting by different articles, this paper has provided a comprehensive overview of the potential developments of these pharmaceutical interventions during the early phase of the outbreak. This systematic review would be useful for cross-check when the quality assessment and meta-analysis of these developments are performed as a follow-up study. Rapid diagnostics, vaccines and therapeutics are key pharmaceutical interventions to limit transmission of respiratory infectious diseases. Many potential developments on these pharmaceutical interventions for 2019-nCoV are ongoing in the containment phase of this outbreak, potentially due to better pandemic preparedness than before. However, lessons from MERS-CoV and SARS-CoV have shown that the journeys for these developments can still be challenging moving ahead. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1 : Example of full search strategy in Pubmed, Table S2 : Google Search: 2019-nCoV diagnostics, Table S3 : Summary of diagnostic assays developed for 2019-nCoV, Table S4
What did the searches yield?
false
3,631
{ "text": [ "A Google search for 2019-nCoV diagnostics (as of 6 February 2020; Table S2 ) yielded five webpage links from government and international bodies with official information and guidelines (WHO, Europe CDC, US CDC, US FDA), three webpage links on diagnostic protocols and scientific commentaries, and five webpage links on market news and press releases. Six protocols for diagnostics using reverse transcriptase polymerase chain reaction (RT-PCR) from six countries were published on WHO's website [9] . Google search for 2019-nCoV vaccines yielded 19 relevant articles." ], "answer_start": [ 6375 ] }
1,719
Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.
What was the benefit of the robust innate immune response to baculovirus vector?
false
1,581
{ "text": [ "non-specific protection from subsequent influenza virus infection" ], "answer_start": [ 20884 ] }
2,683
Estimating the number of infections and the impact of non- pharmaceutical interventions on COVID-19 in 11 European countries 30 March 2020 Imperial College COVID-19 Response Team Seth Flaxmani Swapnil Mishra*, Axel Gandy*, H JulietteT Unwin, Helen Coupland, Thomas A Mellan, Harrison Zhu, Tresnia Berah, Jeffrey W Eaton, Pablo N P Guzman, Nora Schmit, Lucia Cilloni, Kylie E C Ainslie, Marc Baguelin, Isobel Blake, Adhiratha Boonyasiri, Olivia Boyd, Lorenzo Cattarino, Constanze Ciavarella, Laura Cooper, Zulma Cucunuba’, Gina Cuomo—Dannenburg, Amy Dighe, Bimandra Djaafara, Ilaria Dorigatti, Sabine van Elsland, Rich FitzJohn, Han Fu, Katy Gaythorpe, Lily Geidelberg, Nicholas Grassly, Wi|| Green, Timothy Hallett, Arran Hamlet, Wes Hinsley, Ben Jeffrey, David Jorgensen, Edward Knock, Daniel Laydon, Gemma Nedjati—Gilani, Pierre Nouvellet, Kris Parag, Igor Siveroni, Hayley Thompson, Robert Verity, Erik Volz, Caroline Walters, Haowei Wang, Yuanrong Wang, Oliver Watson, Peter Winskill, Xiaoyue Xi, Charles Whittaker, Patrick GT Walker, Azra Ghani, Christl A. Donnelly, Steven Riley, Lucy C Okell, Michaela A C Vollmer, NeilM.Ferguson1and Samir Bhatt*1 Department of Infectious Disease Epidemiology, Imperial College London Department of Mathematics, Imperial College London WHO Collaborating Centre for Infectious Disease Modelling MRC Centre for Global Infectious Disease Analysis Abdul LatifJameeI Institute for Disease and Emergency Analytics, Imperial College London Department of Statistics, University of Oxford *Contributed equally 1Correspondence: nei|[email protected], [email protected] Summary Following the emergence of a novel coronavirus (SARS-CoV-Z) and its spread outside of China, Europe is now experiencing large epidemics. In response, many European countries have implemented unprecedented non-pharmaceutical interventions including case isolation, the closure of schools and universities, banning of mass gatherings and/or public events, and most recently, widescale social distancing including local and national Iockdowns. In this report, we use a semi-mechanistic Bayesian hierarchical model to attempt to infer the impact of these interventions across 11 European countries. Our methods assume that changes in the reproductive number— a measure of transmission - are an immediate response to these interventions being implemented rather than broader gradual changes in behaviour. Our model estimates these changes by calculating backwards from the deaths observed over time to estimate transmission that occurred several weeks prior, allowing for the time lag between infection and death. One of the key assumptions of the model is that each intervention has the same effect on the reproduction number across countries and over time. This allows us to leverage a greater amount of data across Europe to estimate these effects. It also means that our results are driven strongly by the data from countries with more advanced epidemics, and earlier interventions, such as Italy and Spain. We find that the slowing growth in daily reported deaths in Italy is consistent with a significant impact of interventions implemented several weeks earlier. In Italy, we estimate that the effective reproduction number, Rt, dropped to close to 1 around the time of Iockdown (11th March), although with a high level of uncertainty. Overall, we estimate that countries have managed to reduce their reproduction number. Our estimates have wide credible intervals and contain 1 for countries that have implemented a|| interventions considered in our analysis. This means that the reproduction number may be above or below this value. With current interventions remaining in place to at least the end of March, we estimate that interventions across all 11 countries will have averted 59,000 deaths up to 31 March [95% credible interval 21,000-120,000]. Many more deaths will be averted through ensuring that interventions remain in place until transmission drops to low levels. We estimate that, across all 11 countries between 7 and 43 million individuals have been infected with SARS-CoV-Z up to 28th March, representing between 1.88% and 11.43% ofthe population. The proportion of the population infected to date — the attack rate - is estimated to be highest in Spain followed by Italy and lowest in Germany and Norway, reflecting the relative stages of the epidemics. Given the lag of 2-3 weeks between when transmission changes occur and when their impact can be observed in trends in mortality, for most of the countries considered here it remains too early to be certain that recent interventions have been effective. If interventions in countries at earlier stages of their epidemic, such as Germany or the UK, are more or less effective than they were in the countries with advanced epidemics, on which our estimates are largely based, or if interventions have improved or worsened over time, then our estimates of the reproduction number and deaths averted would change accordingly. It is therefore critical that the current interventions remain in place and trends in cases and deaths are closely monitored in the coming days and weeks to provide reassurance that transmission of SARS-Cov-Z is slowing. SUGGESTED CITATION Seth Flaxman, Swapnil Mishra, Axel Gandy et 0/. Estimating the number of infections and the impact of non— pharmaceutical interventions on COVID—19 in 11 European countries. Imperial College London (2020), doi: https://doi.org/10.25561/77731 1 Introduction Following the emergence of a novel coronavirus (SARS-CoV-Z) in Wuhan, China in December 2019 and its global spread, large epidemics of the disease, caused by the virus designated COVID-19, have emerged in Europe. In response to the rising numbers of cases and deaths, and to maintain the capacity of health systems to treat as many severe cases as possible, European countries, like those in other continents, have implemented or are in the process of implementing measures to control their epidemics. These large-scale non-pharmaceutical interventions vary between countries but include social distancing (such as banning large gatherings and advising individuals not to socialize outside their households), border closures, school closures, measures to isolate symptomatic individuals and their contacts, and large-scale lockdowns of populations with all but essential internal travel banned. Understanding firstly, whether these interventions are having the desired impact of controlling the epidemic and secondly, which interventions are necessary to maintain control, is critical given their large economic and social costs. The key aim ofthese interventions is to reduce the effective reproduction number, Rt, ofthe infection, a fundamental epidemiological quantity representing the average number of infections, at time t, per infected case over the course of their infection. Ith is maintained at less than 1, the incidence of new infections decreases, ultimately resulting in control of the epidemic. If Rt is greater than 1, then infections will increase (dependent on how much greater than 1 the reproduction number is) until the epidemic peaks and eventually declines due to acquisition of herd immunity. In China, strict movement restrictions and other measures including case isolation and quarantine began to be introduced from 23rd January, which achieved a downward trend in the number of confirmed new cases during February, resulting in zero new confirmed indigenous cases in Wuhan by March 19th. Studies have estimated how Rt changed during this time in different areas ofChina from around 2-4 during the uncontrolled epidemic down to below 1, with an estimated 7-9 fold decrease in the number of daily contacts per person.1'2 Control measures such as social distancing, intensive testing, and contact tracing in other countries such as Singapore and South Korea have successfully reduced case incidence in recent weeks, although there is a riskthe virus will spread again once control measures are relaxed.3'4 The epidemic began slightly laterin Europe, from January or later in different regions.5 Countries have implemented different combinations of control measures and the level of adherence to government recommendations on social distancing is likely to vary between countries, in part due to different levels of enforcement. Estimating reproduction numbers for SARS-CoV-Z presents challenges due to the high proportion of infections not detected by health systems”7 and regular changes in testing policies, resulting in different proportions of infections being detected over time and between countries. Most countries so far only have the capacity to test a small proportion of suspected cases and tests are reserved for severely ill patients or for high-risk groups (e.g. contacts of cases). Looking at case data, therefore, gives a systematically biased view of trends. An alternative way to estimate the course of the epidemic is to back-calculate infections from observed deaths. Reported deaths are likely to be more reliable, although the early focus of most surveillance systems on cases with reported travel histories to China may mean that some early deaths will have been missed. Whilst the recent trends in deaths will therefore be informative, there is a time lag in observing the effect of interventions on deaths since there is a 2-3-week period between infection, onset of symptoms and outcome. In this report, we fit a novel Bayesian mechanistic model of the infection cycle to observed deaths in 11 European countries, inferring plausible upper and lower bounds (Bayesian credible intervals) of the total populations infected (attack rates), case detection probabilities, and the reproduction number over time (Rt). We fit the model jointly to COVID-19 data from all these countries to assess whether there is evidence that interventions have so far been successful at reducing Rt below 1, with the strong assumption that particular interventions are achieving a similar impact in different countries and that the efficacy of those interventions remains constant over time. The model is informed more strongly by countries with larger numbers of deaths and which implemented interventions earlier, therefore estimates of recent Rt in countries with more recent interventions are contingent on similar intervention impacts. Data in the coming weeks will enable estimation of country-specific Rt with greater precision. Model and data details are presented in the appendix, validation and sensitivity are also presented in the appendix, and general limitations presented below in the conclusions. 2 Results The timing of interventions should be taken in the context of when an individual country’s epidemic started to grow along with the speed with which control measures were implemented. Italy was the first to begin intervention measures, and other countries followed soon afterwards (Figure 1). Most interventions began around 12th-14th March. We analyzed data on deaths up to 28th March, giving a 2-3-week window over which to estimate the effect of interventions. Currently, most countries in our study have implemented all major non-pharmaceutical interventions. For each country, we model the number of infections, the number of deaths, and Rt, the effective reproduction number over time, with Rt changing only when an intervention is introduced (Figure 2- 12). Rt is the average number of secondary infections per infected individual, assuming that the interventions that are in place at time t stay in place throughout their entire infectious period. Every country has its own individual starting reproduction number Rt before interventions take place. Specific interventions are assumed to have the same relative impact on Rt in each country when they were introduced there and are informed by mortality data across all countries. Figure l: Intervention timings for the 11 European countries included in the analysis. For further details see Appendix 8.6. 2.1 Estimated true numbers of infections and current attack rates In all countries, we estimate there are orders of magnitude fewer infections detected (Figure 2) than true infections, mostly likely due to mild and asymptomatic infections as well as limited testing capacity. In Italy, our results suggest that, cumulatively, 5.9 [1.9-15.2] million people have been infected as of March 28th, giving an attack rate of 9.8% [3.2%-25%] of the population (Table 1). Spain has recently seen a large increase in the number of deaths, and given its smaller population, our model estimates that a higher proportion of the population, 15.0% (7.0 [18-19] million people) have been infected to date. Germany is estimated to have one of the lowest attack rates at 0.7% with 600,000 [240,000-1,500,000] people infected. Imperial College COVID-19 Response Team Table l: Posterior model estimates of percentage of total population infected as of 28th March 2020. Country % of total population infected (mean [95% credible intervall) Austria 1.1% [0.36%-3.1%] Belgium 3.7% [1.3%-9.7%] Denmark 1.1% [0.40%-3.1%] France 3.0% [1.1%-7.4%] Germany 0.72% [0.28%-1.8%] Italy 9.8% [3.2%-26%] Norway 0.41% [0.09%-1.2%] Spain 15% [3.7%-41%] Sweden 3.1% [0.85%-8.4%] Switzerland 3.2% [1.3%-7.6%] United Kingdom 2.7% [1.2%-5.4%] 2.2 Reproduction numbers and impact of interventions Averaged across all countries, we estimate initial reproduction numbers of around 3.87 [3.01-4.66], which is in line with other estimates.1'8 These estimates are informed by our choice of serial interval distribution and the initial growth rate of observed deaths. A shorter assumed serial interval results in lower starting reproduction numbers (Appendix 8.4.2, Appendix 8.4.6). The initial reproduction numbers are also uncertain due to (a) importation being the dominant source of new infections early in the epidemic, rather than local transmission (b) possible under-ascertainment in deaths particularly before testing became widespread. We estimate large changes in Rt in response to the combined non-pharmaceutical interventions. Our results, which are driven largely by countries with advanced epidemics and larger numbers of deaths (e.g. Italy, Spain), suggest that these interventions have together had a substantial impact on transmission, as measured by changes in the estimated reproduction number Rt. Across all countries we find current estimates of Rt to range from a posterior mean of 0.97 [0.14-2.14] for Norway to a posterior mean of2.64 [1.40-4.18] for Sweden, with an average of 1.43 across the 11 country posterior means, a 64% reduction compared to the pre-intervention values. We note that these estimates are contingent on intervention impact being the same in different countries and at different times. In all countries but Sweden, under the same assumptions, we estimate that the current reproduction number includes 1 in the uncertainty range. The estimated reproduction number for Sweden is higher, not because the mortality trends are significantly different from any other country, but as an artefact of our model, which assumes a smaller reduction in Rt because no full lockdown has been ordered so far. Overall, we cannot yet conclude whether current interventions are sufficient to drive Rt below 1 (posterior probability of being less than 1.0 is 44% on average across the countries). We are also unable to conclude whether interventions may be different between countries or over time. There remains a high level of uncertainty in these estimates. It is too early to detect substantial intervention impact in many countries at earlier stages of their epidemic (e.g. Germany, UK, Norway). Many interventions have occurred only recently, and their effects have not yet been fully observed due to the time lag between infection and death. This uncertainty will reduce as more data become available. For all countries, our model fits observed deaths data well (Bayesian goodness of fit tests). We also found that our model can reliably forecast daily deaths 3 days into the future, by withholding the latest 3 days of data and comparing model predictions to observed deaths (Appendix 8.3). The close spacing of interventions in time made it statistically impossible to determine which had the greatest effect (Figure 1, Figure 4). However, when doing a sensitivity analysis (Appendix 8.4.3) with uninformative prior distributions (where interventions can increase deaths) we find similar impact of Imperial College COVID-19 Response Team interventions, which shows that our choice of prior distribution is not driving the effects we see in the main analysis. Figure 2: Country-level estimates of infections, deaths and Rt. Left: daily number of infections, brown bars are reported infections, blue bands are predicted infections, dark blue 50% credible interval (CI), light blue 95% CI. The number of daily infections estimated by our model drops immediately after an intervention, as we assume that all infected people become immediately less infectious through the intervention. Afterwards, if the Rt is above 1, the number of infections will starts growing again. Middle: daily number of deaths, brown bars are reported deaths, blue bands are predicted deaths, CI as in left plot. Right: time-varying reproduction number Rt, dark green 50% CI, light green 95% CI. Icons are interventions shown at the time they occurred. Imperial College COVID-19 Response Team Table 2: Totalforecasted deaths since the beginning of the epidemic up to 31 March in our model and in a counterfactual model (assuming no intervention had taken place). Estimated averted deaths over this time period as a result of the interventions. Numbers in brackets are 95% credible intervals. 2.3 Estimated impact of interventions on deaths Table 2 shows total forecasted deaths since the beginning of the epidemic up to and including 31 March under ourfitted model and under the counterfactual model, which predicts what would have happened if no interventions were implemented (and R, = R0 i.e. the initial reproduction number estimated before interventions). Again, the assumption in these predictions is that intervention impact is the same across countries and time. The model without interventions was unable to capture recent trends in deaths in several countries, where the rate of increase had clearly slowed (Figure 3). Trends were confirmed statistically by Bayesian leave-one-out cross-validation and the widely applicable information criterion assessments —WA|C). By comparing the deaths predicted under the model with no interventions to the deaths predicted in our intervention model, we calculated the total deaths averted up to the end of March. We find that, across 11 countries, since the beginning of the epidemic, 59,000 [21,000-120,000] deaths have been averted due to interventions. In Italy and Spain, where the epidemic is advanced, 38,000 [13,000- 84,000] and 16,000 [5,400-35,000] deaths have been averted, respectively. Even in the UK, which is much earlier in its epidemic, we predict 370 [73-1,000] deaths have been averted. These numbers give only the deaths averted that would have occurred up to 31 March. lfwe were to include the deaths of currently infected individuals in both models, which might happen after 31 March, then the deaths averted would be substantially higher. Figure 3: Daily number of confirmed deaths, predictions (up to 28 March) and forecasts (after) for (a) Italy and (b) Spain from our model with interventions (blue) and from the no interventions counterfactual model (pink); credible intervals are shown one week into the future. Other countries are shown in Appendix 8.6. 03/0 25% 50% 753% 100% (no effect on transmissibility) (ends transmissibility Relative % reduction in R. Figure 4: Our model includes five covariates for governmental interventions, adjusting for whether the intervention was the first one undertaken by the government in response to COVID-19 (red) or was subsequent to other interventions (green). Mean relative percentage reduction in Rt is shown with 95% posterior credible intervals. If 100% reduction is achieved, Rt = 0 and there is no more transmission of COVID-19. No effects are significantly different from any others, probably due to the fact that many interventions occurred on the same day or within days of each other as shown in Figure l. 3 Discussion During this early phase of control measures against the novel coronavirus in Europe, we analyze trends in numbers of deaths to assess the extent to which transmission is being reduced. Representing the COVlD-19 infection process using a semi-mechanistic, joint, Bayesian hierarchical model, we can reproduce trends observed in the data on deaths and can forecast accurately over short time horizons. We estimate that there have been many more infections than are currently reported. The high level of under-ascertainment of infections that we estimate here is likely due to the focus on testing in hospital settings rather than in the community. Despite this, only a small minority of individuals in each country have been infected, with an attack rate on average of 4.9% [l.9%-ll%] with considerable variation between countries (Table 1). Our estimates imply that the populations in Europe are not close to herd immunity ("50-75% if R0 is 2-4). Further, with Rt values dropping substantially, the rate of acquisition of herd immunity will slow down rapidly. This implies that the virus will be able to spread rapidly should interventions be lifted. Such estimates of the attack rate to date urgently need to be validated by newly developed antibody tests in representative population surveys, once these become available. We estimate that major non-pharmaceutical interventions have had a substantial impact on the time- varying reproduction numbers in countries where there has been time to observe intervention effects on trends in deaths (Italy, Spain). lfadherence in those countries has changed since that initial period, then our forecast of future deaths will be affected accordingly: increasing adherence over time will have resulted in fewer deaths and decreasing adherence in more deaths. Similarly, our estimates of the impact ofinterventions in other countries should be viewed with caution if the same interventions have achieved different levels of adherence than was initially the case in Italy and Spain. Due to the implementation of interventions in rapid succession in many countries, there are not enough data to estimate the individual effect size of each intervention, and we discourage attributing associations to individual intervention. In some cases, such as Norway, where all interventions were implemented at once, these individual effects are by definition unidentifiable. Despite this, while individual impacts cannot be determined, their estimated joint impact is strongly empirically justified (see Appendix 8.4 for sensitivity analysis). While the growth in daily deaths has decreased, due to the lag between infections and deaths, continued rises in daily deaths are to be expected for some time. To understand the impact of interventions, we fit a counterfactual model without the interventions and compare this to the actual model. Consider Italy and the UK - two countries at very different stages in their epidemics. For the UK, where interventions are very recent, much of the intervention strength is borrowed from countries with older epidemics. The results suggest that interventions will have a large impact on infections and deaths despite counts of both rising. For Italy, where far more time has passed since the interventions have been implemented, it is clear that the model without interventions does not fit well to the data, and cannot explain the sub-linear (on the logarithmic scale) reduction in deaths (see Figure 10). The counterfactual model for Italy suggests that despite mounting pressure on health systems, interventions have averted a health care catastrophe where the number of new deaths would have been 3.7 times higher (38,000 deaths averted) than currently observed. Even in the UK, much earlier in its epidemic, the recent interventions are forecasted to avert 370 total deaths up to 31 of March. 4 Conclusion and Limitations Modern understanding of infectious disease with a global publicized response has meant that nationwide interventions could be implemented with widespread adherence and support. Given observed infection fatality ratios and the epidemiology of COVlD-19, major non-pharmaceutical interventions have had a substantial impact in reducing transmission in countries with more advanced epidemics. It is too early to be sure whether similar reductions will be seen in countries at earlier stages of their epidemic. While we cannot determine which set of interventions have been most successful, taken together, we can already see changes in the trends of new deaths. When forecasting 3 days and looking over the whole epidemic the number of deaths averted is substantial. We note that substantial innovation is taking place, and new more effective interventions or refinements of current interventions, alongside behavioral changes will further contribute to reductions in infections. We cannot say for certain that the current measures have controlled the epidemic in Europe; however, if current trends continue, there is reason for optimism. Our approach is semi-mechanistic. We propose a plausible structure for the infection process and then estimate parameters empirically. However, many parameters had to be given strong prior distributions or had to be fixed. For these assumptions, we have provided relevant citations to previous studies. As more data become available and better estimates arise, we will update these in weekly reports. Our choice of serial interval distribution strongly influences the prior distribution for starting R0. Our infection fatality ratio, and infection-to-onset-to-death distributions strongly influence the rate of death and hence the estimated number of true underlying cases. We also assume that the effect of interventions is the same in all countries, which may not be fully realistic. This assumption implies that countries with early interventions and more deaths since these interventions (e.g. Italy, Spain) strongly influence estimates of intervention impact in countries at earlier stages of their epidemic with fewer deaths (e.g. Germany, UK). We have tried to create consistent definitions of all interventions and document details of this in Appendix 8.6. However, invariably there will be differences from country to country in the strength of their intervention — for example, most countries have banned gatherings of more than 2 people when implementing a lockdown, whereas in Sweden the government only banned gatherings of more than 10 people. These differences can skew impacts in countries with very little data. We believe that our uncertainty to some degree can cover these differences, and as more data become available, coefficients should become more reliable. However, despite these strong assumptions, there is sufficient signal in the data to estimate changes in R, (see the sensitivity analysis reported in Appendix 8.4.3) and this signal will stand to increase with time. In our Bayesian hierarchical framework, we robustly quantify the uncertainty in our parameter estimates and posterior predictions. This can be seen in the very wide credible intervals in more recent days, where little or no death data are available to inform the estimates. Furthermore, we predict intervention impact at country-level, but different trends may be in place in different parts of each country. For example, the epidemic in northern Italy was subject to controls earlier than the rest of the country. 5 Data Our model utilizes daily real-time death data from the ECDC (European Centre of Disease Control), where we catalogue case data for 11 European countries currently experiencing the epidemic: Austria, Belgium, Denmark, France, Germany, Italy, Norway, Spain, Sweden, Switzerland and the United Kingdom. The ECDC provides information on confirmed cases and deaths attributable to COVID-19. However, the case data are highly unrepresentative of the incidence of infections due to underreporting as well as systematic and country-specific changes in testing. We, therefore, use only deaths attributable to COVID-19 in our model; we do not use the ECDC case estimates at all. While the observed deaths still have some degree of unreliability, again due to changes in reporting and testing, we believe the data are ofsufficient fidelity to model. For population counts, we use UNPOP age-stratified counts.10 We also catalogue data on the nature and type of major non-pharmaceutical interventions. We looked at the government webpages from each country as well as their official public health division/information webpages to identify the latest advice/laws being issued by the government and public health authorities. We collected the following: School closure ordered: This intervention refers to nationwide extraordinary school closures which in most cases refer to both primary and secondary schools closing (for most countries this also includes the closure of otherforms of higher education or the advice to teach remotely). In the case of Denmark and Sweden, we allowed partial school closures of only secondary schools. The date of the school closure is taken to be the effective date when the schools started to be closed (ifthis was on a Monday, the date used was the one of the previous Saturdays as pupils and students effectively stayed at home from that date onwards). Case-based measures: This intervention comprises strong recommendations or laws to the general public and primary care about self—isolation when showing COVID-19-like symptoms. These also include nationwide testing programs where individuals can be tested and subsequently self—isolated. Our definition is restricted to nationwide government advice to all individuals (e.g. UK) or to all primary care and excludes regional only advice. These do not include containment phase interventions such as isolation if travelling back from an epidemic country such as China. Public events banned: This refers to banning all public events of more than 100 participants such as sports events. Social distancing encouraged: As one of the first interventions against the spread of the COVID-19 pandemic, many governments have published advice on social distancing including the recommendation to work from home wherever possible, reducing use ofpublictransport and all other non-essential contact. The dates used are those when social distancing has officially been recommended by the government; the advice may include maintaining a recommended physical distance from others. Lockdown decreed: There are several different scenarios that the media refers to as lockdown. As an overall definition, we consider regulations/legislations regarding strict face-to-face social interaction: including the banning of any non-essential public gatherings, closure of educational and public/cultural institutions, ordering people to stay home apart from exercise and essential tasks. We include special cases where these are not explicitly mentioned on government websites but are enforced by the police (e.g. France). The dates used are the effective dates when these legislations have been implemented. We note that lockdown encompasses other interventions previously implemented. First intervention: As Figure 1 shows, European governments have escalated interventions rapidly, and in some examples (Norway/Denmark) have implemented these interventions all on a single day. Therefore, given the temporal autocorrelation inherent in government intervention, we include a binary covariate for the first intervention, which can be interpreted as a government decision to take major action to control COVID-19. A full list of the timing of these interventions and the sources we have used can be found in Appendix 8.6. 6 Methods Summary A Visual summary of our model is presented in Figure 5 (details in Appendix 8.1 and 8.2). Replication code is available at https://github.com/|mperia|CollegeLondon/covid19model/releases/tag/vl.0 We fit our model to observed deaths according to ECDC data from 11 European countries. The modelled deaths are informed by an infection-to-onset distribution (time from infection to the onset of symptoms), an onset-to-death distribution (time from the onset of symptoms to death), and the population-averaged infection fatality ratio (adjusted for the age structure and contact patterns of each country, see Appendix). Given these distributions and ratios, modelled deaths are a function of the number of infections. The modelled number of infections is informed by the serial interval distribution (the average time from infection of one person to the time at which they infect another) and the time-varying reproduction number. Finally, the time-varying reproduction number is a function of the initial reproduction number before interventions and the effect sizes from interventions. Figure 5: Summary of model components. Following the hierarchy from bottom to top gives us a full framework to see how interventions affect infections, which can result in deaths. We use Bayesian inference to ensure our modelled deaths can reproduce the observed deaths as closely as possible. From bottom to top in Figure 5, there is an implicit lag in time that means the effect of very recent interventions manifest weakly in current deaths (and get stronger as time progresses). To maximise the ability to observe intervention impact on deaths, we fit our model jointly for all 11 European countries, which results in a large data set. Our model jointly estimates the effect sizes of interventions. We have evaluated the effect ofour Bayesian prior distribution choices and evaluate our Bayesian posterior calibration to ensure our results are statistically robust (Appendix 8.4). 7 Acknowledgements Initial research on covariates in Appendix 8.6 was crowdsourced; we thank a number of people across the world for help with this. This work was supported by Centre funding from the UK Medical Research Council under a concordat with the UK Department for International Development, the NIHR Health Protection Research Unit in Modelling Methodology and CommunityJameel. 8 Appendix: Model Specifics, Validation and Sensitivity Analysis 8.1 Death model We observe daily deaths Dam for days t E 1, ...,n and countries m E 1, ...,p. These daily deaths are modelled using a positive real-Valued function dam = E(Dam) that represents the expected number of deaths attributed to COVID-19. Dam is assumed to follow a negative binomial distribution with The expected number of deaths (1 in a given country on a given day is a function of the number of infections C occurring in previous days. At the beginning of the epidemic, the observed deaths in a country can be dominated by deaths that result from infection that are not locally acquired. To avoid biasing our model by this, we only include observed deaths from the day after a country has cumulatively observed 10 deaths in our model. To mechanistically link ourfunction for deaths to infected cases, we use a previously estimated COVID- 19 infection-fatality-ratio ifr (probability of death given infection)9 together with a distribution oftimes from infection to death TE. The ifr is derived from estimates presented in Verity et al11 which assumed homogeneous attack rates across age-groups. To better match estimates of attack rates by age generated using more detailed information on country and age-specific mixing patterns, we scale these estimates (the unadjusted ifr, referred to here as ifr’) in the following way as in previous work.4 Let Ca be the number of infections generated in age-group a, Na the underlying size of the population in that age group and AR“ 2 Ca/Na the age-group-specific attack rate. The adjusted ifr is then given by: ifra = fififié, where AR50_59 is the predicted attack-rate in the 50-59 year age-group after incorporating country-specific patterns of contact and mixing. This age-group was chosen as the reference as it had the lowest predicted level of underreporting in previous analyses of data from the Chinese epidemic“. We obtained country-specific estimates of attack rate by age, AR“, for the 11 European countries in our analysis from a previous study which incorporates information on contact between individuals of different ages in countries across Europe.12 We then obtained overall ifr estimates for each country adjusting for both demography and age-specific attack rates. Using estimated epidemiological information from previous studies,“'11 we assume TE to be the sum of two independent random times: the incubation period (infection to onset of symptoms or infection- to-onset) distribution and the time between onset of symptoms and death (onset-to-death). The infection-to-onset distribution is Gamma distributed with mean 5.1 days and coefficient of variation 0.86. The onset-to-death distribution is also Gamma distributed with a mean of 18.8 days and a coefficient of va riation 0.45. ifrm is population averaged over the age structure of a given country. The infection-to-death distribution is therefore given by: um ~ ifrm ~ (Gamma(5.1,0.86) + Gamma(18.8,0.45)) Figure 6 shows the infection-to-death distribution and the resulting survival function that integrates to the infection fatality ratio. Figure 6: Left, infection-to-death distribution (mean 23.9 days). Right, survival probability of infected individuals per day given the infection fatality ratio (1%) and the infection-to-death distribution on the left. Using the probability of death distribution, the expected number of deaths dam, on a given day t, for country, m, is given by the following discrete sum: The number of deaths today is the sum of the past infections weighted by their probability of death, where the probability of death depends on the number of days since infection. 8.2 Infection model The true number of infected individuals, C, is modelled using a discrete renewal process. This approach has been used in numerous previous studies13'16 and has a strong theoretical basis in stochastic individual-based counting processes such as Hawkes process and the Bellman-Harris process.”18 The renewal model is related to the Susceptible-Infected-Recovered model, except the renewal is not expressed in differential form. To model the number ofinfections over time we need to specify a serial interval distribution g with density g(T), (the time between when a person gets infected and when they subsequently infect another other people), which we choose to be Gamma distributed: g ~ Gamma (6.50.62). The serial interval distribution is shown below in Figure 7 and is assumed to be the same for all countries. Figure 7: Serial interval distribution g with a mean of 6.5 days. Given the serial interval distribution, the number of infections Eamon a given day t, and country, m, is given by the following discrete convolution function: _ t—1 Cam — Ram ZT=0 Cr,mgt—‘r r where, similarto the probability ofdeath function, the daily serial interval is discretized by fs+0.5 1.5 gs = T=s—0.Sg(T)dT fors = 2,3, and 91 = fT=Og(T)dT. Infections today depend on the number of infections in the previous days, weighted by the discretized serial interval distribution. This weighting is then scaled by the country-specific time-Varying reproduction number, Ram, that models the average number of secondary infections at a given time. The functional form for the time-Varying reproduction number was chosen to be as simple as possible to minimize the impact of strong prior assumptions: we use a piecewise constant function that scales Ram from a baseline prior R0,m and is driven by known major non-pharmaceutical interventions occurring in different countries and times. We included 6 interventions, one of which is constructed from the other 5 interventions, which are timings of school and university closures (k=l), self—isolating if ill (k=2), banning of public events (k=3), any government intervention in place (k=4), implementing a partial or complete lockdown (k=5) and encouraging social distancing and isolation (k=6). We denote the indicator variable for intervention k E 1,2,3,4,5,6 by IkI’m, which is 1 if intervention k is in place in country m at time t and 0 otherwise. The covariate ”any government intervention” (k=4) indicates if any of the other 5 interventions are in effect,i.e.14’t’m equals 1 at time t if any of the interventions k E 1,2,3,4,5 are in effect in country m at time t and equals 0 otherwise. Covariate 4 has the interpretation of indicating the onset of major government intervention. The effect of each intervention is assumed to be multiplicative. Ram is therefore a function ofthe intervention indicators Ik’t’m in place at time t in country m: Ram : R0,m eXp(— 212:1 O(Rheum)- The exponential form was used to ensure positivity of the reproduction number, with R0,m constrained to be positive as it appears outside the exponential. The impact of each intervention on Ram is characterised by a set of parameters 0(1, ...,OL6, with independent prior distributions chosen to be ock ~ Gamma(. 5,1). The impacts ock are shared between all m countries and therefore they are informed by all available data. The prior distribution for R0 was chosen to be R0,m ~ Normal(2.4, IKI) with K ~ Normal(0,0.5), Once again, K is the same among all countries to share information. We assume that seeding of new infections begins 30 days before the day after a country has cumulatively observed 10 deaths. From this date, we seed our model with 6 sequential days of infections drawn from cl’m,...,66’m~EXponential(T), where T~Exponential(0.03). These seed infections are inferred in our Bayesian posterior distribution. We estimated parameters jointly for all 11 countries in a single hierarchical model. Fitting was done in the probabilistic programming language Stan,19 using an adaptive Hamiltonian Monte Carlo (HMC) sampler. We ran 8 chains for 4000 iterations with 2000 iterations of warmup and a thinning factor 4 to obtain 2000 posterior samples. Posterior convergence was assessed using the Rhat statistic and by diagnosing divergent transitions of the HMC sampler. Prior-posterior calibrations were also performed (see below). 8.3 Validation We validate accuracy of point estimates of our model using cross-Validation. In our cross-validation scheme, we leave out 3 days of known death data (non-cumulative) and fit our model. We forecast what the model predicts for these three days. We present the individual forecasts for each day, as well as the average forecast for those three days. The cross-validation results are shown in the Figure 8. Figure 8: Cross-Validation results for 3-day and 3-day aggregatedforecasts Figure 8 provides strong empirical justification for our model specification and mechanism. Our accurate forecast over a three-day time horizon suggests that our fitted estimates for Rt are appropriate and plausible. Along with from point estimates we all evaluate our posterior credible intervals using the Rhat statistic. The Rhat statistic measures whether our Markov Chain Monte Carlo (MCMC) chains have converged to the equilibrium distribution (the correct posterior distribution). Figure 9 shows the Rhat statistics for all of our parameters Figure 9: Rhat statistics - values close to 1 indicate MCMC convergence. Figure 9 indicates that our MCMC have converged. In fitting we also ensured that the MCMC sampler experienced no divergent transitions - suggesting non pathological posterior topologies. 8.4 SensitivityAnalysis 8.4.1 Forecasting on log-linear scale to assess signal in the data As we have highlighted throughout in this report, the lag between deaths and infections means that it ta kes time for information to propagate backwa rds from deaths to infections, and ultimately to Rt. A conclusion of this report is the prediction of a slowing of Rt in response to major interventions. To gain intuition that this is data driven and not simply a consequence of highly constrained model assumptions, we show death forecasts on a log-linear scale. On this scale a line which curves below a linear trend is indicative of slowing in the growth of the epidemic. Figure 10 to Figure 12 show these forecasts for Italy, Spain and the UK. They show this slowing down in the daily number of deaths. Our model suggests that Italy, a country that has the highest death toll of COVID-19, will see a slowing in the increase in daily deaths over the coming week compared to the early stages of the epidemic. We investigated the sensitivity of our estimates of starting and final Rt to our assumed serial interval distribution. For this we considered several scenarios, in which we changed the serial interval distribution mean, from a value of 6.5 days, to have values of 5, 6, 7 and 8 days. In Figure 13, we show our estimates of R0, the starting reproduction number before interventions, for each of these scenarios. The relative ordering of the Rt=0 in the countries is consistent in all settings. However, as expected, the scale of Rt=0 is considerably affected by this change — a longer serial interval results in a higher estimated Rt=0. This is because to reach the currently observed size of the epidemics, a longer assumed serial interval is compensated by a higher estimated R0. Additionally, in Figure 14, we show our estimates of Rt at the most recent model time point, again for each ofthese scenarios. The serial interval mean can influence Rt substantially, however, the posterior credible intervals of Rt are broadly overlapping. Figure 13: Initial reproduction number R0 for different serial interval (SI) distributions (means between 5 and 8 days). We use 6.5 days in our main analysis. Figure 14: Rt on 28 March 2020 estimated for all countries, with serial interval (SI) distribution means between 5 and 8 days. We use 6.5 days in our main analysis. 8.4.3 Uninformative prior sensitivity on or We ran our model using implausible uninformative prior distributions on the intervention effects, allowing the effect of an intervention to increase or decrease Rt. To avoid collinearity, we ran 6 separate models, with effects summarized below (compare with the main analysis in Figure 4). In this series of univariate analyses, we find (Figure 15) that all effects on their own serve to decrease Rt. This gives us confidence that our choice of prior distribution is not driving the effects we see in the main analysis. Lockdown has a very large effect, most likely due to the fact that it occurs after other interventions in our dataset. The relatively large effect sizes for the other interventions are most likely due to the coincidence of the interventions in time, such that one intervention is a proxy for a few others. Figure 15: Effects of different interventions when used as the only covariate in the model. 8.4.4 To assess prior assumptions on our piecewise constant functional form for Rt we test using a nonparametric function with a Gaussian process prior distribution. We fit a model with a Gaussian process prior distribution to data from Italy where there is the largest signal in death data. We find that the Gaussian process has a very similartrend to the piecewise constant model and reverts to the mean in regions of no data. The correspondence of a completely nonparametric function and our piecewise constant function suggests a suitable parametric specification of Rt. Nonparametric fitting of Rf using a Gaussian process: 8.4.5 Leave country out analysis Due to the different lengths of each European countries’ epidemic, some countries, such as Italy have much more data than others (such as the UK). To ensure that we are not leveraging too much information from any one country we perform a ”leave one country out” sensitivity analysis, where we rerun the model without a different country each time. Figure 16 and Figure 17 are examples for results for the UK, leaving out Italy and Spain. In general, for all countries, we observed no significant dependence on any one country. Figure 16: Model results for the UK, when not using data from Italy for fitting the model. See the Figure 17: Model results for the UK, when not using data from Spain for fitting the model. See caption of Figure 2 for an explanation of the plots. 8.4.6 Starting reproduction numbers vs theoretical predictions To validate our starting reproduction numbers, we compare our fitted values to those theoretically expected from a simpler model assuming exponential growth rate, and a serial interval distribution mean. We fit a linear model with a Poisson likelihood and log link function and extracting the daily growth rate r. For well-known theoretical results from the renewal equation, given a serial interval distribution g(r) with mean m and standard deviation 5, given a = mZ/S2 and b = m/SZ, and a subsequently R0 = (1 + %) .Figure 18 shows theoretically derived R0 along with our fitted estimates of Rt=0 from our Bayesian hierarchical model. As shown in Figure 18 there is large correspondence between our estimated starting reproduction number and the basic reproduction number implied by the growth rate r. R0 (red) vs R(FO) (black) Figure 18: Our estimated R0 (black) versus theoretically derived Ru(red) from a log-linear regression fit. 8.5 Counterfactual analysis — interventions vs no interventions Figure 19: Daily number of confirmed deaths, predictions (up to 28 March) and forecasts (after) for all countries except Italy and Spain from our model with interventions (blue) and from the no interventions counterfactual model (pink); credible intervals are shown one week into the future. DOI: https://doi.org/10.25561/77731 Page 28 of 35 30 March 2020 Imperial College COVID-19 Response Team 8.6 Data sources and Timeline of Interventions Figure 1 and Table 3 display the interventions by the 11 countries in our study and the dates these interventions became effective. Table 3: Timeline of Interventions. Country Type Event Date effective School closure ordered Nationwide school closures.20 14/3/2020 Public events banned Banning of gatherings of more than 5 people.21 10/3/2020 Banning all access to public spaces and gatherings Lockdown of more than 5 people. Advice to maintain 1m ordered distance.22 16/3/2020 Social distancing encouraged Recommendation to maintain a distance of 1m.22 16/3/2020 Case-based Austria measures Implemented at lockdown.22 16/3/2020 School closure ordered Nationwide school closures.23 14/3/2020 Public events All recreational activities cancelled regardless of banned size.23 12/3/2020 Citizens are required to stay at home except for Lockdown work and essential journeys. Going outdoors only ordered with household members or 1 friend.24 18/3/2020 Public transport recommended only for essential Social distancing journeys, work from home encouraged, all public encouraged places e.g. restaurants closed.23 14/3/2020 Case-based Everyone should stay at home if experiencing a Belgium measures cough or fever.25 10/3/2020 School closure Secondary schools shut and universities (primary ordered schools also shut on 16th).26 13/3/2020 Public events Bans of events >100 people, closed cultural banned institutions, leisure facilities etc.27 12/3/2020 Lockdown Bans of gatherings of >10 people in public and all ordered public places were shut.27 18/3/2020 Limited use of public transport. All cultural Social distancing institutions shut and recommend keeping encouraged appropriate distance.28 13/3/2020 Case-based Everyone should stay at home if experiencing a Denmark measures cough or fever.29 12/3/2020 School closure ordered Nationwide school closures.30 14/3/2020 Public events banned Bans of events >100 people.31 13/3/2020 Lockdown Everybody has to stay at home. Need a self- ordered authorisation form to leave home.32 17/3/2020 Social distancing encouraged Advice at the time of lockdown.32 16/3/2020 Case-based France measures Advice at the time of lockdown.32 16/03/2020 School closure ordered Nationwide school closures.33 14/3/2020 Public events No gatherings of >1000 people. Otherwise banned regional restrictions only until lockdown.34 22/3/2020 Lockdown Gatherings of > 2 people banned, 1.5 m ordered distance.35 22/3/2020 Social distancing Avoid social interaction wherever possible encouraged recommended by Merkel.36 12/3/2020 Advice for everyone experiencing symptoms to Case-based contact a health care agency to get tested and Germany measures then self—isolate.37 6/3/2020 School closure ordered Nationwide school closures.38 5/3/2020 Public events banned The government bans all public events.39 9/3/2020 Lockdown The government closes all public places. People ordered have to stay at home except for essential travel.40 11/3/2020 A distance of more than 1m has to be kept and Social distancing any other form of alternative aggregation is to be encouraged excluded.40 9/3/2020 Case-based Advice to self—isolate if experiencing symptoms Italy measures and quarantine if tested positive.41 9/3/2020 Norwegian Directorate of Health closes all School closure educational institutions. Including childcare ordered facilities and all schools.42 13/3/2020 Public events The Directorate of Health bans all non-necessary banned social contact.42 12/3/2020 Lockdown Only people living together are allowed outside ordered together. Everyone has to keep a 2m distance.43 24/3/2020 Social distancing The Directorate of Health advises against all encouraged travelling and non-necessary social contacts.42 16/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a Norway measures cough or fever symptoms.44 15/3/2020 ordered Nationwide school closures.45 13/3/2020 Public events banned Banning of all public events by lockdown.46 14/3/2020 Lockdown ordered Nationwide lockdown.43 14/3/2020 Social distancing Advice on social distancing and working remotely encouraged from home.47 9/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a Spain measures cough or fever symptoms.47 17/3/2020 School closure ordered Colleges and upper secondary schools shut.48 18/3/2020 Public events banned The government bans events >500 people.49 12/3/2020 Lockdown ordered No lockdown occurred. NA People even with mild symptoms are told to limit Social distancing social contact, encouragement to work from encouraged home.50 16/3/2020 Case-based Advice to self—isolate if experiencing a cough or Sweden measures fever symptoms.51 10/3/2020 School closure ordered No in person teaching until 4th of April.52 14/3/2020 Public events banned The government bans events >100 people.52 13/3/2020 Lockdown ordered Gatherings of more than 5 people are banned.53 2020-03-20 Advice on keeping distance. All businesses where Social distancing this cannot be realised have been closed in all encouraged states (kantons).54 16/3/2020 Case-based Advice to self—isolate if experiencing a cough or Switzerland measures fever symptoms.55 2/3/2020 Nationwide school closure. Childminders, School closure nurseries and sixth forms are told to follow the ordered guidance.56 21/3/2020 Public events banned Implemented with lockdown.57 24/3/2020 Gatherings of more than 2 people not from the Lockdown same household are banned and police ordered enforceable.57 24/3/2020 Social distancing Advice to avoid pubs, clubs, theatres and other encouraged public institutions.58 16/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a UK measures cough or fever symptoms.59 12/3/2020 9 References 1. Li, R. et al. Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV2). Science (2020) doi:10.1126/science.abb3221. 2. Zhang, J. et al. Patterns of human social contact and contact with animals in Shanghai, China. 5cLRep.9,1—11(2019) 3. Worldometers.info. Hong Kong: coronavirus cases. https://www.wo rldometers.info/co ronavirus/country/china-hong-kong-sar/. 4. Ferguson, N. et al. Impact of non-pharmaceutical interventions (NPIs) to reduce COVID-19 mortality and healthcare demand (Report 9). https://www.imperial.ac.uk/mrc-global-infectious- disease-analysis/news--wuhan-coronavirus/. 5. Cereda, D. et al. The early phase of the COVID-19 outbreak in Lombardy, Italy. arXiv (2020). 6. Zhao, A. J. et al. Title: Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 Brief Title : Antibody responses in COVID-19 patients. (2020). 7. Jombart, T. et al. Inferring the number of COVID-19 cases from recently reported deaths. medRXiV 2020.03.10.20033761(2020)doi:10.1101/2020.03.10.20033761. 8. Zhang, J. et al. Age profile of susceptibility, mixing, and social distancing shape the dynamics of the novel coronavirus disease 2019 outbreak in China. (2020) doi:10.1101/2020.03.19.20039107. 9. Lourenco, J. et al. Fundamental principles of epidemic spread highlight the immediate need for large-scale serological surveys to assess the stage of the SARS-CoV-2 epidemic. doi:10.1101/2020.03.24.20042291 10. United Nations, Department of Economic and Social Affairs, Population Division. World Population Prospects 2019: Data Booket. ST/ESA/SER.A/424. (2019). 11. Verity, R. et al. Estimates ofthe severity of COVID-19 disease. Lancet Infect Dis in press, (2020). 12. Walker, P. G. T. et al. Report 12: The Global Impact of COVID-19 and Strategies for Mitigation and Suppression. 13. Fraser, C. Estimating Individual and Household Reproduction Numbers in an Emerging Epidemic. PL05 ONE 2, e758 (2007). 14. Cori, A., Ferguson, N. M., Fraser, C. & Cauchemez, S. A New Framework and Software to Estimate Time-Varying Reproduction Numbers During Epidemics. Am. J. Epidemiol. 178, 1505—1512 (20131 15. Nouvellet, P. et al. A simple approach to measure transmissibility and forecast incidence. Epidemics 22, 29—35 (2018). 16. Cauchemez, 8., Valleron, A. J., Boelle, P. Y., Flahault, A. & Ferguson, N. M. Estimating the impact of school closure on influenza transmission from Sentinel data. Nature 452, 750—754 (2008). 17. Bellman, R. & Harris, T. On Age-Dependent Binary Branching Processes. Ann. Math. 55, 280— 295(19521 18. Bellman, R. & Harris, T. E. On the Theory of Age-Dependent Stochastic Branching Processes. Proc. Natl. Acad. Sci. 34, 601—604 (1948). 19. Stan Development Team. 2018. The Stan Core Library, Version 2.18.0. http://mc-stan.org. 20. Bundesministerium. Coronavirus (COVID-19): Status quo — Schulen, Hochschulen, Universitaten und Forschungsinstitutionen. https://www.bmbwf.gv.at/Ministerium/Informationspflicht/corona/corona_status.html. 21. Henley, J. Coronavirus: EU states enact tough measures to stem spread. The Guardian https://www.theguardian.com/world/2020/mar/10/coronavirus-several-eu-states-ban-mass-events- after-italian-lockdown (2020). 22. Bundesministerium. Coronavirus - Aktuelle MaBnahmen. https://www.sozialministerium.at/Informationen-zum-Coronavirus/Coronavirus—Aktuelle- MaBnahmen.html (2020). 23. Federal Public Service. Coronavirus : Phase 2 maintained, transition to the federal phase and additional measures. https://www.info-coronavirus.be/en/2020/03/12/phase-2-maintained- transition-to-the-federal-phase-and-additional-measures/ (2020). 24. Belgium.be. Coronavirus: reinforced measures | Belgium.be. https://www.belgium.be/en/news/2020/coronavirus_reinforced_measures (2020). 25. Federal Public Service. Protect yourself and protect the others. https://www.info- coronavirus.be/en/2020/03/10/protect-yourself—and-protect-the-others/ (2020). 26. Wikipedia. 2020 coronavirus pandemic in Denmark. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_Denmark. 27. Stephensen, Emma K|inker; Hansen, T. S. Danmark lukker ned: Her er regeringens nye tiltag. TV2 https://nyheder.tv2.dk/samfund/2020-03-11-danmark-lukker-ned-her-er-regeringens-nye-tiltag (20201 28. Politi. Nye tiltag mod covid-19. Politi https://politi.dk/coronavirus-i-danmark/seneste-nyt-fra- myndighederne/nye-tiltag-mod-covid-19 (2020). 29. Styrelsen for Patientsikkerhed. Indberetning om covid-19zlnformation om mulighed for p\aabud til enkeltpersoner (coronavirus/covid-19). https://stps.dk/da/ansvar-og- retningslinjer/vejledning/indberetning-om-covid-19/#. 30. Wikipedia. 2020 coronavirus pandemic in France. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_France. 31. The Local. France bans gatherings of more than 100 people as coronavirus death toll rises - The Local. The Local https://www.thelocal.fr/20200313/france-bans-gatherings-of—over-100-people- to-fight-coronavirus-pandemic (2020). 32. Henley, Jon; Willsher, Kim; Kassam, A. Coronavirus: France imposes lockdown as EU calls for 30-day travel ban. The Guardian https://www.theguardian.com/world/2020/mar/16/coronavirus- spain-takes-over-private-healthcare-amid-more-european-lockdowns (2020). 33. Wikipedia. 2020 coronavirus pandemic in Germany. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_Germany. 34. BMI. Coronavirus: Fragen und Antworten. Bundesministerium des Innern,fur Bau und Heimat https://web.archive.org/web/20200317073042/https://www.bmi.bund.de/SharedDocs/faqs/DE/the men/bevoelkerungsschutz/coronavirus/coronavirus-faqs.htmI#doc13738352bodyText7. 35. BBC News. Coronavirus: Germany tightens curbs and bans meetings of more than two. BBC News https://www.bbc.co.uk/news/world-europe-51999080 (2020). 36. Bundesregierung. Kanzlerin trifft Regierungschefs der Lander Sozialkontakte vermeiden, Ausbreitung verlangsamen. https://www.bundesregierung.de/breg-de/themen/coronavirus/mpk- 1730186(2020) 37. Robert Koch Institut. Antworten auf haufig gestellte Fragen zum Coronavirus SARS-CoV-2. Robert Koch Institut https://web.archive.org/web/20200312004624/https://www.rki.de/SharedDocs/FAQ/NCOV2019/F AQ_Liste.html (2020). 38. Ministero della Salute. Governo annuncia sospensione dell’attivita didattica dal 5 a|15 marzo. Ministero della Salute http://www.salute.gov.it/portale/nuovocoronavirus/dettaglioVideoNuovoCoronavirus.jsp?lingua=ita liano&menu=multimedia&p=video&id=2052 (2020). 39. CNN. Italy prohibits travel and cancels all public events in its northern region. CNN https://edition.cnn.com/2020/03/08/europe/italy-coronavirus-lockdown-europe-intl/index.html (2020). 40. Attualita. Coronavirus: stop a pub, cinema, teatro e discoteche anche a Roma. Ecco cosa prevede il nuovo decreto. Roma Today https://www.romatoday.it/attualita/coronavirus-pub-cinema- teatri-locali-chiusi-nuovo-decreto.html (2020). 41. Gazzetta Ufficiale. DECRETO DEL PRESIDENTE DEL CONSIGLIO DEl MINISTRI. Gazzetta Ufflclale https://www.gazzettaufficiale.it/eli/id/2020/03/08/20A01522/sg (2020). 42. Helsedirektoratet. The Norwegian Directorate of Health has issued a decision to close schools and other educational institutions. Helsedirektoratet https://www.helsedirektoratet.no/nyheter/the- norwegian-directorate-of—health-has-issued-a-decision-to-close-schools-and-other-educationa|- institutions (2020). 43. Krostensen, Mette; Hellem-Hansen, Viktoria L.; Tandstad, B. Folkehelseinstituttet mener 23.000 kan vaere smittet. NRK https://www.nrk.no/norge/folkehelseinstituttet-mener-23.000-kan- vaere-smittet-1.14958149 (2020). 44. Norweigen Government. The Government is establishing clear quarantine and isolation rules. regjeringen.no https://www.regjeringen.no/en/aktuelt/the-government-is-establishing-clear- quarantine-and-isolation-rules/id2693647/ (2020). 45. Wikipedia. 2020 coronavirus pandemic in Spain. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_Spain. 46. Gabinete de Prensa. El Gobierno anuncia nuevas medidas para evitar la extension del nuevo coronavirus COVID-19. Gobierno de Espana https://www.mscbs.gob.es/gabinete/notasPrensa.do?id=4807 (2020). 47. Gabinete de Prensa. El Consejo Interterritorial del SNS acuerda medidas concretas para zonas con transmision comunitaria significativa de coronavirus. Gobierno de Espana https://www.mscbs.gob.es/gabinete/notasPrensa.do?id=4806 (2020). 48. Folkhalsomyndigheten. Larosaten och gymnasieskolor uppmanas nu att bedriva distansundervisning. Folkhdlsomyndigheten https://www.folkhalsomyndigheten.se/nyheter-och- press/nyhetsarkiv/2020/mars/larosaten-och-gymnasieskolor-uppmanas-nu-att-bedriva- distansundervisning(2020). 49. The Local. Sweden bans large events to halt coronavirus spread. The Local https://www.theloca|.se/20200311/sweden-to-ban-large-public-gatherings-over-coronavirus (2020). 50. Radosevich. Stockholmers urged to work from home as COVID-19 community spread confirmed. Sveriges Radio https://sverigesradio.se/sida/artikel.aspx?programid=2054&artikel=7430511(2020). 51. Folkhalsomyndigheten. Flera tecken p\aa samhallsspridning av covid-19 i Sverige. Folkhdlsomyndigheten https://www.folkhalsomyndigheten.se/nyheter-och- press/nyhetsarkiv/2020/mars/flera-tecken-pa-samhallsspridning-av-covid-19-i-sverige/ (2020). 52. Bundesamt fur Gesendheit BAG. Bundesrat verscharft Massnahmen gegen das Coronavirus zum Schutz der Gesundheit und unterstUtzt betroffene Branchen. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das-bag/aktuell/medienmitteilungen.msg-id-78437.html (20201 53. Bundesamt fur Gesundheit BAG. Coronavirus: Bundesrat verbietet Ansammlungen von mehr als fUnf Personen. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das- bag/aktuell/medienmitteilungen.msg-id-78513.html (2020). 54. Bundesamt fur Gesundheit BAG. Coronavirus: Bundesrat erklart die «ausserordentliche Lage» und verscharft die Massnahmen. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das-bag/aktuell/medienmitteilungen.msg-id-78454.html (20201 55. Bundesamt fur Gesundheit BAG. Neue Hygiene- und Verhaltensregeln zum Schutz gegen das neue Coronavirus. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das- bag/a ktuell/medienmitteilungen.msg-id-78304.html (2020). 56. UK Government, D. for E. Schools, colleges and early years settings to close. UK Government https://www.gov.uk/government/news/schools-colleges-and-early-years-settings-to-close (2020). 57. UK Government. PM address to the nation on coronavirus: 23 March 2020. UK Government https://www.gov.uk/government/speeches/pm-address-to-the-nation-on-coronavirus-23-march- 2020(20201 58. Boycott-Owen, Mason; Bowman, Verity; Kelly-Linden, Jordan; Gartner, A. G. H. S. T. Coronavirus: Boris Johnson puts UK in lockdown as death tolls reaches 55. The Telegraph https://www.telegraph.co.uk/global-health/science-and-disease/coronavirus-news-uk-latest- update-covid-19-death-toll-cases/ (2020). 59. BBC News. Coronavirus: People with fever or ’continuous’ cough told to self—isolate. BBC News https://www.bbc.co.uk/news/uk-51857856 (2020).
An example of social distancing
false
1,080
{ "text": [ "work from home wherever possible" ], "answer_start": [ 30719 ] }
2,565
Interferon-Induced Transmembrane Protein 3 Inhibits Hantaan Virus Infection, and Its Single Nucleotide Polymorphism rs12252 Influences the Severity of Hemorrhagic Fever with Renal Syndrome https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206578/ SHA: 4328e18bdf9b52875c87f3f5ddb1911636a192d2 Authors: Xu-yang, Zheng; Pei-yu, Bian; Chuan-tao, Ye; Wei, Ye; Hong-wei, Ma; Kang, Tang; Chun-mei, Zhang; Ying-feng, Lei; Xin, Wei; Ping-zhong, Wang; Chang-xing, Huang; Xue-fan, Bai; Ying, Zhang; Zhan-sheng, Jia Date: 2017-01-03 DOI: 10.3389/fimmu.2016.00535 License: cc-by Abstract: Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS). Previous studies have identified interferon-induced transmembrane proteins (IFITMs) as an interferon-stimulated gene family. However, the role of IFITMs in HTNV infection is unclear. In this study, we observed that IFITM3 single nucleotide polymorphisms (SNP) rs12252 C allele and CC genotype associated with the disease severity and HTNV load in the plasma of HFRS patients. In vitro experiments showed that the truncated protein produced by the rs12252 C allele exhibited an impaired anti-HTNV activity. We also proved that IFITM3 was able to inhibit HTNV infection in both HUVEC and A549 cells by overexpression and RNAi assays, likely via a mechanism of inhibiting virus entry demonstrated by binding and entry assay. Localization of IFITM3 in late endosomes was also observed. In addition, we demonstrated that the transcription of IFITM3 is negatively regulated by an lncRNA negative regulator of interferon response (NRIR). Taken together, we conclude that IFITM3, negatively regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the plasma HTNV load and the disease severity of patients with HFRS. Text: associates with the severity of disease, indicating the importance of viremia in the pathogenesis of HFRS (2) . Therefore, further studies of host factors limiting HTNV infection and influencing antiviral response as well as disease progression are clinically significant and timely. The human family of interferon-induced transmembrane proteins (IFITMs) was discovered 25 years ago to consist of interferon-stimulated genes (ISGs) (3) . This family includes five members, namely, IFITM1, 2, 3, 5, and 10, among which IFITM1, 2, and 3 possess antiviral activity (4) . Different IFITM proteins have different antiviral spectrum (5) . For example, IFITM3 has been shown to prevent influenza virus infection in vitro and in mice (6, 7) , and it also inhibits multiple viruses, including filoviruses, rhabdoviruses, flaviviruses, and even Ebola and Zika virus (7) (8) (9) (10) (11) . The antiviral mechanism of IFITM3 is thought to be the restriction of viral entry into cells (4, 12) . Single nucleotide polymorphisms (SNPs) are single nucleotide variations in a genetic sequence that occur at an appreciable frequency in the population. Several SNPs has been identified in IFITM3, among which the rs12252 site with C allele results in a N-terminal truncation of IFITM3 protein, leading to impaired inhibition of influenza virus in vitro (13, 14) . Notably, the frequencies of rs12252 C allele and CC genotype correlate with disease severity in patients infected with influenza virus (13, 15) . HTNV has been shown to induce a type I interferon response (though in later time postinfection) (16, 17) . While overexpression of IFITM1, 2, and 3 in Vero E6 cells has been reported to inhibit HTNV infection (18) , however, the effect of IFITMs on HTNV infection in human cell lines and its role in HFRS still remain unknown. LncRNA comprises a group of non-coding RNAs longer than 200 nt that function as gene regulators. Some lncRNAs have been shown to play a role in innate immunity (19) . Among them, negative regulator of interferon response (NRIR) (lncRNA NRIR, also known as lncRNA-CMPK2) is a non-coding ISG that negatively regulates IFITM1 and Mx1 expression in HCV infection (20) . Notably, IFITM3 is largely homologous to IFITM1, but the role of NRIR in the regulation of IFITM3 in HTNV infection remains unclear. In the present study, we investigate the effect of IFTTM3 on the replication of HTNV and its role in the development of HFRS in humans. We provide primary evidence suggesting that IFITM3, regulated by NRIR, can inhibit HTNV infection and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. This study expands our understanding of the antiviral activity of IFITM3 and enriches our knowledge of innate immune responses to HTNV infection. This study was conducted in accordance with the recommendations of the biomedical research guidelines involving human participants established by the National Health and Family Planning Commission of China. The Institutional Ethics Committee of Tangdu Hospital approved this study. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Before inclusion, all participants were informed of the study objectives and signed the consent form before blood samples and medical records were obtained. Sixty-nine HFRS patients admitted into the Department of Infectious Diseases, Tangdu Hospital between October 2014 and March 2016 were enrolled in this study. All patients were Han Chinese. The diagnosis of HFRS was made based on typical symptoms and signs as well as positive IgM and IgG antibodies against HTNV in the serum assessed by enzyme linked immunosorbent assay (ELISA) in our department. The classification of HFRS severity and the exclusion criteria were described as follows (21) : white blood cells (WBC), platelets (PLT), blood urea nitrogen (BUN), serum creatinine (Scr), and heteromorphic lymphocytes that were tested by the Department of Clinical Laboratory (shown in Table 1 ). According to clinical symptoms and signs, such as fever, effusion, hemorrhage, edema, and renal function, the severity of HFRS can be classified as previously described (21): (1) mild patients were identified with mild renal failure without an obvious oliguric stage; (2) moderate patients were those with obvious symptoms of uremia, effusion (bulbar conjunctiva), hemorrhage (skin and mucous membrane), and renal failure with a typical oliguric stage; (3) severe patients had severe uremia, effusion (bulbar conjunctiva and either peritoneum or pleura), hemorrhage (skin and mucous membrane), and renal failure with oliguria (urine output, 50-500 ml/day) for ≤5 days or anuria (urine output, <50 ml/day) for ≤2 days; and (4) critical patients exhibited ≥1 of the following signs during the illness: refractory shock, visceral hemorrhage, heart failure, pulmonary edema, brain edema, severe secondary infection, and severe renal failure with oliguria (urine output, 50-500 ml/day) for >5 days, anuria (urine output, <50 ml/day) for >2 days, or a BUN level of >42.84 mmol/l. Due to the sample quantity required for SNP typing, the mild and moderate patients were assessed together in the mild group, and we combined severe and critical patients as severe group. The exclusion criteria for this study were patients with: (1) any other kidney disease, (2) diabetes mellitus, (3) autoimmune disease, (4) hematological disease, (5) cardiovascular disease, (6) viral hepatitis (types A, B, C, D, or E), or (7) any other liver disease. In addition, no patients received corticosteroids or other immunomodulatory drugs during the study period (21) . Genomic DNA was extracted from the peripheral blood of patients using the PureGene DNA Isolation kit (Gentra Systems, Minneapolis, MN, USA). The region encompassing the human IFITM3 rs12252 were amplified by PCR (forward primer, 5′-GGAAACTGTTGAGAAACCGAA-3′ and reverse primer, 5′-CATACGCACCTTCACGGAGT-3′). The PCR products were purified and sequenced using an Applied Biosystems 3730xl DNA Analyzer (Thermo Scientific, Waltham, MA, USA). The allele frequencies and genotypes of healthy Han Chinese and other groups were obtained from the 1,000 genomes project (http:// www.1000genomes.org). The HTNV load in plasma samples (collected during the acute phase) from 24 age-and sex-matched HFRS patients with different genotypes were measured using previously reported methods (2) . Briefly, viral RNA was extracted from the plasma of HFRS patients using Purelink Viral RNA/DNA Kits (Invitrogen, Carlsbad, CA, USA). The SuperScript III Platinum One-Step Quantitative RT-PCR System kit (Invitrogen, Carlsbad, CA, USA) was employed for the real-time RT-PCR assay. The primers and probe (provided by Sangon Biotech, Shanghai, China) were as follows: forward, 5′-TACAGAGGGAAATCAATGCC-3′, reverse, 5′-TGTTCAACTCATCTGGATCCTT-3′, and probe, 5′-(FAM) ATCCCTCACCTTCTGCCTGGCTATC (TAMRA)-3′. The synthetic S segment of the HTNV standard strain 76-118 RNA transcript was used as the quantitative calibrator. The external standard was the culture supernatant of Vero E6 cells infected with HTNV 76-118, which was quantified using synthetic quantitative calibrators. For each experiment, one aliquot of calibrated 76-118 standard was extracted in parallel with the clinical samples and serially 10-fold diluted with concentrations ranging from 10.56 to 2.56 log10 copies/ml. PCR was performed using an iQ5 Cycler (Bio-Rad, Hercules, CA, USA) with following conditions: 42°C for 15 min, 95°C for 2 min, and 50 cycles of 15 s at 95°C, 30 s at 53°C, and 30 s at 72°C. Fluorescence was read during the 72°C step of the final segment of every cycling program. HUVEC cells (ScienCell Research Laboratories, Carlsbad, CA, USA) were grown in ECM BulletKit (ScienCell Research Laboratories, Carlsbad, CA, USA) in a 5% CO2 incubator. A549 cells (ATCC Cat# CRM-CCL-185, RRID:CVCL_0023) were grown in our laboratory in DMEM with 10% FBS (Thermo Scientific, Waltham, MA, USA) in a 5% CO2 incubator. Cells were used within passage 10 after primary culture. HTNV strain 76-118 was cultured in Vero E6 cells (ATCC Cat# CRL-1586, RRID:CVCL_0574) in our laboratory and titrated using an immunofluorescence staining assay for HTNV nucleocapsid protein (NP) as previously described (22) . The TCID50 was 10 5 /ml, which was calculated using the Reed-Muench method. The recombinant human IFN-α2a was obtained from PBL Interferon Source (Piscataway, NJ, USA) and dissolved in the buffer provided by the manufacturer (composition not disclosed). HUVEC and A549 cells were infected by incubation with HTNV as indicated moi at 37°C for 60 mins. Subsequently, the virus solution was removed and fresh medium added to the cell culture. Cells were transfected with lentiviral vectors of c-myc-tagged IFITM1, IFITM2, IFITM3, and IFITM3 NΔ21 (purchased from GENECHEM, Shanghai, China) at a moi of 10. Puromycin (2 μg/ ml for HUVEC and 6 μg/ml for A549 cells) was used to create cell lines stably expressing IFITMs. Cells were transfected with control (scrambled) short interfering RNA (siRNA), IFITM1 siRNA, IFITM2 siRNA, or IFITM3 siRNA (10 nM) using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA). SiRNAs were purchased from Origene (Rockville, MD, USA), and the sequences were not disclosed. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using the K1622 kit (Thermo Scientific, Waltham, MA, USA). Quantitative realtime PCR (qPCR) was performed using SYBR Premix Ex Taq II (Takara Biotechnology Co., Dalian, China) with a Bio-Rad iQ5 cycler (Bio-Rad, Hercules, CA, USA). β-actin was used as the reference gene. The primers (Sangon Biotech, Shanghai, China) were as follows: IFITM1 (forward, 5′-ACTCCGTGAAGTCTAGGGACA-3′ and reverse, 5′-TGTCACAGAGCCGAATACCAG-3′); IFITM2 (forward, 5′-ATCCCGGTAACCCGATCAC-3′ and reverse, 5′-CTTCCTGTCCCTAGACTTCAC-3′); IFITM3 (forward, 5′-GGTCTTCGCTGGACACCAT-3′ and reverse, 5′-TGTCCCTAGACTTCACGGAGTA-3′); IFITM3 pre-mRNA (forward, 5′-CATAGCACGCGGCTCT CAG-3′ and reverse, 5′-CGTCGCCAACCATCTTCCTG-3′); HTNV S segment (forward, 5′-GCCTGGAGACCATCTGA AAG-3′ and reverse, 5′-AGTATCGGGACGACAAAGGA-3′); β-actin (forward, 5′-GCTACGTCGCCCTGGACTTC-3′ and reverse, 5′-GTCATAGTCCGCCTAGAAGC-3′); NRIR (forward, 5′-ATGGTTTTCTGGTGCCTTG-3′ and reverse, 5′-GGAGGTTAGAGGTGTCTGCTG-3′); NRAV (forward, 5′-TCACTACTGCCCCAGGATCA-3′ and reverse, 5′-GGTGGTCACAGGACTCATGG-3′). For detection of miR-130a, cDNA was synthesized using the TaqMan microRNA reverse transcription kit (Invitrogen, Carlsbad, CA, USA) with a specific primer in gene-specific TaqMan assay kit (000454, Invitrogen, Carlsbad, CA, USA). MiR-130a level was determined using the gene-specific TaqMan assay kit (000454, Invitrogen, Carlsbad, CA, USA). U6 (001973, Invitrogen, Carlsbad, CA, USA) was used as an endogenous control (23) . Because the pre-mRNA levels can represent the initial transcription rate (24) , the primers used to detect the pre-mRNA of IFITM3 were designed targeting the intron of IFITM3 as previously described (25) . IFITM3 has two exons and one intron. For qPCR of IFITM3 pre-mRNA, the forward primers were positioned in the intron, and the reverse primer was positioned at the beginning of the second exon. For qPCR of IFITM3 mRNA, the forward primers were positioned in the first exon, and the reverse primer was positioned at the beginning of the second exon (24) . Because the basal expression of IFITM3 is low in A549 cells, we detected IFITM3 mRNA and pre-mRNA in A549 cells following IFN-α2a treatment (20 IU/ml for 12 h) after the overexpression of NRIR. Cell lysates were prepared using Radio Immunoprecipitation Assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein (20 μg protein/lane) were electrophoresed on a 10%-SDS-polyacrylamide gel and electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membranes were incubated with antibodies against IFITM1 (Proteintech Group Cat# 60074-1-Ig Lot# RRID:AB_2233405), IFITM2, IFITM3 (Proteintech Group Cat# 66081-1-Ig Lot# RRID:AB_11182821), and β-actin (Proteintech, Wuhan, Hubei, China) or HTNV NP (provided by the Department of Microbiology, The Fourth Military Medical University) overnight at 4°C. The membranes were then washed and incubated with HRP-conjugated IgG antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The blots were developed using an enhanced chemiluminescence detection kit (Millipore, Billerica, MA, USA) and visualized using X-ray film. The blot densities were analyzed using the Quantity One software (Bio-Rad, Hercules, CA, USA). In addition, the RIPA buffer contains 50mM Tris (pH = 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS. Protease inhibitor cocktail (Roche, Basel, Switzerland) was added before use. The cells were cultured on glass coverslips (Millipore, Billerica, MA, USA) until they were semi-confluence and then incubated with HTNV for 60 min (moi = 1). At the indicated times post-HTNV infection, the cells were fixed with 4% PFA, incubated with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), and blocked with 5% BSA for 1 h. Following incubation with a mouse monoclonal antibody against c-myc-tag (Sigma-Aldrich, St. Louis, MO, USA, Sigma-Aldrich Cat# M5546), IFITM3, lysosome-associated membrane glycoprotein 1 (LAMP1, Cell Signaling Technology, Danvers, MA, USA), or HTNV NP at 37°C for 2 h, the cells were washed and incubated with anti-rabbit Ig conjugated to Alexa 555 and anti-mouse Ig conjugated to Alexa 488 (Abcam, Cambridge, MA, USA) secondary antibodies at room temperature for 1 h. The nuclei were counterstained with DAPI. An Olympus BX51 fluorescence microscope system and FV1000 confocal microscopy system (Olympus, Tokyo, Japan) were used to capture the images. hTnV binding and entry assay Cells transduced with IFITM3 or the empty vector were detached and washed extensively with cold PBS. The cells and HTNV were pre-chilled on ice for 30 min, mixed at a moi of 1 and incubated at 4°C for 1 h with rotation. Part of cells were washed extensively with ice-cold PBS and harvested for binding assay. Another part of cells were switched to 37°C for 2 h to allow HTNV entry. The HTNV that remained on the cell surface was removed by treatment with proteinase K (0.1 mg/ml, Thermo Scientific, Waltham, MA, USA). To achieve direct entry of HTNV into cells by virus-plasma membrane fusion as a positive control, cells were pre-chilled on ice for 10 min with 20 mM NH4Cl. Adsorption of HTNV (moi = 1) was performed at 4°C for 1 h. The cells were then washed, and fusion of the virus with the plasma membrane was triggered by incubation in low pH medium (20 mM sodium succinate, pH = 5.5) for 10 min at 37°C. Infection was followed by incubation for 2 h at 37°C in the presence of 20 mM NH4Cl (26) . qPCR analysis of the HTNV S segment was conducted to evaluate the influence of IFITMs on HTNV cell adhesion and HTNV entry. All data were expressed as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). For association analysis of the rs12252 allele and genotype, Fisher's exact test was used. Independent samples t-tests were used for normally distributed data. Differences among groups were determined by one-way analysis of variance (ANOVA) with repeated measures, followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. The iFiTM3 snP rs12252 c allele and cc genotype associated with severe hFrs Disease and a higher Plasma hTnV load To determine the clinical significance of IFITM3 SNP in HTNV infection, the relationship between rs12252 SNP and the severity of HFRS in 69 patients were examined. We sequenced 300 bp of the IFITM3 locus encompassing SNP rs12252 in all enrolled patients. Then, we stratified these patients into mild and severe, based on the clinical assessment as described in Section "Material and Methods. " We found a significantly higher frequency of the C allele among severe HFRS patients compared with the healthy Han Chinese in the 1,000 genomes sequence database (68.29 vs. 52.16%, P = 0.0076). The frequency of rs12252 C in severe patients was also higher than those mild patients (68.29 vs. 46.43%, P = 0.013, Figures 1A,B; Table 2 ). These data suggest that harboring rs12252 C allele increases the risk of suffering severe disease in HTNV-infected individuals, with an odds ratio (95% CI) of 2.124 (1.067-4.230). For genotypes, 43.90% of the severe patients carried the CC genotype, a significantly higher frequency than the control Han Chinese per 1,000 genomes sequence database (26.92% CC genotype, P = 0.03) as well as mildly infected patients (14.29%, P = 0.02, Figures 1A,B ; Table 2 ). However, mildly ill individuals did not exhibit a Fisher's exact test was used to test the association between rs12252 allele/genotype and HFRS severity. (c) The plasma HTNV load in CC genotype patients and CT/TT genotype patients, tested by qRCR analysis. Each symbol represents one individual patient. Independent samples t-test was used to test the difference of HTNV load between groups. *P < 0.05, **P < 0.01. significantly different genotype frequencies compared with the Han Chinese population. In addition, we also found that patients with CC genotype had higher plasma viral load in acute phase ( Figure 1C) . These results support the notion that the normal function of IFITM3 plays a critical role in the immune response to HTNV infection in vivo, which has a substantial influence on the clinical manifestation of HFRS. Previous studies reveal that the truncated IFITM3 protein produced by SNP rs12252 C allele (Figure 2A , the missing part stands for the truncated 21 amino acids from N-terminal of IFITM3, the intramembrane helix, and transmembrane helix was presented as boxes) leads to an impaired anti-influenza activity (14) . To test the functional significance of this polymorphism in HTNV infection, we transfected the majority T or minority C variant IFITM3 alleles that produce full-length or N-terminally truncated (NΔ21) proteins (Figure 2A ) with c-myc-tag to HUVEC and A549 cell using lentivirus vectors ( Figure 2B) . Then, we challenged the cells with HTNV at moi = 1 for 24 h and found that cells with the minority C variant were more susceptible to HTNV infection with higher expression of HTNV S segment ( Figure 2C ) and more positive of HTNV NP ( Figure S3 in Supplementary Material). Indeed, compared with the mock (empty vector)-infected control, the NΔ21 protein almost lost the ability to inhibit HTNV infection in both HUVEC and A549 cells (Figures 2C,D ; Figure S3 in Supplementary Material). To determine the role of HTNV infection in inducing IFITMs, qPCR as well as Western blot of IFITMs were conducted in HUVEC and A549 cells (Figures 3A,B ; Figure S1 in Supplementary Material). While we observed only a moderate upregulation of IFITM1, 2, and 3 mRNA and protein in HUVECs after more than 24 h postinfection; IFITM1, 2, and 3 mRNA, however, were only transiently upregulated in A549 cells and caused no significant change in protein level. We knocked down the IFITM1, 2, and 3 expression by transfection of their siRNAs individually. The effect of siRNAs on the expression of target IFITMs was tested by qPCR in HUVECs ( Figure S2 in Supplementary Material), and the effect of the best oligo against each IFITMs (IFITM1C, IFITM2A, IFITM3B) was tested by Western blot in A549 ( Figure 4A ) and HUVEC cells ( Figure 4B) . To assess the role of IFITMs in anti-HTNV effect of IFN-α2a, IFITM1, 2, and 3 were knocked down respectively by transfecting the above-tested oligoes for 12 h, followed by IFN-α2a treatment (20 IU/ml for another 12 h). The cells were then challenged with HTNV (moi = 1) for 24 h. The HTNV S segment and NP levels were significantly suppressed in both HUVEC and A549 cells in response to IFN-α2a treatment. Notably, knockdown of IFITM3 significantly restored the levels of HTNV S segment and NP in HUVEC and A549 cells. Knockdown of IFITM1 also partially restored the HTNV level in A549 cells (Figures 4C,D) . These results demonstrate that To assess the anti-HTNV effects of IFITMs, we tested the effect of overexpressed IFITM1, 2, and 3 on HTNV infection. c-myc-tagged IFITM1, 2, and 3 were expressed in both HUVEC and A549 cells (Figure 5A) , and the cells were then challenged with HTNV (moi = 1) for 24 h. The HTNV S segment and NP levels were suppressed by IFITM3 overexpression in HUVEC cells (Figures 5B-D) . They were also suppressed by expressing IFITM1 and IFITM3 in A549 cells (Figures 5B-D) . The inhibitory effect of IFITM3 was further confirmed by immunofluorescence analysis of HTNV NP ( Figure S3 in Supplementary Material). These results were in accordance with the above-described RNAi results. To determine whether IFITM3 inhibited HTNV binding or entry, HUVEC and A549 cells were incubated with HTNV (moi = 1) at 4°C for 1 h, unbound virus was washed away, and HTNV RNA collected at this time point represents HTNV bound to the cell surface. After virus binding, the cells were shifted to 37°C for 2 h to allow HTNV internalization, and HTNV RNA collected at this time point represents cell-internalized virus. As a positive control for inhibition of virus entry, we incubated a parallel group of cells with HTNV at pH = 5.5 as described in Section "Materials and Methods." Expression of IFITM3 did not affect HTNV binding ( Figure 6A ) but significantly suppressed HTNV entry in both HUVEC and A549 cells (Figure 6B ). iFiTM3 Was Partially localized to laMP1 + late endosomes in the host cells To elucidate the mechanism of IFITM3 function, we investigated the subcellular localization of IFTIM3 in the host cells. IFITM3 was found partially localized to LAMP1 + late endosomes in HUVECs analyzed by confocal microscopy (Figure 6C) . The co-localization of IFITM3 and LAMP1 + late endosomes had also been found in A549 cells (27) . Because the transfer into LAMP1 + late endosomes is a necessary step for HTNV entry (28) , this result provides an evidence for the anti-HTNV mechanism of IFITM3. LncRNA-and microRNA-mediated regulation of IFITM3 has been reported in several studies. We tested the change of previously reported regulators of IFITMs, such as NRAV, NRIR, and miR-130a after HTNV infection, among which NRIR was the only changed one (downregulated) after HTNV infection ( Figure 7A ; Figure S4 in Supplementary Material) in HUVEC. However, the expression of NRIR was unchanged in A549 cells. We overexpressed NRIR in HUVEC and A549 cells using the pcDNA3.1 vector ( Figure 7B) . Importantly, overexpression of NRIR significantly suppressed IFITM3 mRNA and pre-mRNA levels and facilitated HTNV infection in HUVEC and A549 cells (Figures 7C-E) . These data suggest that lncRNA NRIR is a negative regulator of IFITM3 transcription. Hantaan virus is an enveloped, negative-sense RNA virus from the genus Hantavirus within the family Bunyaviridae. It causes HFRS, which is an important threat to public health worldwide. It is also a potential weapon for biological terrorism. Reservoir animals, usually rodents, are asymptomatic during persistent infection. Unlike in rodents, Hantavirus infection leads to HFRS and Hantavirus pulmonary syndrome (HPS) in humans (21) . The major clinical characteristics of HFRS include fever, hemorrhage, hypotension, and renal injury (1, 21) , causing severe manifestations and death in some cases. The current standard of care for HFRS relies on symptomatic and supportive treatment. It has been confirmed that the plasma viral load is associated with the severity of HFRS, implicating the importance of viremia in the pathogenesis of HFRS (2). However, no direct antiviral medications are currently available for this illness. Interferon is the key molecule for the antiviral response and has been used as an antiviral medicine in many diseases. It has been reported that HTNV infection induces a late type I interferon response (16) . However, the set of ISGs required for IFN-mediated inhibition of HTNV has not yet been identified. Therefore, identification of ISGs that are effective against HTNV is an attractive strategy to identify novel therapeutic targets. In this study, we demonstrated a significantly high frequency of the rs12252 C allele and CC genotype among HFRS patients with severe illness compared with mildly infected individuals and the healthy Han Chinese. The rs12252 C allele and CC genotype are also found to be associated with higher plasma viral load in the early stage of HFRS. We also discovered that HTNV infection induces IFITMs, and the truncated IFITM3 produced by rs12252 C allele exhibits significantly decreased anti-HTNV activity. Interestingly, IFITM3 is found to restrict HTNV infection with a mechanism of cellular entry inhibition. Indeed, IFITM3 is localized to the late endosome in the host cells, which is a necessary structure for HTNV entry. In addition, we find that HTNV infection downregulated lncRNA NRIR 48 h post infection, which negatively regulates the transcription of IFITM3. Collectively, these results suggest that IFITM3, regulated by NRIR, inhibits HTNV infection, and its SNP rs12252 correlates with the disease severity and viral load in patients with HFRS. The antiviral properties of IFITM proteins were identified in 2009 in an RNAi screen for host factors that influence influenza virus replication (29) . IFITM1, 2, and 3 have been demonstrated to possess antiviral activity in several studies. Everitt et al. demonstrated that the severity of influenza virus infection was greatly increased in IFITM3-knockout mice compared with wild-type animals (15) . Different IFITM members have also been confirmed to inhibit the cellular entry of multiple virus families (including filoviruses, rhabdoviruses, and flaviviruses) (7, (9) (10) (11) 30) . For example, HIV-1 and HCV infection are inhibited by IFITM1 (31) (32) (33) (34) . It is commonly believed that IFITMs restrict viral infection at the stage of cellular entry (12) . Recent studies suggested that the cellular location of different IFITMs may influence the range of viruses restricted by each protein (5) . IFITM1 prevents HCV entry because it colocalizes with CD81 on the cell membrane, interrupting the endocytosis of HCV particles (32) , whereas IFITM3 confines influenza virus in acidified endosomal compartments (27) . Notably, retrovirus subvirus particles (ISVPs), which do not require endosomal acidification for entry, are not inhibited by IFITM3 expression, suggesting that IFITM3 may function at the stage of endosomal entry (35) . Studies utilizing cell-cell fusion assays have suggested that IFITM3 blocks the entry of enveloped virus by preventing the fusion of the viral membrane with a limiting membrane of the host cell, either the plasma membrane and/or the endosomal membranes. The results obtained using two-photon laser scanning and fluorescence lifetime imaging (FLIM) suggest that IFITM proteins may reduce membrane fluidity and increase the spontaneous positive curvature in the outer leaflet of membranes (36) . In the present study, we demonstrated that IFN-α2a (20 U/ ml) significantly inhibited HTNV infection, siRNA-mediated depletion of IFITM3 alone significantly mitigated the antiviral effect of IFN-α2a in both HUVEC and A549 cells, whereas depletion of IFITM1 alone alleviated the antiviral effect of IFN-α2a in A549 cells. Overexpression of IFITM3 inhibited HTNV infection to HUVEC and A549 cells. IFITM1 overexpression was also effective in inhibition of HTNV in A549 cells. All these results suggest that IFITM3 is an important control factor under natural infection of HTNV. Our results also demonstrate that the effectiveness of IFITM3 is cell type-independent, which is in accordance with the results from similar viruses, such as RVFV (18) . Binding and entry assays, conducted by controlling the temperature and pH, showed that IFITM3 did not significantly influence HTNV binding but inhibited HTNV entry into HUVEC and A549 cells. Indeed, IFITM3 partially localizes to the late endosome of the host cells, which is a necessary site for the HTNV entry. However, we failed in tracking the transportation of HTNV in infected cells possibly due to the lack of fluorescence-labeled virus. In addition, IFITM1 also suppressed HTNV infection in A549 cells. The mechanism underlying anti-HTNV effect of IFITM1 remains undetermined and deserves to be further explored. According to a recent study on the three-dimensional structure of IFITM3, there is a C-terminal transmembrane α-helix and a two-N-terminal intramembrane α-helices (shown in Figure 2A as black boxes) in IFITM3 (14) . There are two splice variants that differ by the presence or absence of the first N-terminal 21 amino acids (deleted part, shown in Figure 2A as red dotted line). Several SNPs including 13 non-synonymous, 13 synonymous, 1 in-frame stop, and 1 splice site acceptoraltering have been reported in the translated IFITM3 sequence (15, 29) . Among them, the rare SNP rs12252C allele of IFITM3 truncates the protein as described above, leading to a reduced inhibition of influenza virus infection in A549 cells (15) . We demonstrated that truncated IFITM3 protein also loses the ability to inhibit HTNV infection in vitro. In Northern European patients hospitalized with seasonal influenza or pandemic influenza A virus, increased homozygosity of the minor C allele of SNP rs12252 in IFITM3 was observed (37) . In Chinese patients infected with influenza A (H1N1) virus, there was also an increased frequency of the C allele and CC genotype of SNP rs12252 (13) . In the present study, we observed an increased frequency of the C allele and CC genotype of SNP rs12252 in severely infected HFRS patients compared with healthy control and mildly affected patients. Patients carrying the CC genotype also had higher plasma viral loads compared with those with the CT/TT genotype. Given the impaired function of the IFITM3 protein produced by the C mutation, and the fact that enrichment of the rs12252 C allele in patients with severe disease and the higher viral load in patients with the CC genotype, this founding suggests that IFITM3 plays a pivotal role in the anti-HTNV response in vivo. We speculate that the much higher level of CC allele at healthy population of Han Chinese compared with Caucasians may place the Chinese at a higher risk for developing severe illness upon HTNV infection, which needs further investigation. LncRNAs are a group of non-coding RNAs longer than 200 nt that function as gene regulators, playing a role in regulating multiple cellular functions, including the innate immunity. For example, lncRNA NEAT1 is reported to be upregulated by influenza virus or PolyI:C stimulation, which promotes IL-8 expression (38) . lncRNA NRAV has been shown to negatively regulate the initial transcription of IFITM3 and Mx1 by affecting the histone modification of these genes (25) . lncRNA NRIR is a non-coding ISG, which has been reported to negatively regulate IFITM1 and Mx1 expression in HCV infection (20) . Mir-130a was also reported as a regulator of IFITM1 (23) . In this analysis, lncRNA NRIR was downregulated in HUVECs after HTNV infection for 48 h, overexpression of NRIR negatively regulates the initial transcription of IFITM3, evidenced by the decreased pre-mRNA as well as mRNA levels. NRIR overexpression also facilitated HTNV infection. These results indicate that the downregulation of NRIR after HTNV infection is possibly involved in the activation of innate immune responses against HTNV infection. We have also evaluated other potential regulators of IFITM3 before we choose NRIR for further study. Another lncRNA that can regulate IFITM3, i.e., NRAV (NR_038854), remained unchanged after HTNV infection ( Figures S4A,B in Supplementary Material). Additionally, miR-130a, which potentially regulate IFITM3, was also unaltered after HTNV infection ( Figures S4C,D in Supplementary Material). In conclusion, this study revealed a critical role for IFITM3 in HTNV infection. We demonstrated, for the first time to our knowledge, that IFITM3 is a newly identified anti-HTNV ISG; its expression is negatively regulated by NRIR; and its antiviral activity seems via a mechanism of inhibiting virus entry into the host cells. In addition, we discovered that the IFITM3 SNP rs12252 C allele and CC genotype correlates with the plasma HTNV load and the severity of HFRS; and the rs12252 C allele produces a truncated IFITM3 protein (NΔ21) that attenuates its anti-HTNV function. These results provide new insights into the role of IFITM3 in regulating innate immunity against HTNV infection, which is the basis for identifying new targets to develop novel agent against this worldwide infectious disease. aUThOr cOnTribUTiOns ZX-y, BP-y, YC-t, and MH-w performed the experiments; WP-z, BX-f, LY-f, ZY, and JZ-s designed the research; HC-x, YW, and WX analyzed the data; TK and ZC-m provided clinical data; ZX-y and BP-y wrote the paper.
What genotype causes truncation of the IFITM3 protein?
false
556
{ "text": [ "rs12252 C allele" ], "answer_start": [ 34574 ] }
2,555
Backcalculating the Incidence of Infection with COVID-19 on the Diamond Princess https://doi.org/10.3390/jcm9030657 SHA: 0938d2fb07611897abf38cea727ddbeea77b73d9 Authors: Nishiura, Hiroshi Date: 2020 DOI: 10.3390/jcm9030657 License: cc-by Abstract: To understand the time-dependent risk of infection on a cruise ship, the Diamond Princess, I estimated the incidence of infection with novel coronavirus (COVID-19). The epidemic curve of a total of 199 confirmed cases was drawn, classifying individuals into passengers with and without close contact and crew members. A backcalculation method was employed to estimate the incidence of infection. The peak time of infection was seen for the time period from 2 to 4 February 2020, and the incidence has abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February on which a movement restriction policy was imposed. Without the intervention from 5 February, it was predicted that the cumulative incidence with and without close contact would have been as large as 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively, while these were kept to be 102 and 47 cases, respectively. Based on an analysis of illness onset data on board, the risk of infection among passengers without close contact was considered to be very limited. Movement restriction greatly reduced the number of infections from 5 February onwards. Text: An outbreak of novel coronavirus disease (COVID-19) has occurred on a cruise ship, the Diamond Princess [1] . The primary case remains unknown, but the index case, defined as the first identified case, is a passenger who started coughing from 19 January 2020 on board, disembarking the ship in Hong Kong on 25 January. As the case was diagnosed on 1 February, the ship was requested to remain in the ocean near Yokohama from 3 February onwards. Subsequently, the movement of all passengers was restricted on board from 5 February, for a matter of 14 days of quarantine. Out of a total of 3711 persons (consisting of 2666 passengers and 1045 crew members), 199 symptomatic cases have been diagnosed on board as of 24 February, and additional asymptomatic infections and symptomatic cases after disembarkation have also been reported. One of the critical issues in infectious disease epidemiology is that the time of infection event is seldom directly observable. For this reason, the time of infection needs to be statistically estimated, employing a backcalculation method [2] . Using a sophisticated statistical model with doubly intervalcensored likelihood and right truncation with an exponential growth of cases, the mean incubation period has been estimated to be about 5.0 days [3] . To understand the time-dependent risk of infection throughout the course of outbreak and estimate the effectiveness of the quarantine measure from 5 to 19 February 2020, I aimed to estimate the incidence of infection with COVID-19 and also predict the likely number of infections prevented by the quarantine measure. I analyzed the epidemic curve, ct, on day t, illustrated by the number of confirmed cases by the date of illness onset. The confirmatory diagnosis was made, using the reverse transcriptase polymerase chain reaction (RT-PCR). The date of illness onset was defined as the first date of fever. In addition to the date of illness onset, cases were classified by contact history inside the cabin and also by the type of membership, i.e., crew or passenger. Close contact was defined as having at least one cabinmate who was confirmed by RT-PCR. We estimate the number of cases by time of infection, it. Using the probability mass function of the incubation period of length s, fs, the incidence of infection is known to satisfy where E(.) represents the expected value. As for fs, it is known that the mean and standard deviation are 5.0 and 3.0 days, respectively, best fitted by lognormal distribution [3] . Employing a step function, the incidence of infection was statistically estimated via a maximum likelihood method. The estimation was implemented independently by the history of contact and type of membership. Regarding the real-time forecasting, we employed the so-called Richards model, an analogue to the generalized logistic model [4, 5] : where is the cumulative incidence on day t, Z is the cumulative incidence at the end of the outbreak, s is the parameter that governs the flexibility of the logistic curve, a is the early growth rate of cases and ti is the inflection point of the cumulative incidence curve. Assuming that the cumulative incidence is Gaussian distributed, four unknown parameters were estimated. The Richards model was fitted to two different datasets, i.e., (i) the dataset of the entire course of the epidemic and (ii) the dataset by 4 February 2020. The latter dataset corresponds to the time period without any impact of movement restriction that was in place from 5 February onwards. Figure 1 shows the epidemic curve by contact history and type of membership. The highest incidence of illness onset was observed on 7 February. The epidemic curve in a latter half period was dominated by crew members whose movement was not strictly controlled due to the need to continue service on the ship. The second dominating group was passengers with close contact history. The last illness onset date on board of a passenger without close contact was on 14 February. Estimating the incidence of infection, the peak incidence was identified for the period from 2 to 4 February among passengers both with and without close contact (Figure 2 ). The incidence of infection abruptly dropped after 5 February, the date of movement restriction. Among passengers without close contact, the incidence was estimated to be zero, except for 8-10 February 2020, during which 0.98 persons (95% confidence intervals (CI): 0, 7.74) per day were estimated to have been infected. The epidemic peak among crew members was seen for the period from 8 to 10 February 2020. Figure 3 compares the cumulative incidence with and without movement restriction policy from 5 February. In the presence of intervention, the cumulative incidence among passengers with and without close contact and crew members were 102, 47 and 48 cases, respectively, as of 24 February 2020. These were well realized by the Richards model. Without intervention from 5 February onwards, it was predicted that the cumulative incidence with and without close contact would have been 1373 (95% CI: 570, 2176) and 766 (95% CI: 587, 946) cases, respectively. A large outbreak of COVID-19 occurred on a cruise ship. Estimating the incidence, the peak time of infection was shown to have been from 2 to 4 February, and the incidence abruptly declined afterwards. The estimated number of new infections among passengers without close contact was very small from 5 February, on which the movement restriction policy was imposed, and at most there was, on average, one case of infection per day from 8 to 10 February. Other than continued exposure among crew members, the estimated incidence in this study indicates that the movement restriction policy from 5 February 2020 was highly successful in greatly reducing the number of secondary transmissions on board. Based on an analysis of illness onset data on board (and before the disembarkation of a large number of passengers), the risk of infection among passengers without close contact was considered to be very limited Among disembarked passengers, symptomatic cases have started to be reported on the ground in and outside of Japan. In particular, cases arising from passengers without close contact indicate a possible pathway of infection via mechanisms that were not covered by the abovementioned analysis that relied on symptomatic cases. Although the transmission via direct human-to-human contact was prevented by movement restrictions, the role of other modes of transmission, e.g., environmental and asymptomatic transmissions, should be further explored. The author declares no conflict of interest.
With the intervention of movement restrictions starting on 5th February 2020, what were the confirmed cases for COVID-19, were limited to?
false
1,190
{ "text": [ "102 and 47 cases, respectively." ], "answer_start": [ 1194 ] }
1,660
Hantaviruses in the Americas and Their Role as Emerging Pathogens https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/ SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101 Authors: Hjelle, Brian; Torres-Pérez, Fernando Date: 2010-11-25 DOI: 10.3390/v2122559 License: cc-by Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] . The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖). Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: (1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] . (2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] . (3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil). Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] . Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] . Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease.
What does the N-protein act as?
false
4,498
{ "text": [ "a peripheral membrane protein" ], "answer_start": [ 10566 ] }
1,574
Population-Based Pertussis Incidence and Risk Factors in Infants Less Than 6 Months in Nepal https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5907881/ SHA: ef821e34873d4752ecae41cd9dfc08a5e6db45e2 Authors: Hughes, Michelle M; Englund, Janet A; Kuypers, Jane; Tielsch, James M; Khatry, Subarna K; Shrestha, Laxman; LeClerq, Steven C; Steinhoff, Mark; Katz, Joanne Date: 2017-03-01 DOI: 10.1093/jpids/piw079 License: cc-by Abstract: BACKGROUND: Pertussis is estimated to cause 2 percent of childhood deaths globally and is a growing public health problem in developed countries despite high vaccination coverage. Infants are at greatest risk of morbidity and mortality. Maternal vaccination during pregnancy may be effective to prevent pertussis in young infants, but population-based estimates of disease burden in infants are lacking, particularly in low-income countries. The objective of this study was to estimate the incidence of pertussis in infants less than 6 months of age in Sarlahi District, Nepal. METHODS: Nested within a population-based randomized controlled trial of influenza vaccination during pregnancy, infants were visited weekly from birth through 6 months to assess respiratory illness in the prior week. If any respiratory symptoms had occurred, a nasal swab was collected and tested with a multitarget pertussis polymerase chain reaction (PCR) assay. The prospective cohort study includes infants observed between May 2011 and August 2014. RESULTS: The incidence of PCR-confirmed Bordetella pertussis was 13.3 cases per 1000 infant-years (95% confidence interval, 7.7–21.3) in a cohort of 3483 infants with at least 1 day of follow-up. CONCLUSIONS: In a population-based active home surveillance for respiratory illness, a low risk for pertussis was estimated among infants in rural Nepal. Nepal’s immunization program, which includes a childhood whole cell pertussis vaccine, may be effective in controlling pertussis in infants. Text: A resurgence of pertussis across age groups has occurred in several countries in recent years [1] . Middle-and high-income countries that use an acellular pertussis vaccine for the primary vaccination series have been particularly affected [2, 3] , and infants and adolescents have experienced the greatest increase [4] . Factors that may contribute to the increased risk of pertussis include rapidly waning immunity from those vaccinated with acellular vaccines [1, 5, 6] , asymptomatic transmission from individuals vaccinated with acellular vaccines [7] , genetic adaption of Bordetella pertussis [8] , vaccination delay or refusal [9] , improved surveillance and laboratory capabilities [2] , and overall increased awareness of the continuing circulation of B pertussis [1] . Some countries experiencing epidemic pertussis, including the United States, United Kingdom, and Argentina, now recommend pertussis immunization in pregnancy and vaccination of close contacts [10, 11] to protect the youngest infants from pertussis before they can be vaccinated themselves [12] . Recent data from maternal vaccination trials demonstrate the ability of antibodies to be transferred from mothers to their infants in pregnancy and their persistence in infants [13] . Global estimates of pertussis show the highest childhood burden in Southeast Asia [14] . In this region, maternal pertussis vaccination during pregnancy may be a way to protect infants, similar to the approach using tetanus toxoid vaccine. However, globally only 1 population-based estimate of pertussis in infants from birth has been conducted (Senegal) [15] , and surveillance and laboratory capabilities in Asia are lacking [16, 17] . The World Health Organization (WHO) recently recommended that countries using whole cell pertussis vaccines continue to do so in light of recent data indicating that acellular pertussis vaccines are less effective than whole cell pertussis vaccines [18] . Population-based data are needed, especially in low-income settings, to provide a more accurate estimate of the burden of pertussis in infants to inform childhood and maternal immunization policies [19, 20] . We report on a prospective cohort study following infants weekly in their homes to monitor for pertussis disease from birth to age 6 months. The objective was to provide a population-based estimate of laboratory-confirmed pertussis incidence in infants less than 6 months of age in the Sarlahi District, Nepal. The study was nested within 2 consecutive randomized controlled trials of maternal influenza vaccination during pregnancy set in the Sarlahi District, located in the central Terai (low-lying plains) region of Nepal [21] . At the start of the trial, prevalent pregnancies were identified through a census of all households in the catchment area. For the duration of the trial, field workers visited all households in the communities, every 5 weeks, where married women (15-40 years) resided, for surveillance of incident pregnancies. Once a pregnancy was identified, women provided consent and were enrolled. From April 25, 2011 through September 9, 2013, women between 17 and 34 weeks gestation were randomized and vaccinated with either an influenza vaccine or placebo. The study was a population-based prospective cohort of infants followed from birth through 6 months postpartum. Approval for the study was obtained from the Institutional Review Boards at the Johns Hopkins Bloomberg School of Public Health, Cincinnati Children's Medical Center, the Institute of Medicine at Tribhuvan University, Kathmandu, and the Nepal Health Research Council. The trials are registered at Clinicaltrials.gov (NCT01034254). At baseline, information was collected on household structure, socioeconomic status, and demographics. At enrollment, date of last menstrual period and pregnancy history data were collected. As soon as possible after delivery, the mother and infant were visited to collect detailed birth information including infant weight and breastfeeding status. From birth through 6 months, postpartum infants were visited weekly by a field worker, who recorded any infant respiratory symptoms in the past 7 days. If an infant had any of the following symptoms, a mid-nasal nylon flocked swab was collected: fever, cough, wheeze, difficulty breathing, or ear infection. Starting on August 17, 2012, new symptoms, more specific for pertussis, were added to the weekly morbidity visit: apnea, cyanosis, cough with vomit, or whoop/whooping cough. The swabs were stored for up to 1 week at room temperature in PrimeStore Molecular Transport Medium (Longhorn Diagnostics LLC, Bethesda, MD). In addition to these signs, mothers were asked which, if any, infant vaccinations were received in the past 7 days, including pertussis vaccination [22] . Mid-nasal swabs were also collected on a weekly basis from mothers from enrollment through 6 months postpartum who reported fever plus one additional morbidity (cough, sore throat, nasal congestion, or myalgia). All nasal swabs collected from infants were tested for B pertussis, Bordetella parapertussis, and Bordetella bronchispetica. Only the nasal swabs of mothers whose infants tested positive for any of these pathogens were tested for the same pathogens. Real-time polymerase chain reaction (PCR) testing was conducted at the University of Washington's Molecular Virology Laboratory according to previously published methods [23] . Two-target PCR was used to assess the presence of 3 Bordetella species: B pertussis, B parapertussis, and B bronchiseptica. The amplified targets were chromosomal repeated insertion sequence IS481 (IS) and the polymorphic pertussis toxin ptxA promoter region (PT). After amplification, the melting points of the amplicons were measured in an iCycler (Bio-Rad). A sample was interpreted as positive when the target(s) had a melting temperature within the species-specific acceptable range and a computed tomography ≤42. A sample was negative if none of the targets tested positive or a single positive target was not reproducible. Maternal nasal swabs were tested for those mothers whose infants tested positive for any Bordetella species Polymerase chain reaction was also performed for several viral infections (influenza, rhinovirus [RV], respiratory syncytial virus [RSV], bocavirus [BoV], human metapneumovirus, coronavirus, adenovirus, and parainfluenza [1] [2] [3] [4] ) as previously described [21] . Of 3693 women enrolled, 3646 infants were live born to 3621 women (Supplementary Figure 1 ). Infants were included in this analysis if they were followed for any length of the follow-up period (0 to 180 days); median total follow-up was 146 days per infant (Supplementary Figure 2) . The final dataset consists of 3483 infants, contributing 1280 infant-years of observation, with at least 1 follow-up visit during the first 6 months. This includes infants from the entire trial period, both before and after more pertussis-specific additions to the weekly symptom questionnaire. At baseline, data on household structure were gathered. At enrollment, women reported their literacy status (binary) and pregnancy history. The field workers identified their ethnicity into 2 broad groups (Pahadi, a group originating from the hills; or Madeshi, a group originating from north India) from names and observation. Women were categorized as nulliparous or multiparous. Responses to 25 questions about household construction, water and sanitation, and household assets were used to develop an index to measure the socioeconomic status of households. Binary variables for each of the 25 questions and a mean SES score were calculated for each household. Gestational age was measured using a woman's report of date of last menstrual period during pregnancy surveillance. Birth weight was collected as soon as possible after birth using a digital scale (Tanita model BD-585, precision to nearest 10 grams). Birth weights collected >72 hours after birth were excluded from the analysis. Small for gestational age (SGA) was calculated using the sex-specific 10th percentile cutoff described by Alexander et al [24] and the INTERGROWTH-21 standards [25] . Women were asked within how many hours of birth breastfeeding was initiated and binary breastfeeding categories were created (≤1 hour versus >1 hour postdelivery). Incidence was calculated as the number of pertussis cases per 1000 infant-years at risk. Poisson exact 95% confidence intervals (CIs) were constructed. Characteristics of infant pertussis cases were compared with nonpertussis cases using bivariate Poisson regression. Characteristics of all pertussis respiratory episodes were compared with nonpertussis respiratory episodes; t tests were used for continuous predictors and Fisher's exact tests were used for categorical associations due to the low number of pertussis episodes. All statistical analyses were conducted in Stata/SE 14.1. A total of 3483 infants had 4283 episodes of respiratory illness between May 18, 2011 and April 30, 2014. Thirty-nine percent (n = 1350) of infants experienced no respiratory episodes. The incidence of respiratory illness was 3.6 episodes per infant-year (95% CI, 3.5-3.7). Mean episode duration was 4.7 days (95% CI, 4.6-4.9). A total of 3930 (92%) episodes were matched to 1 or more pertussis-tested nasal swabs from 2026 infants (Supplementary Figure 1) . Seventeen cases of B pertussis were identified from 19 nasal swabs (nasal swabs were positive on 2 consecutive weeks for 2 infants). The incidence of PCR-confirmed B pertussis was 13.3 cases per 1000-infant years (95% CI, 7.7-21.3). Five cases of B parapertussis were detected with an incidence of 3.9 cases per 1000 infant-years (95% CI, 1.3-9.1). No cases of B bronchiseptica were identified. The average pertussis episode duration was 8 days (range, 2-33) ( Table 1 ). Mean age of onset of symptoms was 83 days (range, 19-137) (median, 80; interquartile range, 63-109). The most common symptoms were cough, difficulty breathing, and cough with vomit. None of the additional symptoms related to pertussis that were added in year 2 (cyanosis, apnea, cough with vomit, and whoop) resulted in collection of nasal swabs based solely on these additional symptoms. Pertussis episodes were statistically significantly more likely to include difficulty breathing, cough with vomit, and whoop compared with other respiratory illness. Six infants had at least 1 pertussis vaccination before pertussis disease onset (three <2 weeks and three >2 weeks before pertussis illness) with a mean of 18 days from vaccination to illness compared with 49 days for nonpertussis episodes (P = .03). Five infants received their first pertussis vaccination postpertussis disease onset, whereas 6 infants received no pertussis vaccination in the first 180 days. Three fourths of pertussis episodes were coinfected with at least 1 virus, with RV and BoV the most common. Cases of pertussis were more likely to be infected with BoV than respiratory cases due to causes other than pertussis. The majority of cases occurred between February 2013 and January 2014 (Figure 1) . No statistically significant differences between risk factors for pertussis and nonpertussis cases ( Table 2) were documented. Given the low number of pertussis cases, the lack of a statistical association is not evidence of nonassociation. No deaths occurred in infants who had pertussis. Of the 8 mothers of B pertussis-positive infants who had a nasal swab collected (14 nasal swabs total) during their own follow-up, none were positive for any pertussis species. The 5 B parapertussis cases were primarily male whose mothers were primiparous, literate, and Pahadi ethnicity (Supplementary Table 1 ). No mothers of infants who had B parapertussis had a nasal swab collected during follow-up. The average B parapertussis episode duration was 4 days (Supplementary Table 2 ). Mean age of onset of symptoms was 58 days with a range of 7-95 days. The most common symptoms were cough and wheeze. Rhinovirus and RSV were the only coinfections observed. All B parapertussis cases occurred between September 2011 and February 2012 ( Figure 1 ). A low incidence of pertussis and generally mild clinical presentation were found in infants <6 months in Nepal. To our knowledge, this represents one of the first population-based active surveillance of PCR-confirmed pertussis among young infants in Asia. Acellular pertussis vaccine trials conducted in the 1990s found the average pertussis incidence in the whole cell vaccine groups ranged from 1 to 37 cases per 1000 infantyears [26] . Our finding of 13 B pertussis cases per 1000 infantyears was on the lower end of this range. In the United States in 2014, the estimated pertussis incidence in infants less than 6 months was 2 cases per 1000 infant-years [27] , much lower than observed in our study; however, this passive surveillance system likely vastly underestimates pertussis incidence. Thus, there is a need for active surveillance data such as ours. Furthermore, given our highly sensitive case detection method, many of our pertussis cases would likely not have been detected in the previous acellular pertussis vaccine trials. More stringent respiratory symptom criteria would have lowered our incidence estimate even further. The low incidence was found in a population where pentavalent vaccine (Pentavac: Diphtheria, Tetanus, Pertussis [Whole Cell], Hepatitis-B and Haemophilus Type b Conjugate Vaccine; Serum Institute of India Pvt. Ltd), scheduled for administration at 6, 10, and 14 weeks, is received with significant delays (7% of infants received all 3 recommended pertussis vaccines by 6 months) [22] . These data support the WHO's recommendation that countries using whole cell pertussis vaccine continue to do so given that the majority of outbreaks have been concentrated in countries using the acellular pertussis vaccine [2] . Recent studies suggest that protection from acellular pertussis vaccine is not as strong or long lasting as that conferred by the whole cell pertussis vaccine [6, 28] . Another contributing factor to the low pertussis incidence observed could be that surveillance was conducted during a period of low pertussis transmission. Pertussis is a cyclical disease, thought to peak every 2 to 4 years, and we may have captured the burden at a low circulation period [6] . We observed over 70% of our B pertussis cases over a 1-year period. This increase from earlier observation periods could indicate a temporary rise in pertussis consistent with its cyclical pattern or a true increase in the baseline burden. Previous research on pertussis seasonality has in different places and time periods demonstrated various periods of peak transmission or no discernable patterns [29, 30] . Although our data do not support a seasonal pattern, the numbers observed are too low to be conclusive. Pertussis symptom duration and severity were mild compared with the classic pertussis case presentation. Only 3 of the 17 cases fulfilled the WHO criteria, which requires a minimum of 2 weeks of cough, whoop, or posttussive vomiting [31] . Studies on pertussis in infants have generally been clinic-based, hospital-based, or in an outbreak, which therefore required a certain severity of illness for parents to recognize a need for medical attention [29, 30, 32] . These study designs and passive surveillance efforts therefore may have missed milder pertussis cases [33] . Our study, which required only 1 respiratory symptom for a nasal swab to be collected, had increased sensitivity to detect a range of pertussis case presentations. An alternative explanation for the mild cases seen could be an increase in the proportion of mild compared with severe pertussis cases in Nepal. Although cough, difficulty breathing, and cough with vomit were the most common symptoms, no symptom was present in all B pertussis cases. During an epidemic period in Washington state, among infants <1 year, who had a minimum of 14 days cough plus an additional symptom, 82% had posttussive emesis, 29% had apnea, 26% had whoop, and 42% had cyanosis [32] . A study of US neonates with pertussis showed the symptom prevalence to be 97% for cough, 91% for cyanosis, 58% for apnea, and 3% for fever [34] . Our study found lower or equal symptom prevalence with the exception of fever. Fever prevalence was higher in our study, similar to that found in Peru [29] . Although not statistically significant, infants with pertussis were more likely to have been born preterm, low birth weight, and SGA, and their mothers were more likely to be primiparous. These findings are similar to previous studies showing no difference in pertussis cases by sex [29, 35, 36] or crowding [35] but showing differences by birth weight [36] . Coinfections were common, consistent with findings from other hospital-based studies [33] . Codetection of B pertussis and B parapertussis with respiratory viruses may be due to asymptomatic pertussis carriage. The incidence of B parapertussis of 4 cases per 1000 person-years was comparable to that of 2 per 1000 person-years found in the Italian acellular pertussis vaccine trial in 1992-1993 [37] . The duration of illness was shorter for B parapertussis with a maximum duration of 6 days compared with a maximum of 33 days for B pertussis. A milder presentation is consistent with clinical knowledge of B parapertussis infection [37, 38] . Bordetella parapertussis cases occurred only during a 5-month period. There were several study design limitations. We cannot be certain whether the reported symptoms were caused by pertussis, another organism, or whether symptoms were related to 2 or more etiologic agents. We were unable to perform multivariate regression modeling for characteristics associated with pertussis disease and pertussis cases due to the small number of cases we detected. Infant respiratory symptoms were reported by parents, who may have missed signs that might have been observed by a healthcare worker. However, the criteria for collection of the nasal swab were broad and did not require sophisticated clinical skills. However, apnea and cyanosis may have been difficult for parents to identify. Although the criteria for specimen collection changed in year 2, no infant experienced a pertussis-specific symptom in isolation without also having one of the originally specified respiratory symptoms. These data support our assumption that we were unlikely to have missed pertussis cases in year 1 with our less sensitive respiratory symptom criteria. Nasal swabs were collected in the mid-nasal region for influenza virus detection, which may have lowered the sensitivity of pertussis detection. In a field site, the acceptability of an additional nasopharyngeal swab would likely have increased the participant refusal rate. This would have decreased the generalizability of our results to the entire population. Although nasopharyngeal swabs or nasopharyngeal aspirates are the recommended specimen collection method [39] , the nasopharyngeal region was established as the collection area of choice when the diagnostic measure was culture, which has low sensitivity. Recent data demonstrated the comparability of using mid-nasal versus nasopharyngeal swabs in PCR pertussis detection [40] . Strengths of the study included being a population-based, prospective study, with very low refusal rates. Risk factors, clinical symptoms, and coinfections were prospectively identified without the potential bias that may occur when these data are collected retrospectively or in clinical settings. The community-based design allows generalizability of these results to the entire population and not just those seeking care at a health facility or in an outbreak situation. The Sarlahi District is located in the Terai region where the majority of Nepalese reside, and it has similar demographics to the entire population of Nepal [41] . Sarlahi's location near sea level and on the border with India supports the generalizability of these results to many populations living on the Indian subcontinent. The weekly active surveillance with sensitive criteria for pertussis testing was able to detect mild and atypical pertussis cases, which may have been missed by previous traditional surveillance. The multitarget PCR method allowed highly sensitive and specific detection of 2 additional Bordetella species beyond the primary B pertussis target. We observed a low incidence of pertussis in infants in a whole cell vaccine environment. Pertussis cases were generally milder than expected compared with traditional pertussis clinical definitions. These data support clinicians considering pertussis in their differential diagnosis of infants with mild respiratory symptoms. Policymakers in Nepal will need to weigh the benefit of an additional prenatal pertussis vaccine or a switch to acellular primary pertussis vaccine with the low burden of pertussis in infants less than 6 months. Our study demonstrated that mid-nasal swabs were able to detect pertussis using a sensitive multitarget PCR. The less invasive mid-nasal nasal swab is an attractive alternative for pertussis nasal swab collection, and further research is needed to compare this collection site with nasopharyngeal swabs. In the future, this method may enhance population-based surveillance efforts.
What are the clinical symptoms of pertussis?
false
2,170
{ "text": [ "apnea, cyanosis, cough with vomit, or whoop/whooping cough" ], "answer_start": [ 6426 ] }
186
Identifying Locations with Possible Undetected Imported Severe Acute Respiratory Syndrome Coronavirus 2 Cases by Using Importation Predictions, https://wwwnc.cdc.gov/eid/article/26/7/20-0250_article Volume 26, Number 7—July 2020 Research Pablo Martinez De Salazar1Comments to Author , René Niehus, Aimee Taylor1, Caroline O’Flaherty Buckee, and Marc LipsitchComments to Author Author affiliations: Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA Suggested citation for this article Abstract Cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection exported from mainland China could lead to self-sustained outbreaks in other countries. By February 2020, several countries were reporting imported SARS-CoV-2 cases. To contain the virus, early detection of imported SARS-CoV-2 cases is critical. We used air travel volume estimates from Wuhan, China, to international destinations and a generalized linear regression model to identify locations that could have undetected imported cases. Our model can be adjusted to account for exportation of cases from other locations as the virus spreads and more information on importations and transmission becomes available. Early detection and appropriate control measures can reduce the risk for transmission in all locations. A novel coronavirus, later named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in December 2019 in the city of Wuhan, capital of Hubei Province, China, where cases were first confirmed (1). During December 2019–February 2020, the number of confirmed cases increased drastically. Model estimates suggested that >75,000 persons were infected by January 25, 2020, and the epidemic had a doubling time of ≈6 days (2). By the end of January 2020, travel restrictions were implemented for Wuhan and neighboring cities. Nonetheless, the virus spread from Wuhan to other cities in China and outside the country. By February 4, 2020, a total of 23 locations outside mainland China reported cases, 22 of which reported imported cases; Spain reported a case caused by secondary transmission (3). Most cases imported to other locations have been linked to recent travel history from China (3), suggesting that air travel plays a major role in exportation of cases to locations outside of China. To prevent other cities and countries from becoming epicenters of the SARS-CoV-2 epidemic, substantial targeted public health interventions are required to detect cases and control local spread of the virus. We collected estimates of air travel volume from Wuhan to 194 international destinations. We then identified 49 countries that had a score of >49.2/100 on category 2, Early Detection and Reporting of Epidemics of Potential International Concern, of the Global Health Security (GHS) Index (4). We assumed these locations would be proficient at detecting SARS-CoV-2 and reporting confirmed imported cases, which we refer to as imported-and-reported cases. We ran a generalized linear regression model on this subset; based on the results, we generated predictions for the remainder of the sample. Using these predictions, we identified locations that might not be detecting imported cases. Methods To identify locations reporting fewer than predicted imported SARS-CoV-2 infected cases, we fit a model to data from 49 locations outside mainland China with high surveillance capacity according to the GHS Index (4). Among these, 17 had high travel connectivity to Wuhan and 32 have low connectivity to Wuhan. We considered locations to be countries without any position on territorial claims. We performed a Poisson regression by using the cumulative number of imported-and-reported SARS-CoV-2 cases in these 49 countries and the estimated number of daily airline passengers from the Wuhan airport. We then compared predictions from this model with imported-and-reported cases across 194 locations from the GHS Index, excluding China as the epicenter of the outbreak. The model requires data on imported-and-reported cases of SARS-CoV-2 infection, daily air travel volume, and surveillance capacity. We obtained data on imported-and-reported cases aggregated by destination from the World Health Organization technical report issued February 4, 2020 (3). We assumed a case count of 0 for locations not listed. We used February 4 as the cutoff for cumulative imported-and-reported case counts because exported cases from Hubei Province dropped rapidly after this date (3), likely because of travel restrictions for the province implement on January 23. We defined imported-and-reported cases as those with known travel history from China; of those, 83% had a travel history from Hubei Province and 17% traveled from unknown locations in China (3). We excluded reported cases likely caused by transmission outside of China or cases in which the transmission source was still under investigation (3). In addition, we excluded Hong Kong, Macau, and Taiwan from our model because locally transmitted and imported cases were not disaggregated in these locations. We obtained data on daily air travel from a network-based modeling study (S. Lai et al., unpub. data, https://doi.org/10.1101/2020.02.04.20020479External Link) that reported monthly air travel volume estimates for the 27 locations outside mainland China that are most connected to Wuhan. These estimates were calculated from International Air Travel Association data from February 2018, which includes direct and indirect flight itineraries from Wuhan. For these 27 locations, estimated air travel volumes are >6 passengers/day. We assumed that travel volumes for locations not among the most connected are censored by a detection limit. We used a common method of dealing with censored data from environmental sampling (5), or metabolomics (6), to set the daily air travel volume to half the minimum previously reported. Therefore, we used 3 passengers/day for estimated travel volumes for the 167 locations from the GHS Index not listed by Lai et al. We tested the robustness of our results by using a set of alternative values of 0.1, 1, and 6 passengers/day for the censored data. We defined high surveillance locations as those with a GHS Index for category 2 above the 75th quantile. We assessed the number of high surveillance locations, those with 0 imported-and-reported cases, and low surveillance locations, those with case counts >1 (Table). For our model, we assumed that the cumulative imported-and-reported case counts across 49 high surveillance locations follow a Poisson distribution from the beginning of the epidemic until February 4, 2020. Then the expected case count is linearly proportional to the daily air travel volume in the following formula:where i denotes location, Ci denotes the imported-and-reported case count in a location, λi denotes the expected case count in a location, β denotes the regression coefficient, and xi denotes the daily air travel volume of a location. The Poisson model assumes cases are independent and that the variance is equal to the expected case count. Imported-and-reported cases likely meet the independence assumption because the value excludes cases with local transmission. We also checked the robustness of our results by using an over dispersed model with a negative binomial likelihood. We computed the p value of the overdispersion parameter as shown in Gelman and Hill (7). Thumbnail of Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by air travel volume from Wuhan, China. Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line indicates the expected imported-and-reported case counts for locations. Dashed lines represent 95% prediction interval bounds smoothed for all locations. Purple dots indicate location Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by air travel volume from Wuhan, China. Air travel volume measured in number of... We used R version 3.6.1 (https://www.r-project.orgExternal Link) to compute , the maximum likelihood estimate of β, and the expected imported-and-reported case count given high surveillance (Figure 1). We also computed the 95% prediction interval (PI) bounds under this model of high surveillance for all 194 values of daily air travel volume (Figure 1). First, we generated a bootstrapped dataset by sampling n locations with replacement among high surveillance locations. Then, we reestimated β by using the bootstrapped dataset. Finally, we simulated imported-and-reported case counts for all 194 locations under our model by using the estimate of β from the bootstrapped dataset. We repeated the 3 steps 50,000 times to generate 50,000 simulated imported-and-reported case counts for each of the locations computed to the lower and upper PI bounds (PI 2.5%–97.5%). We smoothed the 95% PI bounds by using ggplot2 in R (8). We fit the imported-and-reported case counts of the 49 high surveillance locations to the model and plotted these alongside 145 locations with low surveillance capacity (Figure 1). We noted some overlap between high and low surveillance locations (Figure 1). Thumbnail of Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 (SARS-CoV-2). Air travel volume measured in number of persons/day. No. cases refers to possible undetected imported SARS-CoV-2 cases. Solid line shows the expected imported-and-reported case counts based on our model fitted to high surveillance locations, indicated by purple dots. Dashed lines indicate the 95% pr Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 (SARS-CoV-2). Air travel volume measured in... To assess the robustness of our results we ran 8 additional regression analyses by implementing a series of changes to the analysis. The changes included the following: set the daily air travel volume to 0.1, 1, or 6 passengers/day for locations not listed by Lai et al. (unpub. data, https://doi.org/10.1101/2020.02.04.20020479External Link) (Figure 2, panels A–C); removed all locations not listed by Lai et al. before fitting (Figure 2, panel D); defined high surveillance locations by using a more lenient GHS Index criterion, 50th quantile (Figure 2, panel E), and a more stringent criterion, 95th quantile (Figure 2, panel F); excluded Thailand from the model because it is a high-leverage point (Figure 2, panel G); or used an overdispersed Poisson likelihood with a negative-binomial likelihood (Figure 2, panel H). We provide code for these analyses on GitHub (https://github.com/c2-d2/cov19flightimportExternal Link). Top Results We found that daily air travel volume positively correlates with imported-and-reported case counts of SARS-CoV-2 infection among high surveillance locations (Figure 1). We noted that increasing flight volume by 31 passengers/day is associated with 1 additional expected imported-and-reported case. In addition, Singapore and India lie above the 95% PI in our model; Singapore had 12 more imported-and-reported cases (95% PI 6–17 cases) than expected and India had 3 (95% PI 1–3 cases) more than expected. Thailand has a relatively high air travel volume compared with other locations, but it lies below the 95% PI, reporting 16 (95% PI 1–40 cases) fewer imported-and-reported cases than expected under the model. Indonesia lies below the PI and has no imported-and-reported cases, but the expected case count is 5 (95% PI 1–10 cases) in our model. Across all 8 robustness regression analyses, we consistently observed that Singapore lies above the 95% PI and Thailand and Indonesia lie below (Figure 2). India remains above the 95% PI in all robustness analyses except when we used the more stringent GHS Index, 95th quantile, for fitting; then India lies on the upper bound of the 95% PI (Figure 2, panel F). Top Discussion We aimed to identify locations with likely undetected or underdetected imported cases of SARS-CoV-2 by fitting a model to the case counts in locations with high surveillance capacity and Wuhan-to-location air travel volumes. Our model can be adjusted to account for exportation of cases from locations other than Wuhan as the outbreak develops and more information on importations and self-sustained transmission becomes available. One key advantage of this model is that it does not rely on estimates of incidence or prevalence in the epicenter of the outbreak. Also, we intentionally used a simple generalized linear model. The linearity of the expected case count means that we have only 1 regression coefficient in the model and no extra parameters. The Poisson likelihood then captures the many 0-counts observed for less highly connected locations but also describes the slope between case-count and flight data among more connected locations. We believe this model provides the most parsimonious phenomenologic description of the data. According to our model, locations above the 95% PI of imported-and-reported cases could have higher case-detection capacity. Locations below the 95% PI might have undetected cases because of expected imported-and-reported case counts under high surveillance. Underdetection of cases could increase the international spread of the outbreak because the transmission chain could be lost, reducing opportunities to deploy case-based control strategies. We recommend rapid strengthening of outbreak surveillance and control efforts in locations below the 95% PI lower bound, particularly Indonesia, to curb potential local transmission. Early detection of cases and implantation of appropriate control measures can reduce the risk for self-sustained transmission in all locations. Top Dr. De Salazar is a research fellow at Harvard T.H. Chan School of Public Health, working on multiscale statistical models of infectious diseases within host, population, and metapopulation models. His research interests include diagnostic laboratory methods and public health response. Top Acknowledgments We thank Pamela Martinez, Nicholas Jewel, and Stephen Kissler for valuable feedback. This work was supported by US National Institute of General Medical Sciences (award no. U54GM088558). P.M.D was supported by the Fellowship Foundation Ramon Areces. A.R.T. and C.O.B. were supported by a Maximizing Investigator’s Research Award (no. R35GM124715-02) from the US National Institute of General Medical Sciences. The authors are solely responsible for this content and it does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. Declaration of interests: Marc Lipsitch has received consulting fees from Merck. All other authors declare no competing interests. Top References Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–3. Wu JT, Leung K, Leung GM. Nowcasting and forecasting the potential domestic and international spread of the 2019-nCoV outbreak originating in Wuhan, China: a modelling study. Lancet. 2020;395:689–97. DOIExternal LinkPubMedExternal Link World Health Organization. Coronavirus disease 2019 (COVID-19) situation report—15, 4 Feb 2020 [cited 2020 Feb 14]. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200204-sitrep-15-ncov.pdfExternal Link Nuclear Threat Initiative and Johns Hopkins Center for Health Security. Global health security index [cited 2020 Feb 14]. https://www.ghsindex.orgExternal Link US Environmental Protection Agency. Data quality assessment: statistical methods for practitioners EPA QA/G9-S [cited 2020 Feb 14]. Washington: The Agency; 2006. https://www.epa.gov/sites/production/files/2015-08/documents/g9s-final.pdfExternal Link Lamichhane S, Sen P, Dickens AM, Hyötyläinen T, Orešič M. An overview of metabolomics data analysis: current tools and future perspectives. In: Jaumot J, Bedia C, Tauler R, editors. Comprehensive analytical chemistry. Vol. 82. Amsterdam: Elsevier; 2018. p. 387–413. Gelman A, Hill J. Analytical methods for social research. In: Data analysis using regression and multilevel/hierarchical models. Cambridge: Cambridge University Press; 2006. p. 235–236. Wickham H. ggplot2: elegant graphics for data analysis. New York: Springer; 2016. Top Figures Figure 1. Regression plot of locations with possible undetected imported cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by air travel volume from Wuhan, China. Air travel volume measured in... Figure 2. Analyses of imported-and-reported cases and daily air travel volume using a model to predict locations with potentially undetected cases of severe acute respiratory virus 2 (SARS-CoV-2). Air travel volume... Table Table. Surveillance capacity of locations with and without imported-and-reported cases of severe acute respiratory syndrome coronavirus 2, 2020 Top Suggested citation for this article: De Salazar PM, Niehus R, Taylor A, O’Flaherty Buckee C, Lipsitch M. Identifying locations with possible undetected imported severe acute respiratory syndrome coronavirus 2 cases by using importation predictions. Emerg Infect Dis. 2020 Jul [date cited]. https://doi.org/10.3201/eid2607.200250 DOI: 10.3201/eid2607.200250 Original Publication Date: 3/24/2020 1These authors contributed equally to this article. Table of Contents – Volume 26, Number 7—July 2020
When was SARS-CoV-2 first identified?
false
245
{ "text": [ "December 2019" ], "answer_start": [ 1428 ] }
1,674
Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523942/ SHA: f00f183d0bce0091a02349ec1eab44a76dad9bc4 Authors: Henry, Kevin A.; Arbabi-Ghahroudi, Mehdi; Scott, Jamie K. Date: 2015-08-04 DOI: 10.3389/fmicb.2015.00755 License: cc-by Abstract: For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage (genera Inovirus and Plectrovirus) are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort (1915) and d 'Hérelle (1917) , the first filamentous phage, f1, was isolated in Loeb (1960) and later characterized as a member of a larger group of phage (Ff, including f1, M13, and fd phage) specific for the E. coli conjugative F pilus (Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964) . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry (If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972) , and over time the list of known filamentous phage has expanded to over 60 members (Fauquet et al., 2005) , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012) . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues (Smith, 1985; Parmley and Smith, 1988) . Based on the ideas described in Parmley and Smith (1988) , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991) . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications (Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010) . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( Table 1) . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pVIII, type 3 display on pIII, etc.), resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith (1988), McConnell et al. (1994) , Rondot et al. (2001) Hybrid (type 33 and 3+3 systems) Type 3+3 system <1 2 Smith and Scott (1993) , Smith and Petrenko (1997) pVI Hybrid (type 6+6 system) Yes <1 2 >25 kDa Hufton et al. (1999) pVII Fully recombinant (type 7 system) No ∼5 >25 kDa Kwasnikowski et al. (2005) Hybrid (type 7+7 system) Yes <1 2 Gao et al. (1999) pVIII Fully recombinant (landscape phage; type 8 system) No 2700 3 ∼5-8 residues Kishchenko et al. (1994) , Petrenko et al. (1996) Hybrid (type 88 and 8+8 systems) Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith (1990) , Greenwood et al. (1991) , Smith and Fernandez (2004) pIX Fully recombinant (type 9+9 * system) Yes ∼5 >25 kDa Gao et al. (2002) Hybrid (type 9+9 system) No <1 2 Gao et al. (1999) , Shi et al. (2010) , Tornetta et al. (2010) 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type 3 * +3 display). By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display (up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number (1-5 per phage particle). While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion (Sidhu et al., 2000) . For the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (Meynell and Lawn, 1968 ) and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies (Pratt et al., 1969; Woolford et al., 1977) . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. (1988) . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pVIII display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII (de la Cruz et al., 1988) . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; Table 2) , including malaria and human immunodeficiency virus type 1 (HIV-1). When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits (de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996) , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants (or mimics thereof) of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di Marzo Veronese et al., 1994; Scala et al., 1999) . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( Table 2) . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (Irving et al., 2010 ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (Van Regenmortel, 2012); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (Frenkel et al., 2000 (Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011) , possibly reduced amyloid plaque formation in mice (Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008) , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease (Lavie et al., 2004) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. (2001) found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules (Rudolf et al., 1998) , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes (Abdennebi et al., 1999) . Phage displaying or chemically Rubinchik and Chow (2000) conjugated to sperm antigen peptides or peptide mimics (Samoylova et al., 2012a,b) and gonadotropin-releasing hormone (Samoylov et al., 2012) are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals ( Table 2) , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions (Greenwood et al., 1991) possibly due to copy number differences (pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996) . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH; Melzer et al., 2003; Su et al., 2007) , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target (Henry et al., 2011) . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. (2010) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (Henry et al., 2011) . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see Immunological Mechanisms of Vaccination with Filamentous Phage below). The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII ( Table 3) . Early work, showing that immunization with phage elicited T-cell help (Kölsch et al., 1971; Willis et al., 1993) , was confirmed by several subsequent studies (De Berardinis et al., 1999; Ulivieri et al., 2008) . From the perspective of vaccination against infectious disease, De Berardinis et al. (2000) showed that a cytotoxic T-cell (CTL) epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins (Mascolo et al., 2007) . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus (Wan et al., 2001) and Candida albicans (Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial (Manoutcharian et al., 2004; Morales et al., 2008) . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (Plotkin, 2010) , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (Gram et al., 1993) . E. coli F17a-G adhesin (Van Gerven et al., 2008) , hepatitis B core antigen (Bahadir et al., 2011) , and hepatitis B surface antigen (Balcioglu et al., 2014) all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (Rao et al., 2003) . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv (mimicking a Vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from Vibrio anguillarum challenge (Xia et al., 2005) . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model (Roehnisch et al., 2013) , and was welltolerated and immunogenic in patients with multiple myeloma (Roehnisch et al., 2014) . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone (Cuesta et al., 2006) , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (Figure 1) . The phage particle is immunogenic without adjuvant in all species tested to date, including mice (Willis et al., 1993) , rats (Dente et al., 1994) , rabbits (de la Cruz et al., 1988) , guinea pigs (Frenkel et al., 2000; Kim et al., 2004) , fish (Coull et al., 1996; Xia et al., 2005) , non-human primates (Chen et al., 2001) , and humans (Roehnisch et al., 2014) . Various routes of immunization have been employed, including oral administration (Delmastro et al., 1997) as well as subcutaneous (Grabowska et al., 2000) , intraperitoneal (van Houten et al., 2006) , intramuscular (Samoylova et al., 2012a) , intravenous (Vaks and Benhar, 2011) , and intradermal injection (Roehnisch et al., 2013) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: (i) the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice (Terry et al., 1997; Kneissel et al., 1999) ; (ii) the N-terminal N1 and N2 domains of pIII (van Houten et al., 2010) ; and (iii) bacterial lipopolysaccharide (LPS) embedded in the phage coat (Henry et al., 2011) . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization (Frenkel et al., 2000) . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes (Hashiguchi et al., 2010) . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van Houten et al., 2010) . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | www.frontiersin.org Although serum anti-phage antibody titers appear to be at least partially T-cell dependent (Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010) , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses (Murira, 2014) . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules (Gaubin et al., 2003; Ulivieri et al., 2008) and can activate T H 1, T H 2, and T H 17 helper T cells (Yang et al., 2005a; Wang et al., 2014d) . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII (Kajihara et al., 2000) . Phage proteins can also be cross-presented on MHC class I molecules (Wan et al., 2005) and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help (Del Pozzo et al., 2010) . The latter CTLs mediate a delayed-type hypersensitivity reaction (Fang et al., 2005; Del Pozzo et al., 2010) . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins (Grabowska et al., 2000) . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors (Hashiguchi et al., 2010) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (Molenaar et al., 2002) , particularly of the liver and spleen, where it is retained for days (Zou et al., 2004) , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe (reviewed in Sulakvelidze et al., 2001) . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "Trojan horse" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease (Hagens and Blasi, 2003; Hagens et al., 2004) , lambda phage S holin (Hagens and Blasi, 2003) or a lethal catabolite gene activator protein (Moradpour et al., 2009) effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS (Hagens and Blasi, 2003; Hagens et al., 2004) . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (Hagens et al., 2006) or phage engineered to repress the cellular SOS response (Lu and Collins, 2009) . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli (May et al., 2011) . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (Figure 2) . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression (Poul and Marks, 1999) . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol (Yacoby et al., 2006; Vaks and Benhar, 2011) . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro (Ghosh et al., 2012a) . Tumorspecific peptide:pVIII fusion proteins selected from "landscape" phage (Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a) were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models (Jayanna et al., 2010b; Wang et al., 2014b,c) . Using the B16-OVA tumor model, Eriksson et al. (2007) showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (Eriksson et al., 2009) . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic (Frenkel and Solomon, 2002) and therapeutic (Solomon, 2008) reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (Ksendzovsky et al., 2012) . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (Rakover et al., 2010) . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units (EU)/mL (Boratynski et al., 2004; Branston et al., 2015) , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (Smith and Gingrich, 2005; Branston et al., 2015) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (Boratynski et al., 2004; Zakharova et al., 2005) , polymyxin B chromatography (Grabowska et al., 2000) , and treatment with detergents such as Triton X-100 or Triton X-114 (Roehnisch et al., 2014; Branston et al., 2015) . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate (LAL) assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in Dalmasso et al., 2014) or for detection of foodborne pathogens post-production (reviewed in Schmelcher and Loessner, 2014) . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance (Nanduri et al., 2007) , piezoelectric transducers (Olsen et al., 2006) , linear dichroism (Pacheco-Gomez et al., 2012) , and magnetoelastic sensor technology (Lakshmanan et al., 2007; Huang et al., 2009) were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (Li et al., 2010b) and eggs (Chai et al., 2012) . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (Henry et al., 2011) . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (Figure 3) . The most commonly used approach is functionalization of amine groups with NHS esters (van Houten et al., 2006 (van Houten et al., , 2010 Yacoby et al., 2006) , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (Li et al., 2010a) . Carrico et al. (2012) developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (Ng et al., 2012) or enzymes (Chen et al., 2007; Hess et al., 2012) , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (Welsh et al., 1996) . Lee et al. (2002) engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012) , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (Mao et al., 2003 (Mao et al., , 2004 , nanoparticles , and nanocomposites (Oh et al., 2012; Chen et al., 2014) . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. (2006) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (Nam et al., 2008) . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides (Lee et al., 2009) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging (Ghosh et al., 2012b; Yi et al., 2012) , photocatalytic water splitting (Nam et al., 2010a; Neltner et al., 2010) , light harvesting (Nam et al., 2010b; Chen et al., 2013) , photoresponsive technologies (Murugesan et al., 2013) , neural electrodes (Kim et al., 2014) , and piezoelectric energy generation (Murugesan et al., 2013) . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions (Hansen et al., 1998 (Hansen et al., , 2000 Dahlke Ojennus et al., 1999) in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013) . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (Marks et al., 1992; Bradbury et al., 2011) . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; Koide and Koide, 2012) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations (Lippow et al., 2007) and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. (2011) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (PACE), which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. (2011) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. (2014) later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis (Meyer and Ellington, 2011) . This approach represents a promising avenue for both basic research in molecular evolution (Dickinson et al., 2013) and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil (Murugaiyan et al., 2011) , coastal fresh water (Xue et al., 2012) , alpine lakes (Hofer and Sommaruga, 2001) and deep sea bacteria (Jian et al., 2012) , but not, perhaps surprisingly, the human gut (Kim et al., 2011) . The environmental "phageome" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles (Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005) . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from 0 to 100% of all viral particles (Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001) . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (Hofer and Sommaruga, 2001) . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (Roux et al., 2012) and reclaimed and potable water (Rosario et al., 2009) but have much higher frequencies in wastewater and sewage (Cantalupo et al., 2011; Alhamlan et al., 2013) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution (reviewed in Canchaya et al., 2003) . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage (Waldor and Mekalanos, 1996) . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 (Hassan et al., 2010) . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis (Derbise et al., 2007) , Neisseria meningitidis (Bille et al., 2005 (Bille et al., , 2008 , Vibrio parahaemolyticus (Iida et al., 2001) , E. coli 018:K1:H7 (Gonzalez et al., 2002) , Xanthomonas campestris (Kamiunten and Wakimoto, 1982) , and P. aeruginosa (Webb et al., 2004) , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum (Yamada, 2013) . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (Lin et al., 2011) . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (Petrenko and Makowski, 1993) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (Curtis et al., 2011 (Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (Curtis et al., 2009 ). The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text.
What is the regularity of the virion major coat protein lattice useful for?
false
1,725
{ "text": [ "enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials" ], "answer_start": [ 1274 ] }
2,504
Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.
What can viral infections cause?
false
3,999
{ "text": [ "the disruption of mucociliary function, an important component of the epithelial barrier." ], "answer_start": [ 25257 ] }
1,689
Chikungunya: A Potentially Emerging Epidemic? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860491/ SHA: f7c3160bef4169d29e2a8bdd79dd6e9056d4774c Authors: Thiboutot, Michelle M.; Kannan, Senthil; Kawalekar, Omkar U.; Shedlock, Devon J.; Khan, Amir S.; Sarangan, Gopalsamy; Srikanth, Padma; Weiner, David B.; Muthumani, Karuppiah Date: 2010-04-27 DOI: 10.1371/journal.pntd.0000623 License: cc-by Abstract: Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts. Text: Chikungunya virus (CHIKV), a mosquito-borne pathogen listed by National Institute of Allergy and Infectious Diseases (NIAID) as a Category C Priority Pathogen that causes Chikungunya fever (CHIKF), has been spreading throughout Asia, Africa, and parts of Europe in recent times [1, 2, 3] . CHIKV is an arthropod-borne virus (arbovirus) and is transmitted to humans primarily by Aedes aegypti, the infamous yellow fever propagator [4, 5] . CHIKV infection is marked by severe joint pain, contorting its victims into unusual postures [6] . The disease gets its name from the Kimakonde vernacular language of Tanzania and Mozambique, and the word chikungunya means ''that which contorts or bends up'' and translates in Swahili to ''the illness of the bended walker'' [7, 8, 9] . In Africa, CHIKV is maintained in a sylvatic cycle among forest-dwelling Aedes spp. mosquitoes, wild primates, squirrels, birds, and rodents ( Figure 1 ) [10] . In Asia, the disease is vectored by Ae. aegypti and Ae. albopictus [11] . Transmission in Asia occurs in an urban cycle whereby the mosquito spreads the disease from an infected human to an uninfected human, following an epidemiological pattern similar to dengue fever [12] . The 2005-2006 epidemic of CHIKV in La Reunion islands in the Indian Ocean, spurred the discovery of a new vector species, Ae. albopictus [5] . Wrecking over one-third of the island's population, this epidemic peaked its devastation between January and February 2006, when over 46,000 cases came into light every week, including 284 deaths [5, 13] . Ae. albopictus is common in urban areas of the United States and is already flourishing in 36 states, raising grave concerns to the immunologically naive populace of the United States [14] . Accordingly, this review elaborately details the epidemiology and global expansion of CHIKV, describes its clinical features and pathogenesis and its symptoms and complications, and finally nominates a possible vaccine approach against CHIKV infection. CHIKV has been isolated into three genotypes based on phylogenetic studies. These genotypes, based on the gene sequences of an Envelope protein (E1), are Asian, East/Central/ South African, and West African [4, 11, 15] . Using phylogenetic models, Cherian et al. estimate that the Asian genotype of CHIKV emerged between 50 and 310 y ago, and the West and East African genotypes diverged between 100 and 840 y ago [15] . Since then, CHIKV has come a long way, with several mutations incorporated, and has continued to wreak epidemics in several regions. Recent activities of CHIKV include the Indian epidemic in 2005-2006, which was followed by a sudden explosion of cases in 2007. An estimated 1.3 million people across 13 states were reported to be infected in India [12, 16] , and CHIKV was also widespread in Malaysia, Sri Lanka, and Indonesia [17] . In July-August of 2007, CHIKV was reported in Italy, probably brought in by travelers from CHIKV-prone regions of India, Africa, and Indian Ocean islands such as Mauritius, Madagascar, and Seychelles. Few of the Italian isolates were found to have evolved from the Kerala isolate, which was associated with a A226V shift in E1 gene that represents a successful evolutionary adaptation in the mosquito vector similar to the ones observed in Reunion Island [2, 18, 19] . In recent times, with an increase in global travel, the risk for spreading CHIKV to non-endemic regions has heightened [1] . Several travelers have brought CHIKV home with them after visiting areas with actively infected populations [12, 20] . Such cases have been documented in European countries, Australia, Asia, and the United States [8, 21] . The United States has already reported at least twelve cases of travel-associated CHIKV, while France has reported 850 cases, and the United Kingdom 93 [8, 14] . Beyond this, CHIKV-infected travelers have also been diagnosed in Australia, Belgium, Canada, Czech Republic, French Guiana, Germany, Hong Kong, Italy, Japan, Kenya, Malaysia, Martinique, Norway, Switzerland, and Sri Lanka [21] . Some travelers were viremic, worrying public health officials about the spread of CHIKV to new areas [1, 8] . The incubation time for CHIKV is relatively short, requiring only 2-6 d with symptoms usually appearing 4-7 d post-infection [22] . Vazeille et al. detected CHIKV in the salivary glands of Ae. albopictus only 2 d after infection [5] . Upon infection, CHIKF tends to present itself in two phases. The first stage is acute, while the second stage, experienced by most but not all, is persistent, causing disabling polyarthritis. Characteristics of the acute phase include an abrupt onset of fever, arthralgia, and in some cases, maculopapular rash [6, 23] . The acute phase causes such intense joint and muscular pain that makes movement very difficult and prostrates its victims [6, 20] . Ninety-five percent of infected adults are symptomatic after infection, and of these, most become disabled for weeks to months as a result of decreased dexterity, loss of mobility, and delayed reaction. Eighteen months after disease onset, 40% of patients are found to still have anti-CHIKV IgM [6, 18, 23, 24] . The chronic stage of CHIKF is characterized by polyarthralgia that can last from weeks to years beyond the acute stage [6] . CHIKV has been shown to attack fibroblasts, explaining the involvement of muscles, joints, and skin connective tissues. The high number of nociceptive nerve endings found within the joints and muscle connective tissues can explain pain associated with CHIKF [25, 26] . More than 50% of patients who suffer from severe CHIKF are over 65 y old, and more than 33% of them die. Most adults who suffer from severe CHIKF have underlying medical conditions [6, 24, 27] . The other group that is disproportionately affected by severe CHIKV is children. Other complications associated with CHIKV, from most common to least common, include respiratory failure, cardiovascular decompensation, meningoencephalitis, severe acute hepatitis, severe cutaneous effects, other central nervous system problems, and kidney failure [6, 18, 20, 23, 24, 26, 27] . CHIKV undertakes a complex replication cycle upon host infection (Figure 2 ), which makes its genome susceptible to mutations [28, 29] . For instance, Ae. aegypti, responsible for epidemics in Kenya, Comoros, and Seychelles, carried CHIKV with an alanine in the 226 position of the E1 gene (E1-A226) [4, 18] . However, when the virus struck La Reunion Islands, a decline in population of Ae. aegypti, due to massive dichlorodiphenyltrichloroethane usage and dearth of Ae. albopictus species' www.plosntds.org population, resulted in an ecological pressure, favoring replacement of alanine at position 226 with valine (E1-A226V) [5] . This mutation allowed CHIKV's secondary vector species, Ae. albopictus, to supplement Ae. aegypti as its primary vector [5] . Within a year, the E1-A226V mutation was present in La Reunion Island, and Ae. albopictus apparently vectored the large epidemic infecting 34% of La Reunion Island's population [5] . All of the CHIKV strains isolated from Mayotte carried the E1-A226V mutation, and the mutation was also found in Madagascar in 2007 [5] . The E1-A226V mutation was not present at the beginning of the Indian Ocean Islands outbreak (before September 2005). However, more than 90% of later viral strains found there had incorporated the mutation (December-March 2006), indicating a genotype switch during the winter season [5, 18, 20] . The E1-A226V mutation also enabled an increase in infectivity of Ae. albopictus when compared to its infectivity of Ae. aegypti [4, 11, 18, 30] , and with several factors taken together, Ae. albopictus has become the new preferred and more lethal vector for CHIKV [4, 5, 11] . In fact, Tsetsarkin et al. found that a Green Fluorescent Protein tagged E1-A226V virus was 100 times more infective to Ae. albopictus than it was to Ae. aegypti [4] . In all the Indian Ocean Islands, Ae. albopictus became the main vector for CHIKV within 1-2 y after CHIKV was introduced to the region [31] . Of note is that Ae. aegypti has most likely been established in North America for over 300 y, while Ae. albopictus has been in many areas of the US, since 1985, primarily in Florida [32] and since then has expanded its range in the country. Reiskind et al. set out to determine if Ae. aegypti and Ae. albopictus mosquitoes captured in Florida were susceptible to CHIKV infection by a La Reunion isolate [32] . Each mosquito tested was highly susceptible to infection by a full-length infectious clone of the La Réunion Island isolate, CHIKV LR2006 OPY1 strain. Even though the Ae. albopictus strains were more susceptible to infection, overall ecology and differences in human biting patterns need to be studied further Characteristically, there are two rounds of translation: (+) sense genomic RNA (49S9 = 11.7 kb) acts directly as mRNA and is partially translated (59 end) to produce non-structural proteins (nsp's). These proteins are responsible for replication and formation of a complementary (2) strand, the template for further (+) strand synthesis. Subgenomic mRNA (26 S = 4.1 kb) replication occurs through the synthesis of full-length (2) intermediate RNA, which is regulated by nsp4 and p123 precursor in early infection and later by mature nsp's. Translation of the newly synthesized sub-genomic RNA results in production of structural proteins such as Capsid and protein E2-6k-E1 (from 39 end of genome). Assembly occurs at the cell surface, and the envelope is acquired as the virus buds from the cell and release and maturation almost simultaneous occurred. Replication occurs in the cytoplasm and is very rapid (,4 h) [28, 29] . doi:10.1371/journal.pntd.0000623.g002 www.plosntds.org to gain a more accurate understanding of a potential CHIKV epidemic in the US [32] . During the 7 d preceding birth, no human mother has been reported to transmit the disease vertically. However, about 50% of newborns delivered while the mother was infected with CHIKV contracted the disease from their mother, despite the method of delivery. Furthermore, there have been instances of CHIKV transmission from mother to fetus causing congenital illness and fetal death [33] . During the 2005-2006 La Reunion Island outbreaks, Ramful et al. discovered that mothers could transmit CHIKV to their progeny during the perinatal period (Day 24 to Day +1) [33, 34] , and it is associated with a high degree of morbidity. By mean Day 4 of life, all of the neonates were symptomatic for CHIKV, exhibiting common CHIKF symptoms. Six neonates were confirmed to have contracted CHIKV and developed mengoencephalitis. Of those mothers who, during the La Reunion Island epidemic, were infected long before delivery, only three fetal deaths were reported [12, 33] . Ramful et al. theorized that motherto-child transmission most likely happens transplacentally shortly before delivery [33] . A similar study by Gerardin et al. reported nineteen cases of neonatal infection associated with intrapartum maternal viremia that progressed to develop encephalitis owing to vertical transmission from infected mothers [34] . Clinical and epidemiological similarities with dengue fever make CHIKV diagnosis difficult, which may lead physicians to misdiagnose CHIKV as dengue fever; therefore, the incidence of CHIKV may actually be higher than currently believed (Table 1 ) [6, 12, 35] . The amount of time elapsed since disease onset is the most critical parameter when choosing a diagnostic test. CHIKV can be detected and isolated by culturing with mosquito cells (C6/36), Vero cells (mammalian), or in mice [26] . However, this method can take at least a week and only achieves a high sensitivity during the viremic phase, which usually only lasts up to 48 h after the bite. Five days post-infection, the viral isolation approach has a low sensitivity but is still the preferred method for detecting the CHIKV strain [12, 26, 31, 35] . RT-PCR on the other hand is a faster and more sensitive method that can be used within the first week of disease onset [26] , and it is currently the most sensitive method for detecting and quantifying viral mRNA [4, 36] . Classic serological detection, by assays such as ELISA [37] , immunofluorescence [5, 38] , complement binding, and haemagglutination inhibition [39] , constitutes the second diagnostic tool used for biological diagnosis of CHIKV infection. These proven techniques are useful for detection of Antigen in mosquitoes during epidemiological studies. These assays detect virus-specific IgM and IgG, however the sensitivity and specificity of these assays has been poorly characterized. Viral competence, or the potential of viral infection and transmission, is an important parameter that can be quantified by ELISA, viral culture, and PCR. A study by Ng et al. showed biomarkers indicative of severe CHIKV infection [40] . They found decreased levels of RANTES and increased levels of Interleukin-6 (IL-6) and Interleukin-1b (IL-1b) that could be sued for CHIKV detection in patients as indicators of CHIKV-driven cytokine storm. Couderc et al. demonstrate another cytokine, type-I IFN, as a key player in the progression to CHIKV infection [26] . Using an IFN-a/b null mouse model, they demonstrated evidence of muscles, joints, and skin as privileged CHIKV targets, which is consistent with human pathology. Although Ng et al. concluded that RANTES levels were significantly suppressed in severe CHIKF patients [40] , interestingly, an increase in levels of RANTES has been observed in dengue infection [41] . Since the symptoms of CHIKF mimic those of dengue fever, results obtained from this study strongly suggest that RANTES could be a potential distinctive biomarker that differentiates between these two clinically similar diseases. There are no approved antiviral treatments currently available for CHIKV [1, 3, 12, 42] . Currently, CHIKF is treated symptomatically, usually with non-steroidal anti-inflammatory drugs or steroids, bed rest, and fluids. Movement and mild exercise are thought to decrease stiffness and morning arthralgia, but heavy exercise may exacerbate rheumatic symptoms. Corticosteroids may be used in cases of debilitating chronic CHIKV infection. There is a debate about the appropriateness of chloroquine as treatment for unresolved, non-steroidal anti-inflammatory drugresistant arthritis [43] . A study showed that viral production was www.plosntds.org drastically reduced at 16 h post-infection after treatment with 100 mM dec-RVKR-cmk (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone), a furine inhibitor [42, 44] . Chloroquine acted by raising the pH, blocking low pH-dependent entry of virus into the cell. It is important to note that dec-RVKR-cmk or chloroquine only inhibited viral spreading from cell to cell, not CHIKV replication once it had entered the cell [43] . However, most would agree that the best weapon against CHIKV is prevention. A live CHIKV vaccine developed by the United States reached phase II clinical trial encompassing 59 healthy volunteers [45] . Eight percent of the volunteers experienced transient arthralgia, while 98% of the volunteers had seroconversion [45] . However, live CHIKV vaccines are still questionable. One cannot discount the risk of a live vaccine possibly inducing chronic rheumatism. Also, there is the question as to whether widespread use among the public could trigger mosquito transmission or lead to chronic infection or viral reversion [1] . An alternative approach would be to produce a chimeric vaccine against CHIKV. Wang et al. developed a chimeric alphavirus vaccine that is uniformly attenuated and does not cause reactogenicity in mice [3] . Three different versions of this vaccine were made using three different backbone vectors: Venezuelan equine encephalitis virus (VEEV) attenuated vaccine strain T-83, naturally attenuated eastern equine encephalitis virus (EEEV), and attenuated Sindbis virus (SINV). In short, CHIKV structural proteins were engineered into the backbones of the aforementioned vaccines to produce the chimeras [3] . These chimeras were found to stimulate a strong humoral immunity, and even at doses of 5.3-5.8 log 10 PFU, they did not trigger reactogenicity. When vaccinated mice were challenged with CHIKV, neither adult nor neonatal mice gained weight, had fever, or displayed signs of neurological illness. Upon comparison of the chimeras with the Army181/25 vaccine, the Army vaccine resulted in higher levels of viremia and replication in the joints of neonatal mice. Because the joints are known targets of CHIKV, Wang et al. noted their vaccine might avoid the negative reactogenic side effects of the Army vaccine. After being subcutaneously vaccinated with 5.3-5.8 log 10 PFU of the chimeric vaccines, mice produced strong neutralizing antibody titers. The VEEV and EEEV chimeras yielded higher neutralizing antibody titers than the SINV chimera without being more virulent. On top of this, the VEEV and EEEV CHIKV chimeras seemed to be more immunogenic than the Army vaccine despite the chimeras' lower viremia and replication in the joints of neonatal mice [3] . Tiwari et al. [46] adopted a different strategy using formalin inactivated CHIKV in combination with alhydrogel (Aluminum Hydroxide) as an adjuvant. This study clearly suggests that this vaccine elicits both humoral and cell-mediated immune responses in mice, providing its immunogenic potential. A recent study by Couderc et al. [47] showed passive immunization as a potential treatment for CHIKV infection. Using purified immunoglobulin extracted from convalescent CHIKV patients, they demonstrated effective neutralizing activity against CHIKV infection both in vitro and in vivo. This thereby establishes a potential preventive and therapeutic approach to combat CHIKV infection. Pathogenesis studies conducted with related alpha virus, like RRV, have shown the role of macrophages in persistence on infection [48] . They also demonstrated the role of RRV-specific CD8 T cells in clearing viral load in infected patients, thereby warranting similar investigations with CHIKV and the importance of investigating a cell-mediated immune response-based vaccine against CHIKV [49] . There are always certain risks associated with live attenuated or inactivated viral vaccines [50] . One way to avoid these potential problems is to construct a consensus-based DNA vaccine. DNA based vaccines have an improved safety profile as compared to live or attenuated vaccines [51, 52] . A consequence of CHIKV's rapid evolution is difficulty in constructing a vaccine that will be able to Figure 3 . Levels of CHIKV-specific IgG in mice immunized with CHIKV vaccines. Each group of C57BL/6 mice (n = 5) was immunized with 12.5 mg of pVax1 control vector or CHIKV vaccine plasmids as indicated at 0 and 2 wk. Mice were bled 2 wk after each immunization, and each group's serum pool was diluted to 1:100 and 1:500 for reaction with specific vaccine constructs. Serum was incubated for 1 h at 37uC on 96-well plates coated with 2 mg/ml of respective CHIKV peptides, and antibody was detected using anti-mouse IgG-HRP and OD was measured at 405 nm. doi:10.1371/journal.pntd.0000623.g003 www.plosntds.org effectively protect large populations from multiple strains of the virus. One of the strengths of DNA consensus vaccines is its ability to induce cross-reactive immune responses against the three distinct phylogenetic groups of CHIKV. Also DNA-based vaccines can be produced more rapidly than protein-based vaccines. Recently, Muthumani et al. constructed a vaccine that was shown to induce both humoral and cellular immunity in vivo in 3-4-wk-old female C57/BL6 mice [49] . These mice were immunized using an in vivo electroporation method to deliver the vaccine into the quadriceps muscle. The consensus construct was designed against E1, E2, and the core protein capsid. To design the construct, they aligned 21 sequences of CHIKV isolated between 1952 and 2006, using strains from differing countries, including La Reunion Island. The most common nucleotide among the sequences was chosen at each position to be used in the consensus construct, taking care not to alter the reading frame. They conducted codon and RNA optimization, added a strong Kozak sequence, and substituted signal peptide with an immunoglobulin E leader sequence to improve vaccine efficacy. After immunizing the mice, spleens were harvested along with serum and tested to determine antibody titer. After three immunizations, consensus E1, E2, and C vaccines were shown to induce T-cell immune responses leading to strong IFN-c responses and proliferation in C57/BL6 mice. Furthermore, when compared with control mice, immunized mice had higher total IgG levels as well as higher anti-E1 specific, anti-E2 specific, and anti-C specific IgG antibodies, suggesting a strong humoral immune response ( Figure 3 ) and also specificity for the antigens encoded in the vaccine constructs ( Figure 4 ). Because of its promising results and the need for a safer vaccine, this consensus DNA vaccine deserves further investigation. Determining longevity of protective effects of the vaccine and persistence of antibody and IFN-c responses could be the next step of investigation. Challenged studies of immunized mice must also be carried out. CHIKV mosquito-borne disease has caused massive outbreaks for at least half a century but is no longer confined to the www.plosntds.org developing nations. It began to encroach into the boundaries of the developing world. As a result, the NIAID has designated CHIKV as a Category C pathogen alongside the influenza and SARS-CoV viruses [3] . Realization of the potential severity of this disease is exigent; for instance, if used as a biological weapon, the world economy could be severely crippled; if enough members of the armed forces were to become infected during a military deployment, military operations could be significantly affected. Efforts to monitor the disease will only provide minimal warning in a global society, and steps to prevent the morbidity and mortality associated with pandemic are imperative [21, 31] . Despite the gravity of its infectious potency and the fear of it being a potential biological weapon, there is currently no vaccine for CHIKV infections. Live attenuated vaccine trials were carried out in 2000, but funding for the project was discontinued. Newer approaches such as DNA vaccines appear promising over conventional strategies like live attenuated or inactivated virus and thus call for further investigation. Recent advances such electroporation delivery and incorporation of adjuvants has boosted DNA vaccine efficacy [51, 53] . Despite the low antibody response to DNA vaccines, other numerous advantages have overshadowed these minor drawbacks (Table 2) , the most important one being the ability to induce both humoral and cellular immune responses [51, 54] . Judging by recent success, such as the immunogenic construct developed by Muthumani et al., DNA vaccines could play a major role in combating CHIKV [49] . Vaccines are literally a critical component of CHIKV disease control and therefore research in this area is highly encouraged. The dramatic spread of dengue viruses (DENV) throughout tropical America since 1980 via the same vectors and human hosts underscores the risk to public health in the Americas. The adverse events associated with the current live vaccine are well documented [55] . Realizing these drawbacks, earnest efforts should be taken to develop new strategies to forestall further spread and complications.
When didthe Asian genotype diverge from African genotype?
false
2,491
{ "text": [ "between 100 and 840 y ago" ], "answer_start": [ 3588 ] }
1,629
The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459876/ SHA: f0b1fa4036434b57c8307d43c39a4193f7e8053a Authors: Morokutti-Kurz, Martina; König-Schuster, Marielle; Koller, Christiane; Graf, Christine; Graf, Philipp; Kirchoff, Norman; Reutterer, Benjamin; Seifert, Jan-Marcus; Unger, Hermann; Grassauer, Andreas; Prieschl-Grassauer, Eva; Nakowitsch, Sabine Date: 2015-06-08 DOI: 10.1371/journal.pone.0128794 License: cc-by Abstract: BACKGROUND: Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated in-vitro and in-vivo. PRINCIPAL FINDINGS: We show in-vitro that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection. CONCLUSION: A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials. Text: The periodic appearance of new influenza variants poses a worldwide pandemic threat. Since the emergence of the new A(H7N9) virus, more than 400 human cases were reported to the WHO with a mortality rate of more than 35%. Most patients with A(H7N9) infections had contact with poultry or visited live animal markets. However, some sporadic cases seemed to be a result of human to human transmissions [1, 2] . In contrast to pandemic viruses which fulminantly enter the human population and cause high mortality rates, seasonal influenza viruses generally cause uncomplicated and transient infections in humans, with virus replication localized to the upper respiratory tract [3, 4] . However, in its fully developed form influenza is an acute respiratory disease resulting in hospitalizations and deaths mainly among high-risk groups. Worldwide, annual epidemics result in about three to five million cases of severe illness, and about 250,000 to 500,000 deaths [5] . For this reason WHO [6] and CDC [7] recommend antiviral treatment for any patient with suspected influenza who is at risk for influenza complications without previous laboratory confirmation. It is known that influenza virus infections are often accompanied by other viral pathogens [8] . Depending on the detection method (qRT-PCR or immunofluorescence) different ratios of co-infections have been found. Analysis by qRT-PCR revealed that 54.5-83.3% of influenza A or B positive patients were found to have at least one concomitant respiratory viral infection [9] [10] [11] [12] . The detection frequency with immunofluorescence was found to be even higher (90-100%) [13, 14] . Potential concomitant viral pathogens of influenza virus infections include human rhinovirus (hRV), respiratory syncytial virus, adenovirus, human coronavirus, human metapneumovirus and parainfluenza virus [14, 15] . As a result of the multiple infections, a specific anti-influenza mono-therapy treats the influenza virus infection only, but not the infection with the concomitant viral pathogen. Hence, the therapy often fails to sufficiently resolve symptoms. This is also reflected by the fact that neuraminidase inhibitors (NI) are highly efficacious in animal models investigating influenza mono-infections [16, 17] but show lower efficacy against influenza symptoms in clinical trials in adults with natural infections [18] . Therefore, there is a high medical need for a broadly acting antiviral therapy in combination with a specific anti-influenza therapy for treatment of patients suffering from upper respiratory tract symptoms. Ideally, the substances present in the combination complement each other by different modes of action, leading to a treatment that provides full protection against a broad range of different respiratory viruses as well as different influenza strains with a low probability to induce escape mutations. One approach for a broad antiviral therapy is the creation of a protective physical barrier in the nasal cavity using carrageenan. Carrageenan is a high molecular weight sulfated polymer derived from red seaweed (Rhodophyceae) that has been extensively used in food, cosmetic and pharmaceutical industry and is generally recognized as safe by the FDA (GRAS) (reviewed in [19] ). Three main forms of carrageenans are commercially used: kappa, iota and lambda. They differ from each other in the degree of sulfation, solubility and gelling properties [20] . The antiviral mechanism of carrageenan is based on the interference with viral attachment; as a consequence, viral entry is inhibited [21, 22] . Its antiviral activity is dependent on the type of polymer as well as the virus and the host cells [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] and has been reviewed in [33] [34] [35] . We published that iota-carrageenan is a potent inhibitor of hRV [36] and influenza A [37] replication and demonstrated the antiviral efficacy of iota-carrageenan against common cold viruses by intranasal application in several randomized, double-blind, parallel group, placebo-controlled clinical trials [38] [39] [40] . The pooled analysis of two studies conducted in 153 children and 203 adults revealed that patients infected with any respiratory virus, who were intranasally treated with iota-carrageenan showed a 1.9 day faster recovery from common cold symptoms than placebo treated patients in the intention-to-treat population [41, 42] . The anti-influenza activity was shown by subgroup analysis of 49 influenza infected patients who benefited from a 3.3 days faster recovery from symptoms. The use of carrageenan nasal spray was associated with a significant reduction of the influenza viral load in nasal fluids and a significant increase in the number of virus free patients within the treatment period of 7 days. In good accordance Prieschl-Grassauer are co-founders of Marinomed Biotechnologie GmbH. Marinomed Biotechnologie GmbH had a role in study design, data collection and analysis, decision to publish, preparation of the manuscript and is financing the processing charge of the manuscript. with the literature [9] [10] [11] [12] [13] [14] we observed that the majority of influenza virus infected patients suffered from a concomitant respiratory viral infection (66%) as determined by real-time PCR. Carrageenan containing nasal sprays are already marketed for the treatment of respiratory viral infections under different brand names in 18 countries. At present the only available effective drugs for treatment and post exposure prevention of influenza are the NI (Oseltamivir and Zanamivir worldwide; Peramivir in Japan and South Korea). Since the large-scale use of M2 blockers for prophylaxis and treatment in humans [43] and farming [44] , the currently circulating influenza viruses already lack sensitivity to this drug group [45] . We have already shown an additive therapeutic effect of a combination therapy with intranasally applied iota-carrageenan and orally administered Oseltamivir in lethally H1N1 A/PR/ 8/34 infected mice and a treatment start 48 hours post infection (hpi) [37] . Due to these very promising results we further developed the concept of combining carrageenan with an NI therapy. In contrast to Oseltamivir, which needs to be activated by metabolic conversion, Zanamivir is directly applied as active drug and can also be administered intranasally [46] [47] [48] [49] [50] [51] [52] . The potential of an intranasal administration of Zanamivir was investigated by GlaxoSmithKline. In seven clinical challenge trials 66 volunteers were infected with influenza B/Yamagata/16/88 and 213 with influenza A/Texas/36/91 (H1N1). 156 of these participants got intranasally applied Zanamivir at different doses (daily dose levels from 6.4 mg to 96 mg) for prophylaxis or therapy [46, 47, 53, 54] . These challenge trials showed that treatment starting before and up to 36 hours post virus inoculation was associated with prevention of laboratory confirmed influenza and febrile illness as well as a reduction in viral titers, duration of shedding and symptoms. In total, safety data from 1092 patients after intranasal application of Zanamivir were published and no evidence for Zanamivir induced adverse events or increased frequencies of local nasal intolerance in comparison to placebo groups was found [46, 49, 52] . Taken together, the combination of a carrageenan nasal spray that provides broad antiviral activity against upper respiratory infections-including influenza-with Zanamivir, a specific anti-influenza drug, meets the existing medical need to treat multiple viral infections. In the present work we investigate the therapeutic effect of a combination of carrageenan and Zanamivir in-vitro and in an animal model. Kappa-carrageenan and iota-carrageenan were purchased from FMC Biopolymers (Philadelphia, PA). The identity, purity (>95%) of carrageenan subtypes and the molecular weight (>100,000) was confirmed by NMR analysis as described elsewhere [55] and the presence of lambda-carrageenan was below the detection limit of 3%. The dry polymer powders were dissolved in aqua bidest (Fresenius Kabi, Austria) to a final concentration of 2.4 mg/ml iota-and 0.8 mg/ml kappa-carrageenan. This 2x stock solution was sterile filtered through a 0.22 μm filter (PAA, Switzerland) and stored at room temperature until use. For further testing the stock solution was diluted to a mixture containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan (hereinafter referred to as "carrageenan"). Zanamivir was purchased as powder (Haosun Pharma, China) and the identity and purity was confirmed by NMR analysis. Zanamivir was either dissolved in carrageenan or placebo solutions, followed by sterile filtration through a 0.22 μm filter (Sarstedt, Germany). For in-vivo studies all Zanamivir containing solutions were freshly prepared. Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in a 37°C incubator (Sanyo, Japan; CO 2 : 5%, relative humidity: >95%). MDCK cells were grown in Dulbecco's minimal essential (DMEM) high glucose medium (PAA, Austria) supplemented with 10% fetal bovine serum (FBS; PAA, Austria; heat inactivated). Influenza virus A/Hansa Hamburg/01/09 (H1N1(09)pdm) was kindly provided by Peter Staeheli Department of Virology, University of Freiburg, Germany and previously described in [56] ; A/Teal/Germany/Wv632/05 (H5N1) previously published in [57] (accession numbers CY061882-9) and A/Turkey/Germany/R11/01 (H7N7) (taxonomy ID 278191, accession number AEZ68716) were supplied by courtesy of Martin Beer, Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Riems, Germany; A/Aichi/2/68 (H3N2) was purchased from the ATCC. All influenza viruses were propagated in MDCK cells at 37°C and 5% CO 2 in influenza medium [Opti-Pro serum free medium (Gibco, Austria) supplemented with 4 mM L-glutamine (PAA, Austria), 1% antibiotic-antimycotic mix (PAA, Austria) and 5 μg/ml trypsin (Sigma Aldrich, Austria)]. To determine the 50% inhibitory concentration (IC 50 ) and the combination effect of carrageenan and Zanamivir, a semi-liquid plaque assay was developed. Into 96 well tissue culture plates 1.7x10 4 MDCK cells/well were seeded and infected at 90% confluence (24-28 hours later). Serial dilutions of carrageenan and Zanamivir were prepared in assay medium (influenza medium without trypsin). For infection, viruses were diluted to an MOI of 0.003 (H1N1(09)pdm and H3N2 Aichi), 0.015 (H5N1) or 0.004 (H7N7), respectively, in assay medium and incubated at room temperature (RT) for 10 min with the serial dilutions of carrageenan and/or Zanamivir, respectively. For evaluation of the combination effect of carrageenan and Zanamivir, viruses were diluted in assay medium containing constant concentrations of either carrageenan or Zanamivir. The other substance was serially diluted and used for virus incubation. Cells were infected in 6 replicates/compound dilution, respectively, and incubated at RT for 45 min before inoculum removal. Cells were further incubated with the respective concentration of the investigated substances present in the overlay [influenza medium with 2.25% Carboxymethylcellulose (CMC, Fluka, Austria)] for 30-42 hours at 37°C. Evolving plaques were evaluated after methanol/acetone cell fixation by immune staining with antibodies either directed against the influenza A nucleoprotein (AbD Serotec, Germany) (for H1N1(09)pdm, H5N1 and H7N7) or the hemagglutinin (AbD Serotec, Germany) (for H3N2). Analysis was done with a HRP labeled detection antibody (Thermo Scientific, Germany) using TMB (Biolegend, Germany) as substrate and a microplate reader at 450 nm. The reduction of detected signal represents a reduction in the number and size of plaques and indicates suppression of viral replication during infection and cultivation. After the immunostaining cells were stained with 0.005% crystal violet solution to assess the condition of the cell layer and the toxicity of the compounds. IC 50 values and standard deviations were calculated for a sigmoidal dose response model using XLfit Excel add-in version 5.3.1.3. All animal experiments were carried out according to the guidelines of the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" and the Austrian law for animal experiments. All animal experiments were approved by the Veterinary University of Vienna institutional ethics committee and performed under the Austrian Federal Ministry of Science and Research experimental animal license numbers BMWF-68.205/0262-II/3b/2011 and BMWF-68.205/0142-II/3b2012. C57BL/6 mice were purchased from Janvier Labs, France and maintained under standard laboratory conditions in the animal facilities of the Veterinary University of Vienna. For euthanasia and anesthesia asphyxiation through CO 2 was used and all efforts were made to minimize suffering. For infection experiments, 3-5 weeks old female mice were intranasally inoculated with 50 μl influenza virus solution (25 μl/nostril) containing 2.27x10 3 or 1.65x10 3 plaque-forming unit of H1N1(09)pdm or H7N7, respectively. Subsequently, treatment started 24, 48 or 72 hpi, as indicated for the different experiments. Treatment was performed intranasally either with 50 μl therapeutic solution or placebo twice per day for 5 days. As therapy either carrageenan (containing 1.2 mg/ml iota-carrageenan and 0.4 mg/ml kappa-carrageenan to provide a daily dose of 12 mg/kg body weight (BW)), Zanamivir (containing either 130 μg/ml or 390 μg/ml Zanamivir, to provide a daily dose of 1 or 3 mg/kg BW, respectively) or a combination of carrageenan and Zanamivir were used. Carrageenan and Zanamivir are used at non-toxic concentrations as shown by [58] and [59] . Mice were monitored twice daily for 15 days for survival and weight loss. Mortality also includes mice that were sacrificed for ethical considerations when they had lost more than 25% of their initial body weight. We confirm the viral infection in these animals by necropsy and scoring of the lung inflammation. As the mechanisms underlying the antiviral activity of NI and carrageenans are fundamentally distinct, they are likely to exhibit different activities towards the individual influenza virus strains. As a result, in combination they could complement each other to provide protection against a broader spectrum of influenza virus strains than the individual compounds. To test this hypothesis, we investigated the sensitivity of various influenza virus strains to Zanamivir and carrageenan in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells [60, 61] . Using this method, we determined the IC 50 of Zanamivir and carrageenan against influenza A viruses of human and animal origin, namely H1N1(09)pdm (A/Hansa Hamburg/01/09), H3N2 (A/Aichi/2/68), low pathogenic (LP) H5N1 (A/Teal/Germany/ Wv632/05) and LP H7N7 (A/Turkey/Germany/R11/01) ( Table 1) . Both substances were nontoxic at the highest tested concentration (400 μM Zanamivir and 533 μg/ml carrageenan), neither was their combination. Furthermore, CMC in the overlay did not show any virus inhibitory effect (data not shown). Inhibition of viral replication of all tested influenza strains was achieved with both substances. However, the IC 50 values varied widely depending on the influenza virus strain. The IC 50 values of Zanamivir ranged between 0.18 μM for H5N1 and 22.97 μM for H7N7 and that of carrageenan from 0.39 μg/ml to 118.48 μg/ml for H1N1(09)pdm and H7N7, respectively (see Table 1 ). These results demonstrate that carrageenan and Zanamivir target individual influenza strains to different extents so that they may complement each other to provide broader anti-influenza activity. The type of compound interaction was characterized by employing isobolograms (Fig 1) . As described in [62] , isobolograms graphically compare the doses of two compounds needed to reach 50% inhibition to the predicted doses calculated based on a model of drug additivity. A curve linearity of~1 is expected for an additive compound interaction whereas a curve progression <1 argue for synergistic and >1 for an antagonistic compound interaction. Two virus strains were selected for those experiments, one being the most sensitive to carrageenan (H1N1(09)pdm) and one being the least sensitive (H7N7). In both cases the isobolograms show a synergistic interaction of carrageenan and Zanamivir (Fig 1) . Thus, it was shown that Zanamivir and carrageenan target individual influenza viruses with different efficiencies, most probably due to their different antiviral strategies. As a result, the combination provides synergistic activity with higher protection against a broader spectrum of influenza virus strains than the individual compounds. In the influenza animal model, C57Bl/6 mice are challenged with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). Infection and treatment (twice a day for 5 days) are done intranasally without anesthesia. We investigated whether the combination of Zanamivir and carrageenan is more efficacious in reducing mortality than the corresponding mono-therapies. First, we determined the minimal effective dose of a Zanamivir mono-therapy that significantly improved survival time of H1N1 and H7N7 infected mice. For the H7N7 lethal infection the minimal effective dose of Zanamivir as mono-therapy ranged between 1 and 3 mg/kg BW/ day (data not shown). Next, we compared the antiviral activity of carrageenan (12 mg/kg BW/ day) and Zanamivir (1 and 3 mg/kg BW/day) mono-therapies with the respective combination versus placebo treatment. Survival rates of mice with treatment starting 24 hpi are shown in Fig 2A. All placebo treated mice died between day 7 and 9 and also in all mono-therapy groups 100% lethality was observed until day 15. In contrast, the combination therapies led to 50% and 90% survival, depending on the Zanamivir concentration. Statistical analysis showed that the Zanamivir mono-therapy 1 mg/kg BW/day did not show a significant benefit (p = 0.1810), whereas the mono-therapy with 3 mg/kg BW/day significantly increased the survival rate compared with placebo treated mice (p = 0.0016). Both Zanamivir concentrations experienced significant benefit in survival by the combination with carrageenan (p<0.0001). Similarly, the combination therapies resulted in remarkably increased survival (p = 0.0421 for 1 mg and p<0.0001 for 3 mg/kg BW/day) when compared to the carrageenan mono-therapy. No statistically significant difference was observed between the combination containing 3 mg/kg BW/day Zanamivir and that containing 1 mg/kg BW/day (p = 0.0525). However, a trend for an increased survival rate with the higher Zanamivir concentration was evident. Therefore, for further investigation the combination therapy containing 3 mg/kg BW/day Zanamivir was evaluated in lethally H7N7 infected mice. Next, the therapeutic potential of the combination with a delayed therapy start 48 or 72 hpi versus placebo treatment was explored. The survival rates of mice are shown in Fig 2B. All placebo treated mice died until day 10 and also in the group with the treatment start 72 hpi 100% lethality was found. In contrast, the combination therapy starting 48 hpi provided a statistically significant enhanced survival rate in comparison to placebo-treated mice (p = 0.0010). In summary, the combination of two effective, established mono-therapies resulted in a significantly enhanced survival in lethally H7N7 infected mice. Additionally, the combination therapy was highly efficient in comparison to placebo treatment even after a treatment onset up to 48 hpi. Intranasal therapy with carrageenan and Zanamivir starting 72 hpi significantly protects lethally influenza H1N1(09)pdm infected mice Next, the minimal effective dose of Zanamivir used as mono-therapy was evaluated in a lethal H1N1(09)pdm mouse model, following the same scheme as described in the H7N7 experiments. The lowest effective dose of Zanamivir after a treatment start 24 hpi was 1 mg/kg BW/ day and its combination with carrageenan was highly effective (data not shown). In the following experiment the therapeutic potential of the combination with a therapy start 48 or 72 hpi was investigated in comparison with the respective placebo treatment. As shown in Fig 3, the survival rates of mice treated with the combination therapy were highly significantly increased in comparison to the placebo group (p<0.0001). There was no difference in survival between the two therapy starting points, 48 or 72 hpi, which both resulted We investigated the antiviral effect of a combination of carrageenan with the NI Zanamivir in cell culture studies and in mouse influenza infection models. We have previously shown that a combined therapy of iota-carrageenan with the NI Oseltamivir led to significantly enhanced survival in mice infected with H1N1 PR/8/34 in comparison with the respective mono-therapies [37] . However, Oseltamivir is an orally administered prodrug, which has to be converted into its active form by metabolic processing. Therefore, a further development of a combination nasal spray was not possible with Oseltamivir. Instead Zanamivir-a NI that is applied as active drug-was chosen for the development of a compound combination. During the evaluation process we found that the binding efficiency of different carrageenan subtypes on different influenza strains varies. The combined use of iota-and kappa-carrageenan for the treatment of lethally influenza infected C57Bl/6 mice revealed a better therapeutic effect than the use of iota-carrageenan alone (S1 Fig). Thus, to provide a broader spectrum of activity against different influenza virus strains, a mixture of iota-and kappa-carrageenan (designated as carrageenan) was used for further evaluation. For investigation of the effect of a compound combination of carrageenan and Zanamivir, we examined their inhibition efficiency, individually and in combination, against influenza viruses in an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. The combination showed a synergistic inhibition of virus replication in in-vitro assays with all tested influenza viruses (Fig 1) . This indicates that the physical interaction of the polymer with the virus does not disturb the inhibition of the neuraminidase by Zanamivir. This was confirmed in in-vitro tests examining a potential influence of the polymer on the neuraminidase inhibiting activity of Zanamivir (data not shown). Hence, the observed synergistic effect is based on the combination of two distinct underlying mechanisms. As a result, in the proposed combination both mechanisms would complement each other to provide more efficient protection against a broader spectrum of influenza virus strains than the individual compounds. The synergistic effect was also shown in lethal mice models (Fig 2 and Fig 3) . The pathogenicity of influenza viruses in mice varies and is dependent on the strain and its adaptation to the host. Depending on virus dose and strain, influenza viruses can induce lethal infections in certain mouse strains usually within two weeks [37, 63] . In our model, C57Bl/6 mice are challenged intranasally with a lethal dose of the respective virus and treated with different regimens in comparison to a vehicle control (placebo). In such a model, early virus replication takes place in the upper respiratory tract. From there, virus spreads to the lung and causes lethal pneumonia. The effect of the treatment on mortality is assessed in comparison to placebotreated control mice. Of all in-vitro tested influenza strains the H1N1(09)pdm and the LP H7N7 are particularly interesting for two reasons. First, they are highly relevant pathogens, as placebo or with the mono-therapies consisting of carrageenan (12 mg/kg BW/day) or Zanamivir (1 and 3 mg/ kg BW/day) or a combination thereof. Treatment started 24 hpi and continued for 5 days. (B) Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan with Zanamivir (3 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival in both experiments (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. both are involved in recent influenza outbreaks. The H1N1(09)pdm is associated with more than 18,400 deaths in the season 2009/2010 while the LP H7N7 carries an HA closely related to that of the avian influenza H7N9 virus which has caused more than 175 deaths until October 2014 [64] . Second, they are of special interest for the carrageenan/Zanamivir combination approach. They have shown to differ in in-vitro susceptibility to carrageenan, Zanamivir (Table 1 ) and the combination thereof (Fig 1) . While H1N1(09)pdm was highly sensitive to inhibition by both substances alone, H7N7 required much higher concentrations of carrageenan and Zanamivir, respectively, to achieve similar inhibition efficiencies. Therefore, both virus strains were chosen to further explore the efficiency of the combination therapy in a mouse model. We established lethal mouse models with both viruses that resulted in 6.8 and 8.5 mean survival days for LP H7N7 and H1N1(09)pdm, respectively. These results are in good accordance to similar already published lethal influenza models [65] [66] [67] . In our models the lowest effective dose for Zanamivir at a treatment start 24 hpi was found to be between 1 to 3 mg/kg BW/day for both viruses. This concentration range is relatively high in comparison to other published studies. However, these studies were done under anesthesia with different viruses and a prophylactic therapy start [65, 66] . The fact that a higher dose of NI is needed for an effective treatment when the therapy starts 24 hpi is already known for Oseltamivir [68] . Nonetheless, also data with much higher effective concentrations (10 mg/kg BW/day [69] ) and with similar concentrations of Zanamivir (2.5 mg/kg BW/day [67] ) were published as well. We found that the combination of carrageenan with 3 mg/kg BW/day Zanamivir used for treatment of H7N7 infected mice resulted in significantly enhanced survival of mice in comparison to both mono-therapies (Fig 2) . The significantly enhanced survival compared to the placebo treated group was also found after a delayed treatment start 48 hpi. Furthermore, in the H1N1(09)pdm model the combination of carrageenan with 1 mg/kg BW/day Zanamivir showed statistically significant enhanced survival in comparison to placebo treatment even after a treatment start 72 hpi. This is a remarkable finding since NIs are normally not effective when applied 72 hpi. The finding supports the development of the Zanamivir and carrageenan combination approach. As the intranasal treatment regime is incapable to effectively treat virus infections of the lung, the primary target of such a product is the prophylaxis and therapy of uncomplicated influenza. Since the majority of influenza infections causes uncomplicated illnesses and practically all cases of influenza start with an infection of the nasal cavity or the upper respiratory tract, the therapeutic potential is huge. However, clinical studies are required to elucidate and demonstrate the potential of the proposed combination therapy. Combination of antiviral strategies has led to impressive achievements in the combat against other viral disease like HIV. In particular the problem of antiviral resistance could be addressed with this strategy. In the last decade concerns have been raised about the increased emergence of Oseltamivir resistant influenza viruses. The augmented appearance of viruses carrying the mutation H275Y in the neuraminidase of H1N1(09)pdm viruses that confers resistance to Oseltamivir left Zanamivir as only treatment option for symptomatic patients infected with an Oseltamivir resistant influenza strain [70] . In contrast to Oseltamivir, resistance to Zanamivir is less frequent. To date, Zanamivir resistant influenza has been detected only once, in an immunocompromised patient [71, 72] . However, lessons should be learned from previous anti-influenza interventions which resulted in occurrence of resistance against currently approved drugs [73] . Therefore, concerns are comprehensible that an increased Zanamivir use may also lead to the rapid emergence of resistances [74] . To overcome this threat, a combination of antivirals which inhibits virus replication by distinct mechanisms is a valid strategy. We checked for the possibility of generating double compound escape mutant viruses while passaging viruses in the presence of increasing concentrations of compound combinations. After 10 passages in MDCK cells no resistance to the compound combination for any tested influenza virus could be found (data not shown). However, this finding does not guarantee that emergence of Zanamivir escape mutants can be completely halted. In summary, we demonstrated that the anti-influenza mechanisms of both single compounds complement each other. The combination provides synergistically better protection against a broader spectrum of influenza viruses than the individual compounds. A nasal spray containing carrageenan together with Zanamivir provides an easy to apply treatment of upper respiratory tract infections in patients under suspicion to be influenza infected. Patients would benefit from the fast and efficient treatment of uncomplicated influenza in the upper respiratory tract. Due to the faster influenza virus clearance from the upper respiratory tract and the independent antiviral mechanism of carrageenan and Zanamivir the likelihood to develop escape mutations against Zanamivir will be reduced. Both individual compounds are able to reduce severity and/or duration of the influenza illness and a combination is expected to work similarly. Additionally, due to the broad antiviral effectiveness of carrageenan, patients will receive in parallel a treatment of concomitant viral infections. Therefore, patients will benefit from a decreased probability to develop complications. In consideration of the complications known to accompany an influenza virus illness this combinational therapy meets an urgent medical need. A second scope of this combination is the protection against newly emerging pandemic viruses during the time until identification of the virus followed by manufacturing and distribution of vaccines [43] . Even if, due to new reverse genetic techniques, less time for production of vaccines is needed, it still takes months before large quantities of vaccine are available [75] . During this time the human population should be protected to decelerate viral spread. At the moment the only available opportunities for personal protection are hygiene measures and the use of Tamiflu (brand name of Oseltamivir). Novel protection and treatment options for influenza are desperately needed. Based on our encouraging results in mice we suggest testing a nasal spray containing carrageenan in combination with the neuraminidase inhibitor Zanamivir in clinical trials for prevention or treatment of uncomplicated influenza infections. Supporting Information S1 Fig. Therapeutic efficacy of iota-carrageenan solely or together with kappa-carrageenan in influenza H7N7 lethal infected mice. Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and accordingly intranasally treated twice per day either with placebo or with iota-carrageenan or with a mixture of iota-and kappa-carrageenan. Treatment started 24 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated, uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant; non-significance (n.s.) was obtained with p-values >0.05. (TIFF)
What is Carrageenan?
false
2,141
{ "text": [ "a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections" ], "answer_start": [ 587 ] }
1,689
Chikungunya: A Potentially Emerging Epidemic? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860491/ SHA: f7c3160bef4169d29e2a8bdd79dd6e9056d4774c Authors: Thiboutot, Michelle M.; Kannan, Senthil; Kawalekar, Omkar U.; Shedlock, Devon J.; Khan, Amir S.; Sarangan, Gopalsamy; Srikanth, Padma; Weiner, David B.; Muthumani, Karuppiah Date: 2010-04-27 DOI: 10.1371/journal.pntd.0000623 License: cc-by Abstract: Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts. Text: Chikungunya virus (CHIKV), a mosquito-borne pathogen listed by National Institute of Allergy and Infectious Diseases (NIAID) as a Category C Priority Pathogen that causes Chikungunya fever (CHIKF), has been spreading throughout Asia, Africa, and parts of Europe in recent times [1, 2, 3] . CHIKV is an arthropod-borne virus (arbovirus) and is transmitted to humans primarily by Aedes aegypti, the infamous yellow fever propagator [4, 5] . CHIKV infection is marked by severe joint pain, contorting its victims into unusual postures [6] . The disease gets its name from the Kimakonde vernacular language of Tanzania and Mozambique, and the word chikungunya means ''that which contorts or bends up'' and translates in Swahili to ''the illness of the bended walker'' [7, 8, 9] . In Africa, CHIKV is maintained in a sylvatic cycle among forest-dwelling Aedes spp. mosquitoes, wild primates, squirrels, birds, and rodents ( Figure 1 ) [10] . In Asia, the disease is vectored by Ae. aegypti and Ae. albopictus [11] . Transmission in Asia occurs in an urban cycle whereby the mosquito spreads the disease from an infected human to an uninfected human, following an epidemiological pattern similar to dengue fever [12] . The 2005-2006 epidemic of CHIKV in La Reunion islands in the Indian Ocean, spurred the discovery of a new vector species, Ae. albopictus [5] . Wrecking over one-third of the island's population, this epidemic peaked its devastation between January and February 2006, when over 46,000 cases came into light every week, including 284 deaths [5, 13] . Ae. albopictus is common in urban areas of the United States and is already flourishing in 36 states, raising grave concerns to the immunologically naive populace of the United States [14] . Accordingly, this review elaborately details the epidemiology and global expansion of CHIKV, describes its clinical features and pathogenesis and its symptoms and complications, and finally nominates a possible vaccine approach against CHIKV infection. CHIKV has been isolated into three genotypes based on phylogenetic studies. These genotypes, based on the gene sequences of an Envelope protein (E1), are Asian, East/Central/ South African, and West African [4, 11, 15] . Using phylogenetic models, Cherian et al. estimate that the Asian genotype of CHIKV emerged between 50 and 310 y ago, and the West and East African genotypes diverged between 100 and 840 y ago [15] . Since then, CHIKV has come a long way, with several mutations incorporated, and has continued to wreak epidemics in several regions. Recent activities of CHIKV include the Indian epidemic in 2005-2006, which was followed by a sudden explosion of cases in 2007. An estimated 1.3 million people across 13 states were reported to be infected in India [12, 16] , and CHIKV was also widespread in Malaysia, Sri Lanka, and Indonesia [17] . In July-August of 2007, CHIKV was reported in Italy, probably brought in by travelers from CHIKV-prone regions of India, Africa, and Indian Ocean islands such as Mauritius, Madagascar, and Seychelles. Few of the Italian isolates were found to have evolved from the Kerala isolate, which was associated with a A226V shift in E1 gene that represents a successful evolutionary adaptation in the mosquito vector similar to the ones observed in Reunion Island [2, 18, 19] . In recent times, with an increase in global travel, the risk for spreading CHIKV to non-endemic regions has heightened [1] . Several travelers have brought CHIKV home with them after visiting areas with actively infected populations [12, 20] . Such cases have been documented in European countries, Australia, Asia, and the United States [8, 21] . The United States has already reported at least twelve cases of travel-associated CHIKV, while France has reported 850 cases, and the United Kingdom 93 [8, 14] . Beyond this, CHIKV-infected travelers have also been diagnosed in Australia, Belgium, Canada, Czech Republic, French Guiana, Germany, Hong Kong, Italy, Japan, Kenya, Malaysia, Martinique, Norway, Switzerland, and Sri Lanka [21] . Some travelers were viremic, worrying public health officials about the spread of CHIKV to new areas [1, 8] . The incubation time for CHIKV is relatively short, requiring only 2-6 d with symptoms usually appearing 4-7 d post-infection [22] . Vazeille et al. detected CHIKV in the salivary glands of Ae. albopictus only 2 d after infection [5] . Upon infection, CHIKF tends to present itself in two phases. The first stage is acute, while the second stage, experienced by most but not all, is persistent, causing disabling polyarthritis. Characteristics of the acute phase include an abrupt onset of fever, arthralgia, and in some cases, maculopapular rash [6, 23] . The acute phase causes such intense joint and muscular pain that makes movement very difficult and prostrates its victims [6, 20] . Ninety-five percent of infected adults are symptomatic after infection, and of these, most become disabled for weeks to months as a result of decreased dexterity, loss of mobility, and delayed reaction. Eighteen months after disease onset, 40% of patients are found to still have anti-CHIKV IgM [6, 18, 23, 24] . The chronic stage of CHIKF is characterized by polyarthralgia that can last from weeks to years beyond the acute stage [6] . CHIKV has been shown to attack fibroblasts, explaining the involvement of muscles, joints, and skin connective tissues. The high number of nociceptive nerve endings found within the joints and muscle connective tissues can explain pain associated with CHIKF [25, 26] . More than 50% of patients who suffer from severe CHIKF are over 65 y old, and more than 33% of them die. Most adults who suffer from severe CHIKF have underlying medical conditions [6, 24, 27] . The other group that is disproportionately affected by severe CHIKV is children. Other complications associated with CHIKV, from most common to least common, include respiratory failure, cardiovascular decompensation, meningoencephalitis, severe acute hepatitis, severe cutaneous effects, other central nervous system problems, and kidney failure [6, 18, 20, 23, 24, 26, 27] . CHIKV undertakes a complex replication cycle upon host infection (Figure 2 ), which makes its genome susceptible to mutations [28, 29] . For instance, Ae. aegypti, responsible for epidemics in Kenya, Comoros, and Seychelles, carried CHIKV with an alanine in the 226 position of the E1 gene (E1-A226) [4, 18] . However, when the virus struck La Reunion Islands, a decline in population of Ae. aegypti, due to massive dichlorodiphenyltrichloroethane usage and dearth of Ae. albopictus species' www.plosntds.org population, resulted in an ecological pressure, favoring replacement of alanine at position 226 with valine (E1-A226V) [5] . This mutation allowed CHIKV's secondary vector species, Ae. albopictus, to supplement Ae. aegypti as its primary vector [5] . Within a year, the E1-A226V mutation was present in La Reunion Island, and Ae. albopictus apparently vectored the large epidemic infecting 34% of La Reunion Island's population [5] . All of the CHIKV strains isolated from Mayotte carried the E1-A226V mutation, and the mutation was also found in Madagascar in 2007 [5] . The E1-A226V mutation was not present at the beginning of the Indian Ocean Islands outbreak (before September 2005). However, more than 90% of later viral strains found there had incorporated the mutation (December-March 2006), indicating a genotype switch during the winter season [5, 18, 20] . The E1-A226V mutation also enabled an increase in infectivity of Ae. albopictus when compared to its infectivity of Ae. aegypti [4, 11, 18, 30] , and with several factors taken together, Ae. albopictus has become the new preferred and more lethal vector for CHIKV [4, 5, 11] . In fact, Tsetsarkin et al. found that a Green Fluorescent Protein tagged E1-A226V virus was 100 times more infective to Ae. albopictus than it was to Ae. aegypti [4] . In all the Indian Ocean Islands, Ae. albopictus became the main vector for CHIKV within 1-2 y after CHIKV was introduced to the region [31] . Of note is that Ae. aegypti has most likely been established in North America for over 300 y, while Ae. albopictus has been in many areas of the US, since 1985, primarily in Florida [32] and since then has expanded its range in the country. Reiskind et al. set out to determine if Ae. aegypti and Ae. albopictus mosquitoes captured in Florida were susceptible to CHIKV infection by a La Reunion isolate [32] . Each mosquito tested was highly susceptible to infection by a full-length infectious clone of the La Réunion Island isolate, CHIKV LR2006 OPY1 strain. Even though the Ae. albopictus strains were more susceptible to infection, overall ecology and differences in human biting patterns need to be studied further Characteristically, there are two rounds of translation: (+) sense genomic RNA (49S9 = 11.7 kb) acts directly as mRNA and is partially translated (59 end) to produce non-structural proteins (nsp's). These proteins are responsible for replication and formation of a complementary (2) strand, the template for further (+) strand synthesis. Subgenomic mRNA (26 S = 4.1 kb) replication occurs through the synthesis of full-length (2) intermediate RNA, which is regulated by nsp4 and p123 precursor in early infection and later by mature nsp's. Translation of the newly synthesized sub-genomic RNA results in production of structural proteins such as Capsid and protein E2-6k-E1 (from 39 end of genome). Assembly occurs at the cell surface, and the envelope is acquired as the virus buds from the cell and release and maturation almost simultaneous occurred. Replication occurs in the cytoplasm and is very rapid (,4 h) [28, 29] . doi:10.1371/journal.pntd.0000623.g002 www.plosntds.org to gain a more accurate understanding of a potential CHIKV epidemic in the US [32] . During the 7 d preceding birth, no human mother has been reported to transmit the disease vertically. However, about 50% of newborns delivered while the mother was infected with CHIKV contracted the disease from their mother, despite the method of delivery. Furthermore, there have been instances of CHIKV transmission from mother to fetus causing congenital illness and fetal death [33] . During the 2005-2006 La Reunion Island outbreaks, Ramful et al. discovered that mothers could transmit CHIKV to their progeny during the perinatal period (Day 24 to Day +1) [33, 34] , and it is associated with a high degree of morbidity. By mean Day 4 of life, all of the neonates were symptomatic for CHIKV, exhibiting common CHIKF symptoms. Six neonates were confirmed to have contracted CHIKV and developed mengoencephalitis. Of those mothers who, during the La Reunion Island epidemic, were infected long before delivery, only three fetal deaths were reported [12, 33] . Ramful et al. theorized that motherto-child transmission most likely happens transplacentally shortly before delivery [33] . A similar study by Gerardin et al. reported nineteen cases of neonatal infection associated with intrapartum maternal viremia that progressed to develop encephalitis owing to vertical transmission from infected mothers [34] . Clinical and epidemiological similarities with dengue fever make CHIKV diagnosis difficult, which may lead physicians to misdiagnose CHIKV as dengue fever; therefore, the incidence of CHIKV may actually be higher than currently believed (Table 1 ) [6, 12, 35] . The amount of time elapsed since disease onset is the most critical parameter when choosing a diagnostic test. CHIKV can be detected and isolated by culturing with mosquito cells (C6/36), Vero cells (mammalian), or in mice [26] . However, this method can take at least a week and only achieves a high sensitivity during the viremic phase, which usually only lasts up to 48 h after the bite. Five days post-infection, the viral isolation approach has a low sensitivity but is still the preferred method for detecting the CHIKV strain [12, 26, 31, 35] . RT-PCR on the other hand is a faster and more sensitive method that can be used within the first week of disease onset [26] , and it is currently the most sensitive method for detecting and quantifying viral mRNA [4, 36] . Classic serological detection, by assays such as ELISA [37] , immunofluorescence [5, 38] , complement binding, and haemagglutination inhibition [39] , constitutes the second diagnostic tool used for biological diagnosis of CHIKV infection. These proven techniques are useful for detection of Antigen in mosquitoes during epidemiological studies. These assays detect virus-specific IgM and IgG, however the sensitivity and specificity of these assays has been poorly characterized. Viral competence, or the potential of viral infection and transmission, is an important parameter that can be quantified by ELISA, viral culture, and PCR. A study by Ng et al. showed biomarkers indicative of severe CHIKV infection [40] . They found decreased levels of RANTES and increased levels of Interleukin-6 (IL-6) and Interleukin-1b (IL-1b) that could be sued for CHIKV detection in patients as indicators of CHIKV-driven cytokine storm. Couderc et al. demonstrate another cytokine, type-I IFN, as a key player in the progression to CHIKV infection [26] . Using an IFN-a/b null mouse model, they demonstrated evidence of muscles, joints, and skin as privileged CHIKV targets, which is consistent with human pathology. Although Ng et al. concluded that RANTES levels were significantly suppressed in severe CHIKF patients [40] , interestingly, an increase in levels of RANTES has been observed in dengue infection [41] . Since the symptoms of CHIKF mimic those of dengue fever, results obtained from this study strongly suggest that RANTES could be a potential distinctive biomarker that differentiates between these two clinically similar diseases. There are no approved antiviral treatments currently available for CHIKV [1, 3, 12, 42] . Currently, CHIKF is treated symptomatically, usually with non-steroidal anti-inflammatory drugs or steroids, bed rest, and fluids. Movement and mild exercise are thought to decrease stiffness and morning arthralgia, but heavy exercise may exacerbate rheumatic symptoms. Corticosteroids may be used in cases of debilitating chronic CHIKV infection. There is a debate about the appropriateness of chloroquine as treatment for unresolved, non-steroidal anti-inflammatory drugresistant arthritis [43] . A study showed that viral production was www.plosntds.org drastically reduced at 16 h post-infection after treatment with 100 mM dec-RVKR-cmk (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone), a furine inhibitor [42, 44] . Chloroquine acted by raising the pH, blocking low pH-dependent entry of virus into the cell. It is important to note that dec-RVKR-cmk or chloroquine only inhibited viral spreading from cell to cell, not CHIKV replication once it had entered the cell [43] . However, most would agree that the best weapon against CHIKV is prevention. A live CHIKV vaccine developed by the United States reached phase II clinical trial encompassing 59 healthy volunteers [45] . Eight percent of the volunteers experienced transient arthralgia, while 98% of the volunteers had seroconversion [45] . However, live CHIKV vaccines are still questionable. One cannot discount the risk of a live vaccine possibly inducing chronic rheumatism. Also, there is the question as to whether widespread use among the public could trigger mosquito transmission or lead to chronic infection or viral reversion [1] . An alternative approach would be to produce a chimeric vaccine against CHIKV. Wang et al. developed a chimeric alphavirus vaccine that is uniformly attenuated and does not cause reactogenicity in mice [3] . Three different versions of this vaccine were made using three different backbone vectors: Venezuelan equine encephalitis virus (VEEV) attenuated vaccine strain T-83, naturally attenuated eastern equine encephalitis virus (EEEV), and attenuated Sindbis virus (SINV). In short, CHIKV structural proteins were engineered into the backbones of the aforementioned vaccines to produce the chimeras [3] . These chimeras were found to stimulate a strong humoral immunity, and even at doses of 5.3-5.8 log 10 PFU, they did not trigger reactogenicity. When vaccinated mice were challenged with CHIKV, neither adult nor neonatal mice gained weight, had fever, or displayed signs of neurological illness. Upon comparison of the chimeras with the Army181/25 vaccine, the Army vaccine resulted in higher levels of viremia and replication in the joints of neonatal mice. Because the joints are known targets of CHIKV, Wang et al. noted their vaccine might avoid the negative reactogenic side effects of the Army vaccine. After being subcutaneously vaccinated with 5.3-5.8 log 10 PFU of the chimeric vaccines, mice produced strong neutralizing antibody titers. The VEEV and EEEV chimeras yielded higher neutralizing antibody titers than the SINV chimera without being more virulent. On top of this, the VEEV and EEEV CHIKV chimeras seemed to be more immunogenic than the Army vaccine despite the chimeras' lower viremia and replication in the joints of neonatal mice [3] . Tiwari et al. [46] adopted a different strategy using formalin inactivated CHIKV in combination with alhydrogel (Aluminum Hydroxide) as an adjuvant. This study clearly suggests that this vaccine elicits both humoral and cell-mediated immune responses in mice, providing its immunogenic potential. A recent study by Couderc et al. [47] showed passive immunization as a potential treatment for CHIKV infection. Using purified immunoglobulin extracted from convalescent CHIKV patients, they demonstrated effective neutralizing activity against CHIKV infection both in vitro and in vivo. This thereby establishes a potential preventive and therapeutic approach to combat CHIKV infection. Pathogenesis studies conducted with related alpha virus, like RRV, have shown the role of macrophages in persistence on infection [48] . They also demonstrated the role of RRV-specific CD8 T cells in clearing viral load in infected patients, thereby warranting similar investigations with CHIKV and the importance of investigating a cell-mediated immune response-based vaccine against CHIKV [49] . There are always certain risks associated with live attenuated or inactivated viral vaccines [50] . One way to avoid these potential problems is to construct a consensus-based DNA vaccine. DNA based vaccines have an improved safety profile as compared to live or attenuated vaccines [51, 52] . A consequence of CHIKV's rapid evolution is difficulty in constructing a vaccine that will be able to Figure 3 . Levels of CHIKV-specific IgG in mice immunized with CHIKV vaccines. Each group of C57BL/6 mice (n = 5) was immunized with 12.5 mg of pVax1 control vector or CHIKV vaccine plasmids as indicated at 0 and 2 wk. Mice were bled 2 wk after each immunization, and each group's serum pool was diluted to 1:100 and 1:500 for reaction with specific vaccine constructs. Serum was incubated for 1 h at 37uC on 96-well plates coated with 2 mg/ml of respective CHIKV peptides, and antibody was detected using anti-mouse IgG-HRP and OD was measured at 405 nm. doi:10.1371/journal.pntd.0000623.g003 www.plosntds.org effectively protect large populations from multiple strains of the virus. One of the strengths of DNA consensus vaccines is its ability to induce cross-reactive immune responses against the three distinct phylogenetic groups of CHIKV. Also DNA-based vaccines can be produced more rapidly than protein-based vaccines. Recently, Muthumani et al. constructed a vaccine that was shown to induce both humoral and cellular immunity in vivo in 3-4-wk-old female C57/BL6 mice [49] . These mice were immunized using an in vivo electroporation method to deliver the vaccine into the quadriceps muscle. The consensus construct was designed against E1, E2, and the core protein capsid. To design the construct, they aligned 21 sequences of CHIKV isolated between 1952 and 2006, using strains from differing countries, including La Reunion Island. The most common nucleotide among the sequences was chosen at each position to be used in the consensus construct, taking care not to alter the reading frame. They conducted codon and RNA optimization, added a strong Kozak sequence, and substituted signal peptide with an immunoglobulin E leader sequence to improve vaccine efficacy. After immunizing the mice, spleens were harvested along with serum and tested to determine antibody titer. After three immunizations, consensus E1, E2, and C vaccines were shown to induce T-cell immune responses leading to strong IFN-c responses and proliferation in C57/BL6 mice. Furthermore, when compared with control mice, immunized mice had higher total IgG levels as well as higher anti-E1 specific, anti-E2 specific, and anti-C specific IgG antibodies, suggesting a strong humoral immune response ( Figure 3 ) and also specificity for the antigens encoded in the vaccine constructs ( Figure 4 ). Because of its promising results and the need for a safer vaccine, this consensus DNA vaccine deserves further investigation. Determining longevity of protective effects of the vaccine and persistence of antibody and IFN-c responses could be the next step of investigation. Challenged studies of immunized mice must also be carried out. CHIKV mosquito-borne disease has caused massive outbreaks for at least half a century but is no longer confined to the www.plosntds.org developing nations. It began to encroach into the boundaries of the developing world. As a result, the NIAID has designated CHIKV as a Category C pathogen alongside the influenza and SARS-CoV viruses [3] . Realization of the potential severity of this disease is exigent; for instance, if used as a biological weapon, the world economy could be severely crippled; if enough members of the armed forces were to become infected during a military deployment, military operations could be significantly affected. Efforts to monitor the disease will only provide minimal warning in a global society, and steps to prevent the morbidity and mortality associated with pandemic are imperative [21, 31] . Despite the gravity of its infectious potency and the fear of it being a potential biological weapon, there is currently no vaccine for CHIKV infections. Live attenuated vaccine trials were carried out in 2000, but funding for the project was discontinued. Newer approaches such as DNA vaccines appear promising over conventional strategies like live attenuated or inactivated virus and thus call for further investigation. Recent advances such electroporation delivery and incorporation of adjuvants has boosted DNA vaccine efficacy [51, 53] . Despite the low antibody response to DNA vaccines, other numerous advantages have overshadowed these minor drawbacks (Table 2) , the most important one being the ability to induce both humoral and cellular immune responses [51, 54] . Judging by recent success, such as the immunogenic construct developed by Muthumani et al., DNA vaccines could play a major role in combating CHIKV [49] . Vaccines are literally a critical component of CHIKV disease control and therefore research in this area is highly encouraged. The dramatic spread of dengue viruses (DENV) throughout tropical America since 1980 via the same vectors and human hosts underscores the risk to public health in the Americas. The adverse events associated with the current live vaccine are well documented [55] . Realizing these drawbacks, earnest efforts should be taken to develop new strategies to forestall further spread and complications.
In the epidemic peak how many cases per week were there on the island?
false
2,485
{ "text": [ "46,000" ], "answer_start": [ 2683 ] }
2,669
Frontiers in antiviral therapy and immunotherapy https://doi.org/10.1002/cti2.1115 SHA: facbfdfa7189ca9ff83dc30e5d241ab22e962dbf Authors: Heaton, Steven M Date: 2020 DOI: 10.1002/cti2.1115 License: cc-by Abstract: nan Text: Globally, recent decades have witnessed a growing disjunction, a 'Valley of Death' 1,2 no less, between broadening strides in fundamental biomedical research and their incommensurate reach into the clinic. Plumbing work on research funding and development pipelines through recent changes in the structure of government funding, 2 new public and private joint ventures and specialist undergraduate and postgraduate courses now aim to incorporate pathways to translation at the earliest stages. Reflecting this shift, the number of biomedical research publications targeting 'translational' concepts has increased exponentially, up 1800% between 2003 and 2014 3 and continuing to rise rapidly up to the present day. Fuelled by the availability of new research technologies, as well as changing disease, cost and other pressing issues of our time, further growth in this exciting space will undoubtedly continue. Despite recent advances in the therapeutic control of immune function and viral infection, current therapies are often challenging to develop, expensive to deploy and readily select for resistance-conferring mutants. Shaped by the hostvirus immunological 'arms race' and tempered in the forge of deep time, the biodiversity of our world is increasingly being harnessed for new biotechnologies and therapeutics. Simultaneously, a shift towards host-oriented antiviral therapies is currently underway. In this Clinical & Translational Immunology Special Feature, I illustrate a strategic vision integrating these themes to create new, effective, economical and robust antiviral therapies and immunotherapies, with both the realities and the opportunities afforded to researchers working in our changing world squarely in mind. Opening this CTI Special Feature, I outline ways these issues may be solved by creatively leveraging the so-called 'strengths' of viruses. Viral RNA polymerisation and reverse transcription enable resistance to treatment by conferring extraordinary genetic diversity. However, these exact processes ultimately restrict viral infectivity by strongly limiting virus genome sizes and their incorporation of new information. I coin this evolutionary dilemma the 'information economy paradox'. Many viruses attempt to resolve this by manipulating multifunctional or multitasking host cell proteins (MMHPs), thereby maximising host subversion and viral infectivity at minimal informational cost. 4 I argue this exposes an 'Achilles Heel' that may be safely targeted via host-oriented therapies to impose devastating informational and fitness barriers on escape mutant selection. Furthermore, since MMHPs are often conserved targets within and between virus families, MMHP-targeting therapies may exhibit both robust and broadspectrum antiviral efficacy. Achieving this through drug repurposing will break the vicious cycle of escalating therapeutic development costs and trivial escape mutant selection, both quickly and in multiple places. I also discuss alternative posttranslational and RNA-based antiviral approaches, designer vaccines, immunotherapy and the emerging field of neo-virology. 4 I anticipate international efforts in these areas over the coming decade will enable the tapping of useful new biological functions and processes, methods for controlling infection, and the deployment of symbiotic or subclinical viruses in new therapies and biotechnologies that are so crucially needed. Upon infection, pathogens stimulate expression of numerous host inflammatory factors that support recruitment and activation of immune cells. On the flip side, this same process also causes immunopathology when prolonged or deregulated. 5 In their contribution to this Special Feature, Yoshinaga and Takeuchi review endogenous RNA-binding proteins (RBPs) that post-transcriptionally control expression of crucial inflammatory factors in various tissues and their potential therapeutic applications. 6 These RBPs include tristetraprolin and AUF1, which promote degradation of AU-rich element (ARE)-containing mRNA; members of the Roquin and Regnase families, which respectively promote or effect degradation of mRNAs harbouring stem-loop structures; and the increasingly apparent role of the RNA methylation machinery in controlling inflammatory mRNA stability. These activities take place in various subcellular compartments and are differentially regulated during infection. In this way, mRNA-destabilising RBPs constitute a 'brake' on the immune system, which may ultimately be toggled therapeutically. I anticipate continued efforts in this area will lead to new methods of regaining control over inflammation in autoimmunity, selectively enhancing immunity in immunotherapy, and modulating RNA synthesis and virus replication during infection. Another mRNA under post-transcriptional regulation by Regnase-1 and Roquin is Furin, which encodes a conserved proprotein convertase crucial in human health and disease. Furin, along with other PCSK family members, is widely implicated in immune regulation, cancer and the entry, maturation or release of a broad array of evolutionarily diverse viruses including human papillomavirus (HPV), influenza (IAV), Ebola (EboV), dengue (DenV) and human immunodeficiency virus (HIV). Here, Braun and Sauter review the roles of furin in these processes, as well as the history and future of furin-targeting therapeutics. 7 They also discuss their recent work revealing how two IFN-cinducible factors exhibit broad-spectrum inhibition of IAV, measles (MV), zika (ZikV) and HIV by suppressing furin activity. 8 Over the coming decade, I expect to see an ever-finer spatiotemporal resolution of host-oriented therapies to achieve safe, effective and broad-spectrum yet costeffective therapies for clinical use. The increasing abundance of affordable, sensitive, high-throughput genome sequencing technologies has led to a recent boom in metagenomics and the cataloguing of the microbiome of our world. The MinION nanopore sequencer is one of the latest innovations in this space, enabling direct sequencing in a miniature form factor with only minimal sample preparation and a consumer-grade laptop computer. Nakagawa and colleagues here report on their latest experiments using this system, further improving its performance for use in resource-poor contexts for meningitis diagnoses. 9 While direct sequencing of viral genomic RNA is challenging, this system was recently used to directly sequence an RNA virus genome (IAV) for the first time. 10 I anticipate further improvements in the performance of such devices over the coming decade will transform virus surveillance efforts, the importance of which was underscored by the recent EboV and novel coronavirus (nCoV / COVID-19) outbreaks, enabling rapid deployment of antiviral treatments that take resistance-conferring mutations into account. Decades of basic immunology research have provided a near-complete picture of the main armaments in the human antiviral arsenal. Nevertheless, this focus on mammalian defences and pathologies has sidelined examination of the types and roles of viruses and antiviral defences that exist throughout our biosphere. One case in point is the CRISPR/Cas antiviral immune system of prokaryotes, which is now repurposed as a revolutionary gene-editing biotechnology in plants and animals. 11 Another is the ancient lineage of nucleocytosolic large DNA viruses (NCLDVs), which are emerging human pathogens that possess enormous genomes of up to several megabases in size encoding hundreds of proteins with unique and unknown functions. 12 Moreover, hundreds of human-and avian-infective viruses such as IAV strain H5N1 are known, but recent efforts indicate the true number may be in the millions and many harbour zoonotic potential. 13 It is increasingly clear that host-virus interactions have generated truly vast yet poorly understood and untapped biodiversity. Closing this Special Feature, Watanabe and Kawaoka elaborate on neo-virology, an emerging field engaged in cataloguing and characterising this biodiversity through a global consortium. 14 I predict these efforts will unlock a vast wealth of currently unexplored biodiversity, leading to biotechnologies and treatments that leverage the host-virus interactions developed throughout evolution. When biomedical innovations fall into the 'Valley of Death', patients who are therefore not reached all too often fall with them. Being entrusted with the resources and expectation to conceive, deliver and communicate dividends to society is both cherished and eagerly pursued at every stage of our careers. Nevertheless, the road to research translation is winding and is built on a foundation of basic research. Supporting industry-academia collaboration and nurturing talent and skills in the Indo-Pacific region are two of the four pillars of the National Innovation and Science Agenda. 2 These frame Australia's Medical Research and Innovation Priorities, which include antimicrobial resistance, global health and health security, drug repurposing and translational research infrastructure, 15 capturing many of the key elements of this CTI Special Feature. Establishing durable international relationships that integrate diverse expertise is essential to delivering these outcomes. To this end, NHMRC has recently taken steps under the International Engagement Strategy 16 to increase cooperation with its counterparts overseas. These include the Japan Agency for Medical Research and Development (AMED), tasked with translating the biomedical research output of that country. Given the reciprocal efforts at accelerating bilateral engagement currently underway, 17 the prospects for new areas of international cooperation and mobility have never been more exciting nor urgent. With the above in mind, all contributions to this CTI Special Feature I have selected from research presented by fellow invitees to the 2018 Awaji International Forum on Infection and Immunity (AIFII) and 2017 Consortium of Biological Sciences (ConBio) conferences in Japan. Both Australia and Japan have strong traditions in immunology and related disciplines, and I predict that the quantity, quality and importance of our bilateral cooperation will accelerate rapidly over the short to medium term. By expanding and cooperatively leveraging our respective research strengths, our efforts may yet solve the many pressing disease, cost and other sustainability issues of our time.
What is predicted these efforts on neo-virology will unlock?
false
4,168
{ "text": [ "a vast wealth of currently unexplored biodiversity, leading to biotechnologies and treatments that leverage the host-virus interactions developed throughout evolution." ], "answer_start": [ 8382 ] }
1,719
Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.
What can provide an improved vaccine regime?
false
1,652
{ "text": [ "Recombinant live-attenuated or replication-deficient influenza viruses" ], "answer_start": [ 42323 ] }
1,719
Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.
What did the clinical trial with CMV VRP show?
false
1,573
{ "text": [ "vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe" ], "answer_start": [ 18812 ] }
1,560
Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
What parameter is used to measure antioxidant levels?
false
2,140
{ "text": [ "superoxide dismutase" ], "answer_start": [ 11390 ] }
2,440
Optimization Method for Forecasting Confirmed Cases of COVID-19 in China https://doi.org/10.3390/jcm9030674 SHA: 1d7f8850c5244fdc9b387038e7eeae9bcbbde6d2 Authors: Al-Qaness, Mohammed A. A.; Ewees, Ahmed A.; Fan, Hong; Abd El Aziz, Mohamed Date: 2020 DOI: 10.3390/jcm9030674 License: cc-by Abstract: In December 2019, a novel coronavirus, called COVID-19, was discovered in Wuhan, China, and has spread to different cities in China as well as to 24 other countries. The number of confirmed cases is increasing daily and reached 34,598 on 8 February 2020. In the current study, we present a new forecasting model to estimate and forecast the number of confirmed cases of COVID-19 in the upcoming ten days based on the previously confirmed cases recorded in China. The proposed model is an improved adaptive neuro-fuzzy inference system (ANFIS) using an enhanced flower pollination algorithm (FPA) by using the salp swarm algorithm (SSA). In general, SSA is employed to improve FPA to avoid its drawbacks (i.e., getting trapped at the local optima). The main idea of the proposed model, called FPASSA-ANFIS, is to improve the performance of ANFIS by determining the parameters of ANFIS using FPASSA. The FPASSA-ANFIS model is evaluated using the World Health Organization (WHO) official data of the outbreak of the COVID-19 to forecast the confirmed cases of the upcoming ten days. More so, the FPASSA-ANFIS model is compared to several existing models, and it showed better performance in terms of Mean Absolute Percentage Error (MAPE), Root Mean Squared Relative Error (RMSRE), Root Mean Squared Relative Error (RMSRE), coefficient of determination ( R 2 ), and computing time. Furthermore, we tested the proposed model using two different datasets of weekly influenza confirmed cases in two countries, namely the USA and China. The outcomes also showed good performances. Text: A large family of viruses, called coronaviruses, are severe pathogens for human beings, which infect respiratory, hepatic, gastrointestinal, and neurologic diseases. They are distributed among humans, birds, livestock, mice, bats, and other wild animals [1] [2] [3] . The outbreaks of two previous coronaviruses, SARS-CoV and MERS-CoV in 2003 and 2012, respectively, have approved the transmission from animal to animal, and human to human [4] . In December 2019, the World Health Organization (WHO) received notifications from China for many cases of respiratory illness that were linked to some people who had visited a seafood market in Wuhan [5] . Currently, Wuhan city suffers from the spreading of a novel coronavirus, called COVID-19 (previously, it was called 2019-nCoV). In [6] , the authors concluded that COVID-19 likely originated in bats, because it is more similar to two bat-derived coronavirus strains. However, the source of the COVID-19 is not confirmed yet, and it communities, Hong Kong and Toronto, were 1.2 and 1.32, respectively. Ong et al. [20] proposed a monitoring and forecasting model for influenza A (H1N1-2009). Furthermore, Nah et al. [21] proposed a probability-based model to predict the spread of the MERS. The Adaptive Neuro-Fuzzy Inference System (ANFIS) [22] is widely applied in time series prediction and forecasting problems, and it showed good performance in many existing applications. It offers flexibility in determining nonlinearity in the time series data, as well as combining the properties of both artificial neural networks (ANN) and fuzzy logic systems. It has been applied in various forecasting applications, for example, in [23] , a stock price forecasting model was proposed using ANFIS and empirical mode decomposition. Chen et al. [24] proposed a TAIEX time series forecasting model based on a hybrid of ANFIS and ordered weighted averaging (OWA). In [25] , another time series forecasting method was presented for electricity prices based on ANFIS. Svalina et al. [26] proposed an ANFIS based forecasting model for close price indices for a stock market for five days. Ekici and Aksoy [27] presented an ANFIS based building energy consumption forecasting model. More so, ANFIS is also applied to forecast electricity loads [28] . Kumar et al. [29] proposed an ANFIS based model to forecast return products. Ho and Tsai [30] applied ANFIS to forecast product development performance. However, estimating ANFIS parameters is a challenge that needs to be improved. Therefore, in previous studies, some individual swarm intelligence (SI) methods have been applied to the ANFIS parameters to enhance time series forecasting because these parameters have a significant effect on the performance of ANFIS. The SI methods include the particle swarm optimization (PSO) [31, 32] , social-spider optimization [33] , sine-cosine algorithm (SCA) [34] , and multi-verse optimizer (MVO) [35] . For example, in [34] SCA algorithm was applied to improve the ANFIS model to forecast oil consumption in three countries, namely, Canada, Germany, and Japan. In the same context, in [35] , The MVO algorithm was used to enhance the ANFIS model to forecast oil consumption in two countries. In addition, in [36] the PSO was used with ANFIS to predict biochar yield. However, individual SI algorithms may stock at local optima. Therefore, one solution is to apply hybrid SI algorithms to avoid this problem. In [37] , a hybrid of two SI algorithms, namely GA and SSA, was presented to improve the ANFIS model. The proposed new model called GA-SSA-ANFIS was applied to forecast crude oil prices for long-term time series data. However, the previously mentioned methods suffer from some limitations that can affect the performance of the forecasting output such as slow convergence and the ability to balance between exploration and exploitation phases can influence the quality of the final output. This motivated us to propose an alternative forecasting method dependent on the hybridization concept. This concept avoids the limitations of traditional SI techniques by combining the strengths of different techniques, and this produces new SI techniques that are better than traditional ones. In the current study, we propose an improved ANFIS model based on a modified flower pollination algorithm (FPA) using the salp swarm algorithm (SSA). The FPA is an optimization algorithm proposed by Yang [38] , which was inspired by the flow pollination process of the flowering plants. The FPA was employed in various optimization applications, for example to estimate solar PV parameter [39, 40] , solving sudoku puzzles [41] , feature selection [42] , antenna design [43] , and other applications [44] [45] [46] [47] . Moreover, SSA is also an optimization algorithm proposed by Mirjalili et al. [48] inspired by the behavior of salp chains. In recent years, the SSA was utilized to solve different optimization problems, such as feature selection [49, 50] , data classification [51] , image segmentation [52] , and others [53, 54] . The proposed method called FPASSA is a hybrid of FPA and SSA, in which the SSA is applied as a local search method for FPA. The proposed FPASSA starts by receiving the historical COVID-19 dataset. Then a set of solutions is generated where each of them represents the value for the parameters of the ANFIS model. Then the quality of each solution is calculated using the fitness value, and the solution that has the best fitness value is chosen to represent the best solution. Then the probability of each solution is computed. Then the current solution will be updated, either using global or local strategy in FPA. However, in the case of local strategy, the operators of SSA or FPA will be used according to the probability of the fitness value for each solution. The process of updating the solutions is repeated until reaching the stop condition, and the best parameter configurations are used to forecast the number of confirmed cases of COVID-19. The main contribution points of the current study are as follows: 1. We propose an efficient forecasting model to forecast the confirmed cases of the COVID-19 in China for the upcoming ten days based on previously confirmed cases. An improved ANFIS model is proposed using a modified FPA algorithm, using SSA. We compare the proposed model with the original ANFIS and existing modified ANFIS models, such as PSO, GA, ABC, and FPA. The rest of this study is organized as follows. The preliminaries of ANFIS, FPA, and SSA are described in Section 2. Section 3 presents the proposed FPASSA, and Section 4 presents the experimental setup and results. We conclude this study in Section 5. The principles of the ANFIS are given in this section. The ANFIS model links the fuzzy logic and neural networks [22] . It generates a mapping between the input and output by applying IF-THEN rules (it is also called Takagi-Sugeno inference model). Figure 1 illustrates the ANFIS model where, y and x define the inputs to Layer 1 whereas, O 1i is its output of node i that is computed as follows: where µ denotes the generalized Gaussian membership functions. A i and B i define the membership values of µ. α i and ρ i denote the premise parameters set. The output of Layer 2 (it is also known as the firing strength of a rule) is calculated as follows: Meanwhile, the output of Layer 3 (it is also known as the normalized firing strength) is calculated as follows: The output of Layer 4 (it is also known as an adaptive node) is calculated as follows: where r i , q i , and p i define the consequent parameters of the node i. Layer 5 contains only one node; its output is computed as: Flower Pollination Algorithm is an optimization method proposed by Yang [38] . It simulates the transfer of flowers' pollen by pollinators in nature. This algorithm utilizes the two types of pollination (i.e., self-pollination and cross-pollination). In self-pollination, the pollination occurs with no pollinators, whereas, in cross-pollination, the pollens are moved between different plants. In more detail, the self-pollination can be represented as a local pollination while the cross-pollination can be called global pollination. The global pollination or cross-pollination can be mathematically formed as follows: where x t i defines the pollen i at iteration t. L denotes the pollination's strength or the step size. F * is the target position or best solution. In some cases, insects can fly with different distance steps for a long space; therefore, Levy fly distribution is applied to simulate this movement. where λ = 1.5. Γ(λ) denotes the gamma function. This distribution is available for large steps s > 0. The self-pollination or local pollination can be mathematically formed as follows: where x t i and x k i represent pollens from different flower in the same plant. in the range [0,1] The process of pollination can be done using cross-pollination or self-pollination. Therefore, the random variable p, in the range [0, 1], is used to determine this process. SSA is an optimization technique introduced by [48] . It simulates the Salps' behavior in nature. This behavior is called salp chain. The mathematical model of SSA begins by splinting its population into a leader group and followers group. The leader is the front salp, whereas, the followers are the other salps. The search space is determined in n-dimensions with n variables. Equation (10) works to update the salps' positions. where x 1 j denotes the leader's position in j-th dimension. F j is the target position. ub j and lb j represent the max and min bounds, respectively. c 2 and c 3 denote random numbers in [0, 1]. c 1 is an important parameter; it balances between the exploration and exploitation phases. It is computed as follows: where the current loop number is t and the max loop' number is t max . Then, the followers' position is updated as follows: where x i j defines the i-th position of the follower in j-th dimension. i > 1. This section explains the proposed FPASSA-ANFIS method. It is a time series method for forecasting the confirmed cases of the COVID-19, as given in Figure 2 . The FPASSA-ANFIS utilizes the improved FPA to train the ANFIS model by optimizing its parameters. The FPASSA-ANFIS contains five layers as the classic ANFIS model. Layer 1 contains the input variables (the historical COVID-19 confirmed cases). Whereas Layer 5 produces the forecasted values. In the learning phase, the FPASSA is used to select the best weights between Layer 4 and Layer 5. The FPASSA-ANFIS starts by formatting the input data in a time series form. In our case, the autocorrelation function (ACF) was considered. ACF is one of the methods applied to find patterns in the data; it presents information about the correlation between points separated by various time lags. Therefore, in this paper, the variables with ACF greater than 0.2 are considered i.e., 5-lags. Besides, the training data contains 75% of the dataset, whereas the testing data contains 25% of them. The number of clusters is defined by the fuzzy c-mean (FCM) method to construct the ANFIS model. The parameters of the ANFIS model are prepared by the FPASSA algorithm. In the training phase, the calculation error (as in Equation (13)) between the real data and the predicted data is used to evaluate the parameters' quality. where T is the real data, and P is the predicted data. N s is the sample length. The smaller values of the objective function indicate good ANFIS's parameter. On the other hand, the updating phase of the followers' positions in the SSA algorithm is applied to improve the global pollination phase in the FPA algorithm. In this improvement, there is a random variable (r) used to switch between both phases. If r > 0.5, then the operators of the SSA is used; otherwise, the operators of the FPA are used. In general, The FPASSA starts by constructing the population (X); afterward, the objective function is calculated for each solution. The solution with the lowest error value is saved to the next iteration. This sequence is repeated until meeting the stop condition, which in this paper, is the maximum number of iterations. Then the best solution is passed to train the parameters of the ANFIS model. After finishing the training phase, the testing phase is started with the best solution to compute the final output. The performance of the proposed method is evaluated by comparing the real data with the predicted data using the performance measures. Finally, the FPASSA produces a foretasted value for confirmed cases of COVID-19 in China in the next day. The steps of the proposed FPASSA are presented in Algorithm 1. Input: Historical COVID-19 dataset, size of population N, total number of iterations t max . Divide the data into training and testing sets. Using Fuzzy c-mean method to determine the number of membership functions. Constructing the ANFIS network. Set the initial value for N solutions (X). Return the best solution that represents the best configuration for ANFIS. Apply the testing set to the best ANFIS model. Forecasting the COVID-19 for the next ten days. This section presents the description of the used dataset, the performance measures, the parameter setting for all methods, the experiment results, and discussions. The main dataset of this study is COVID-19 dataset. It was collected from the WHO website (https: //www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/). It contains the daily confirmed cases in China from 21 January 2020 to 18 February 2020, as shown in Table 1 . We used 75% from the dataset to train the model while the rest is used to test it. Moreover, we evaluated the performance of the proposed method using two datasets of weekly influenza confirmed cases. The first one is called DS1; it was collected from the Centers for Disease Control and Prevention (CDC) (https://www.cdc.gov/flu/weekly/). It starts from week number 40 in 2015 and continues until week number 6 in 2020. Whereas, the second one is called DS2. It was collected from the WHO website (https://www.who.int/influenza). It contains the data of weekly influenza confirmed cases in China from week number 1 in 2016 to week number 8 in 2020. The quality of the proposed method is evaluated using a set of performance metrics as follows: • Root Mean Square Error (RMSE): where Yp and Y are the predicted and original values, respectively. • Mean Absolute Error (MAE): • Mean Absolute Percentage Error (MAPE): • Root Mean Squared Relative Error (RMSRE): N s represents the sample size of the data. • Coefficient of Determination (R 2 ): where Y represents the average of Y. The lowest value of RMSE, MAE, MAPE, and RMSRE refers to the best method. The higher value of R 2 indicates better correlation for the method. This paper aims to assess the ability of the FPASSA to forecast the COVID-19 by comparing its performance with other methods, namely the ANFIS and the trained ANFIS models using PSO, GA, ABC, FPA, and FPASSA. The parameters' setting for these models is listed in Table 2 . The common parameters, such as population size, are set to 25 and 100 iterations are applied. Besides, each algorithm is performed for 30 independent runs to fair comparisons. The selected parameters are chosen because they produced good behavior in previous experiments, such as [34, 35, 55, 56] . Table 2 . Parameters' setting. Parameters Setting Max. epochs = 100, Error goal = 0, Initial step = 0.01, Decrease rate = 0.9, Increase rate = 1. In this section, the performance of the proposed FPASSA to predict the DS1 and DS2 is discussed. It can be concluded from Table 3 that the performance of FPASSA outperformed the compared methods in all measures, whereas the FPA is ranked second. The results of DS2 indicate that the FPASSA is ranked first in terms of RMSE, MAPE, R 2 , and the CPU time. Whereas, the PSO is ranked second, followed by the FPA, GA, then ABC. These results denote that the proposed method can optimize the parameters of the ANFIS model effectively and produce good results in terms of the performance measures. Comparison results between the proposed FPASSA and other models to forecast COVID-19 are given in Table 4 . It can be concluded that the FPASSA outperforms other models. For example, by analyzing the results of RMSE, MAE, MAPE, RMSRE, and CPU time(s) it can be observed that the FPASSA achieves the smallest value among the comparison algorithms, and this indicates the high quality of the FPASSA. Meanwhile, the FPA allocates the second rank, which provides better results than the rest of the methods. Moreover, the value of R 2 refers to the high correlation between the prediction obtained by the proposed FPASSA method and the original COVID-19, which has nearly 0.97. This can also be noticed from Figure 3 , which depicts the training of the algorithms using the historical data of the COVID-19 as well as their forecasting values for ten days. Table 5 depicts the forecasting value for the confirmed cases of the COVID-19 in China from 19/2/2020 to 28/2/2020. From these results, it can be noticed that the outbreak will reach its highest level on the day 28/2/2020. The average percentage of the increase over the forecasted period is 10%, the highest percentage is 12% on 28/2/2020, and the lowest percentage is 8.7% on 19/2/2020. From the previous results, it can be concluded that the proposed FPASSA-ANFIS has a high ability to forecast the COVID-19 dataset. These results avoid the limitations of traditional ANFIS because of the combination with the modified FPA method. Moreover, the operators of SSA are combined with the local strategy of FPA to enhance their exploitation ability. However, the time computational of the proposed FPASSA method still requires more improvements. This paper proposed a modified version for the flower pollination algorithm (FPA) using the salp swarm algorithm (SSA). This modified version, called FPASSA, is applied to improve the performance of the ANFIS through determining the optimal value for its parameters. The developed FPASSA-ANFIS model is applied as a forecasting technique for a novel coronavirus, called COVID-19, that was discovered in Wuhan, China at the end of last year and January of the current year. The proposed FPASSA-ANFIS model has a high ability to predict the number of confirmed cases within ten days. Besides, FPASSA-ANFIS outperforms other forecasting models in terms of RMSE, MAE, MAPE, RMSRE, and R 2 . Furthermore, two datasets of weekly influenza confirmed cases in the USA and China were used to evaluate the proposed method, and the evaluation outcomes showed its good performance. According to the promising results obtained by the proposed FPASSA-ANFIS, it can be applied in different forecasting applications.
In which applications has it been applied?
false
4,408
{ "text": [ "in various forecasting applications, for example, in [23] , a stock price forecasting model was proposed using ANFIS and empirical mode decomposition" ], "answer_start": [ 3509 ] }
2,504
Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.
What is this disease characterized by ?
false
3,862
{ "text": [ "airway inflammation leading to complications such as coughing, wheezing and shortness of breath." ], "answer_start": [ 2230 ] }
1,689
Chikungunya: A Potentially Emerging Epidemic? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860491/ SHA: f7c3160bef4169d29e2a8bdd79dd6e9056d4774c Authors: Thiboutot, Michelle M.; Kannan, Senthil; Kawalekar, Omkar U.; Shedlock, Devon J.; Khan, Amir S.; Sarangan, Gopalsamy; Srikanth, Padma; Weiner, David B.; Muthumani, Karuppiah Date: 2010-04-27 DOI: 10.1371/journal.pntd.0000623 License: cc-by Abstract: Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts. Text: Chikungunya virus (CHIKV), a mosquito-borne pathogen listed by National Institute of Allergy and Infectious Diseases (NIAID) as a Category C Priority Pathogen that causes Chikungunya fever (CHIKF), has been spreading throughout Asia, Africa, and parts of Europe in recent times [1, 2, 3] . CHIKV is an arthropod-borne virus (arbovirus) and is transmitted to humans primarily by Aedes aegypti, the infamous yellow fever propagator [4, 5] . CHIKV infection is marked by severe joint pain, contorting its victims into unusual postures [6] . The disease gets its name from the Kimakonde vernacular language of Tanzania and Mozambique, and the word chikungunya means ''that which contorts or bends up'' and translates in Swahili to ''the illness of the bended walker'' [7, 8, 9] . In Africa, CHIKV is maintained in a sylvatic cycle among forest-dwelling Aedes spp. mosquitoes, wild primates, squirrels, birds, and rodents ( Figure 1 ) [10] . In Asia, the disease is vectored by Ae. aegypti and Ae. albopictus [11] . Transmission in Asia occurs in an urban cycle whereby the mosquito spreads the disease from an infected human to an uninfected human, following an epidemiological pattern similar to dengue fever [12] . The 2005-2006 epidemic of CHIKV in La Reunion islands in the Indian Ocean, spurred the discovery of a new vector species, Ae. albopictus [5] . Wrecking over one-third of the island's population, this epidemic peaked its devastation between January and February 2006, when over 46,000 cases came into light every week, including 284 deaths [5, 13] . Ae. albopictus is common in urban areas of the United States and is already flourishing in 36 states, raising grave concerns to the immunologically naive populace of the United States [14] . Accordingly, this review elaborately details the epidemiology and global expansion of CHIKV, describes its clinical features and pathogenesis and its symptoms and complications, and finally nominates a possible vaccine approach against CHIKV infection. CHIKV has been isolated into three genotypes based on phylogenetic studies. These genotypes, based on the gene sequences of an Envelope protein (E1), are Asian, East/Central/ South African, and West African [4, 11, 15] . Using phylogenetic models, Cherian et al. estimate that the Asian genotype of CHIKV emerged between 50 and 310 y ago, and the West and East African genotypes diverged between 100 and 840 y ago [15] . Since then, CHIKV has come a long way, with several mutations incorporated, and has continued to wreak epidemics in several regions. Recent activities of CHIKV include the Indian epidemic in 2005-2006, which was followed by a sudden explosion of cases in 2007. An estimated 1.3 million people across 13 states were reported to be infected in India [12, 16] , and CHIKV was also widespread in Malaysia, Sri Lanka, and Indonesia [17] . In July-August of 2007, CHIKV was reported in Italy, probably brought in by travelers from CHIKV-prone regions of India, Africa, and Indian Ocean islands such as Mauritius, Madagascar, and Seychelles. Few of the Italian isolates were found to have evolved from the Kerala isolate, which was associated with a A226V shift in E1 gene that represents a successful evolutionary adaptation in the mosquito vector similar to the ones observed in Reunion Island [2, 18, 19] . In recent times, with an increase in global travel, the risk for spreading CHIKV to non-endemic regions has heightened [1] . Several travelers have brought CHIKV home with them after visiting areas with actively infected populations [12, 20] . Such cases have been documented in European countries, Australia, Asia, and the United States [8, 21] . The United States has already reported at least twelve cases of travel-associated CHIKV, while France has reported 850 cases, and the United Kingdom 93 [8, 14] . Beyond this, CHIKV-infected travelers have also been diagnosed in Australia, Belgium, Canada, Czech Republic, French Guiana, Germany, Hong Kong, Italy, Japan, Kenya, Malaysia, Martinique, Norway, Switzerland, and Sri Lanka [21] . Some travelers were viremic, worrying public health officials about the spread of CHIKV to new areas [1, 8] . The incubation time for CHIKV is relatively short, requiring only 2-6 d with symptoms usually appearing 4-7 d post-infection [22] . Vazeille et al. detected CHIKV in the salivary glands of Ae. albopictus only 2 d after infection [5] . Upon infection, CHIKF tends to present itself in two phases. The first stage is acute, while the second stage, experienced by most but not all, is persistent, causing disabling polyarthritis. Characteristics of the acute phase include an abrupt onset of fever, arthralgia, and in some cases, maculopapular rash [6, 23] . The acute phase causes such intense joint and muscular pain that makes movement very difficult and prostrates its victims [6, 20] . Ninety-five percent of infected adults are symptomatic after infection, and of these, most become disabled for weeks to months as a result of decreased dexterity, loss of mobility, and delayed reaction. Eighteen months after disease onset, 40% of patients are found to still have anti-CHIKV IgM [6, 18, 23, 24] . The chronic stage of CHIKF is characterized by polyarthralgia that can last from weeks to years beyond the acute stage [6] . CHIKV has been shown to attack fibroblasts, explaining the involvement of muscles, joints, and skin connective tissues. The high number of nociceptive nerve endings found within the joints and muscle connective tissues can explain pain associated with CHIKF [25, 26] . More than 50% of patients who suffer from severe CHIKF are over 65 y old, and more than 33% of them die. Most adults who suffer from severe CHIKF have underlying medical conditions [6, 24, 27] . The other group that is disproportionately affected by severe CHIKV is children. Other complications associated with CHIKV, from most common to least common, include respiratory failure, cardiovascular decompensation, meningoencephalitis, severe acute hepatitis, severe cutaneous effects, other central nervous system problems, and kidney failure [6, 18, 20, 23, 24, 26, 27] . CHIKV undertakes a complex replication cycle upon host infection (Figure 2 ), which makes its genome susceptible to mutations [28, 29] . For instance, Ae. aegypti, responsible for epidemics in Kenya, Comoros, and Seychelles, carried CHIKV with an alanine in the 226 position of the E1 gene (E1-A226) [4, 18] . However, when the virus struck La Reunion Islands, a decline in population of Ae. aegypti, due to massive dichlorodiphenyltrichloroethane usage and dearth of Ae. albopictus species' www.plosntds.org population, resulted in an ecological pressure, favoring replacement of alanine at position 226 with valine (E1-A226V) [5] . This mutation allowed CHIKV's secondary vector species, Ae. albopictus, to supplement Ae. aegypti as its primary vector [5] . Within a year, the E1-A226V mutation was present in La Reunion Island, and Ae. albopictus apparently vectored the large epidemic infecting 34% of La Reunion Island's population [5] . All of the CHIKV strains isolated from Mayotte carried the E1-A226V mutation, and the mutation was also found in Madagascar in 2007 [5] . The E1-A226V mutation was not present at the beginning of the Indian Ocean Islands outbreak (before September 2005). However, more than 90% of later viral strains found there had incorporated the mutation (December-March 2006), indicating a genotype switch during the winter season [5, 18, 20] . The E1-A226V mutation also enabled an increase in infectivity of Ae. albopictus when compared to its infectivity of Ae. aegypti [4, 11, 18, 30] , and with several factors taken together, Ae. albopictus has become the new preferred and more lethal vector for CHIKV [4, 5, 11] . In fact, Tsetsarkin et al. found that a Green Fluorescent Protein tagged E1-A226V virus was 100 times more infective to Ae. albopictus than it was to Ae. aegypti [4] . In all the Indian Ocean Islands, Ae. albopictus became the main vector for CHIKV within 1-2 y after CHIKV was introduced to the region [31] . Of note is that Ae. aegypti has most likely been established in North America for over 300 y, while Ae. albopictus has been in many areas of the US, since 1985, primarily in Florida [32] and since then has expanded its range in the country. Reiskind et al. set out to determine if Ae. aegypti and Ae. albopictus mosquitoes captured in Florida were susceptible to CHIKV infection by a La Reunion isolate [32] . Each mosquito tested was highly susceptible to infection by a full-length infectious clone of the La Réunion Island isolate, CHIKV LR2006 OPY1 strain. Even though the Ae. albopictus strains were more susceptible to infection, overall ecology and differences in human biting patterns need to be studied further Characteristically, there are two rounds of translation: (+) sense genomic RNA (49S9 = 11.7 kb) acts directly as mRNA and is partially translated (59 end) to produce non-structural proteins (nsp's). These proteins are responsible for replication and formation of a complementary (2) strand, the template for further (+) strand synthesis. Subgenomic mRNA (26 S = 4.1 kb) replication occurs through the synthesis of full-length (2) intermediate RNA, which is regulated by nsp4 and p123 precursor in early infection and later by mature nsp's. Translation of the newly synthesized sub-genomic RNA results in production of structural proteins such as Capsid and protein E2-6k-E1 (from 39 end of genome). Assembly occurs at the cell surface, and the envelope is acquired as the virus buds from the cell and release and maturation almost simultaneous occurred. Replication occurs in the cytoplasm and is very rapid (,4 h) [28, 29] . doi:10.1371/journal.pntd.0000623.g002 www.plosntds.org to gain a more accurate understanding of a potential CHIKV epidemic in the US [32] . During the 7 d preceding birth, no human mother has been reported to transmit the disease vertically. However, about 50% of newborns delivered while the mother was infected with CHIKV contracted the disease from their mother, despite the method of delivery. Furthermore, there have been instances of CHIKV transmission from mother to fetus causing congenital illness and fetal death [33] . During the 2005-2006 La Reunion Island outbreaks, Ramful et al. discovered that mothers could transmit CHIKV to their progeny during the perinatal period (Day 24 to Day +1) [33, 34] , and it is associated with a high degree of morbidity. By mean Day 4 of life, all of the neonates were symptomatic for CHIKV, exhibiting common CHIKF symptoms. Six neonates were confirmed to have contracted CHIKV and developed mengoencephalitis. Of those mothers who, during the La Reunion Island epidemic, were infected long before delivery, only three fetal deaths were reported [12, 33] . Ramful et al. theorized that motherto-child transmission most likely happens transplacentally shortly before delivery [33] . A similar study by Gerardin et al. reported nineteen cases of neonatal infection associated with intrapartum maternal viremia that progressed to develop encephalitis owing to vertical transmission from infected mothers [34] . Clinical and epidemiological similarities with dengue fever make CHIKV diagnosis difficult, which may lead physicians to misdiagnose CHIKV as dengue fever; therefore, the incidence of CHIKV may actually be higher than currently believed (Table 1 ) [6, 12, 35] . The amount of time elapsed since disease onset is the most critical parameter when choosing a diagnostic test. CHIKV can be detected and isolated by culturing with mosquito cells (C6/36), Vero cells (mammalian), or in mice [26] . However, this method can take at least a week and only achieves a high sensitivity during the viremic phase, which usually only lasts up to 48 h after the bite. Five days post-infection, the viral isolation approach has a low sensitivity but is still the preferred method for detecting the CHIKV strain [12, 26, 31, 35] . RT-PCR on the other hand is a faster and more sensitive method that can be used within the first week of disease onset [26] , and it is currently the most sensitive method for detecting and quantifying viral mRNA [4, 36] . Classic serological detection, by assays such as ELISA [37] , immunofluorescence [5, 38] , complement binding, and haemagglutination inhibition [39] , constitutes the second diagnostic tool used for biological diagnosis of CHIKV infection. These proven techniques are useful for detection of Antigen in mosquitoes during epidemiological studies. These assays detect virus-specific IgM and IgG, however the sensitivity and specificity of these assays has been poorly characterized. Viral competence, or the potential of viral infection and transmission, is an important parameter that can be quantified by ELISA, viral culture, and PCR. A study by Ng et al. showed biomarkers indicative of severe CHIKV infection [40] . They found decreased levels of RANTES and increased levels of Interleukin-6 (IL-6) and Interleukin-1b (IL-1b) that could be sued for CHIKV detection in patients as indicators of CHIKV-driven cytokine storm. Couderc et al. demonstrate another cytokine, type-I IFN, as a key player in the progression to CHIKV infection [26] . Using an IFN-a/b null mouse model, they demonstrated evidence of muscles, joints, and skin as privileged CHIKV targets, which is consistent with human pathology. Although Ng et al. concluded that RANTES levels were significantly suppressed in severe CHIKF patients [40] , interestingly, an increase in levels of RANTES has been observed in dengue infection [41] . Since the symptoms of CHIKF mimic those of dengue fever, results obtained from this study strongly suggest that RANTES could be a potential distinctive biomarker that differentiates between these two clinically similar diseases. There are no approved antiviral treatments currently available for CHIKV [1, 3, 12, 42] . Currently, CHIKF is treated symptomatically, usually with non-steroidal anti-inflammatory drugs or steroids, bed rest, and fluids. Movement and mild exercise are thought to decrease stiffness and morning arthralgia, but heavy exercise may exacerbate rheumatic symptoms. Corticosteroids may be used in cases of debilitating chronic CHIKV infection. There is a debate about the appropriateness of chloroquine as treatment for unresolved, non-steroidal anti-inflammatory drugresistant arthritis [43] . A study showed that viral production was www.plosntds.org drastically reduced at 16 h post-infection after treatment with 100 mM dec-RVKR-cmk (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone), a furine inhibitor [42, 44] . Chloroquine acted by raising the pH, blocking low pH-dependent entry of virus into the cell. It is important to note that dec-RVKR-cmk or chloroquine only inhibited viral spreading from cell to cell, not CHIKV replication once it had entered the cell [43] . However, most would agree that the best weapon against CHIKV is prevention. A live CHIKV vaccine developed by the United States reached phase II clinical trial encompassing 59 healthy volunteers [45] . Eight percent of the volunteers experienced transient arthralgia, while 98% of the volunteers had seroconversion [45] . However, live CHIKV vaccines are still questionable. One cannot discount the risk of a live vaccine possibly inducing chronic rheumatism. Also, there is the question as to whether widespread use among the public could trigger mosquito transmission or lead to chronic infection or viral reversion [1] . An alternative approach would be to produce a chimeric vaccine against CHIKV. Wang et al. developed a chimeric alphavirus vaccine that is uniformly attenuated and does not cause reactogenicity in mice [3] . Three different versions of this vaccine were made using three different backbone vectors: Venezuelan equine encephalitis virus (VEEV) attenuated vaccine strain T-83, naturally attenuated eastern equine encephalitis virus (EEEV), and attenuated Sindbis virus (SINV). In short, CHIKV structural proteins were engineered into the backbones of the aforementioned vaccines to produce the chimeras [3] . These chimeras were found to stimulate a strong humoral immunity, and even at doses of 5.3-5.8 log 10 PFU, they did not trigger reactogenicity. When vaccinated mice were challenged with CHIKV, neither adult nor neonatal mice gained weight, had fever, or displayed signs of neurological illness. Upon comparison of the chimeras with the Army181/25 vaccine, the Army vaccine resulted in higher levels of viremia and replication in the joints of neonatal mice. Because the joints are known targets of CHIKV, Wang et al. noted their vaccine might avoid the negative reactogenic side effects of the Army vaccine. After being subcutaneously vaccinated with 5.3-5.8 log 10 PFU of the chimeric vaccines, mice produced strong neutralizing antibody titers. The VEEV and EEEV chimeras yielded higher neutralizing antibody titers than the SINV chimera without being more virulent. On top of this, the VEEV and EEEV CHIKV chimeras seemed to be more immunogenic than the Army vaccine despite the chimeras' lower viremia and replication in the joints of neonatal mice [3] . Tiwari et al. [46] adopted a different strategy using formalin inactivated CHIKV in combination with alhydrogel (Aluminum Hydroxide) as an adjuvant. This study clearly suggests that this vaccine elicits both humoral and cell-mediated immune responses in mice, providing its immunogenic potential. A recent study by Couderc et al. [47] showed passive immunization as a potential treatment for CHIKV infection. Using purified immunoglobulin extracted from convalescent CHIKV patients, they demonstrated effective neutralizing activity against CHIKV infection both in vitro and in vivo. This thereby establishes a potential preventive and therapeutic approach to combat CHIKV infection. Pathogenesis studies conducted with related alpha virus, like RRV, have shown the role of macrophages in persistence on infection [48] . They also demonstrated the role of RRV-specific CD8 T cells in clearing viral load in infected patients, thereby warranting similar investigations with CHIKV and the importance of investigating a cell-mediated immune response-based vaccine against CHIKV [49] . There are always certain risks associated with live attenuated or inactivated viral vaccines [50] . One way to avoid these potential problems is to construct a consensus-based DNA vaccine. DNA based vaccines have an improved safety profile as compared to live or attenuated vaccines [51, 52] . A consequence of CHIKV's rapid evolution is difficulty in constructing a vaccine that will be able to Figure 3 . Levels of CHIKV-specific IgG in mice immunized with CHIKV vaccines. Each group of C57BL/6 mice (n = 5) was immunized with 12.5 mg of pVax1 control vector or CHIKV vaccine plasmids as indicated at 0 and 2 wk. Mice were bled 2 wk after each immunization, and each group's serum pool was diluted to 1:100 and 1:500 for reaction with specific vaccine constructs. Serum was incubated for 1 h at 37uC on 96-well plates coated with 2 mg/ml of respective CHIKV peptides, and antibody was detected using anti-mouse IgG-HRP and OD was measured at 405 nm. doi:10.1371/journal.pntd.0000623.g003 www.plosntds.org effectively protect large populations from multiple strains of the virus. One of the strengths of DNA consensus vaccines is its ability to induce cross-reactive immune responses against the three distinct phylogenetic groups of CHIKV. Also DNA-based vaccines can be produced more rapidly than protein-based vaccines. Recently, Muthumani et al. constructed a vaccine that was shown to induce both humoral and cellular immunity in vivo in 3-4-wk-old female C57/BL6 mice [49] . These mice were immunized using an in vivo electroporation method to deliver the vaccine into the quadriceps muscle. The consensus construct was designed against E1, E2, and the core protein capsid. To design the construct, they aligned 21 sequences of CHIKV isolated between 1952 and 2006, using strains from differing countries, including La Reunion Island. The most common nucleotide among the sequences was chosen at each position to be used in the consensus construct, taking care not to alter the reading frame. They conducted codon and RNA optimization, added a strong Kozak sequence, and substituted signal peptide with an immunoglobulin E leader sequence to improve vaccine efficacy. After immunizing the mice, spleens were harvested along with serum and tested to determine antibody titer. After three immunizations, consensus E1, E2, and C vaccines were shown to induce T-cell immune responses leading to strong IFN-c responses and proliferation in C57/BL6 mice. Furthermore, when compared with control mice, immunized mice had higher total IgG levels as well as higher anti-E1 specific, anti-E2 specific, and anti-C specific IgG antibodies, suggesting a strong humoral immune response ( Figure 3 ) and also specificity for the antigens encoded in the vaccine constructs ( Figure 4 ). Because of its promising results and the need for a safer vaccine, this consensus DNA vaccine deserves further investigation. Determining longevity of protective effects of the vaccine and persistence of antibody and IFN-c responses could be the next step of investigation. Challenged studies of immunized mice must also be carried out. CHIKV mosquito-borne disease has caused massive outbreaks for at least half a century but is no longer confined to the www.plosntds.org developing nations. It began to encroach into the boundaries of the developing world. As a result, the NIAID has designated CHIKV as a Category C pathogen alongside the influenza and SARS-CoV viruses [3] . Realization of the potential severity of this disease is exigent; for instance, if used as a biological weapon, the world economy could be severely crippled; if enough members of the armed forces were to become infected during a military deployment, military operations could be significantly affected. Efforts to monitor the disease will only provide minimal warning in a global society, and steps to prevent the morbidity and mortality associated with pandemic are imperative [21, 31] . Despite the gravity of its infectious potency and the fear of it being a potential biological weapon, there is currently no vaccine for CHIKV infections. Live attenuated vaccine trials were carried out in 2000, but funding for the project was discontinued. Newer approaches such as DNA vaccines appear promising over conventional strategies like live attenuated or inactivated virus and thus call for further investigation. Recent advances such electroporation delivery and incorporation of adjuvants has boosted DNA vaccine efficacy [51, 53] . Despite the low antibody response to DNA vaccines, other numerous advantages have overshadowed these minor drawbacks (Table 2) , the most important one being the ability to induce both humoral and cellular immune responses [51, 54] . Judging by recent success, such as the immunogenic construct developed by Muthumani et al., DNA vaccines could play a major role in combating CHIKV [49] . Vaccines are literally a critical component of CHIKV disease control and therefore research in this area is highly encouraged. The dramatic spread of dengue viruses (DENV) throughout tropical America since 1980 via the same vectors and human hosts underscores the risk to public health in the Americas. The adverse events associated with the current live vaccine are well documented [55] . Realizing these drawbacks, earnest efforts should be taken to develop new strategies to forestall further spread and complications.
What is CHIKV marked by?
false
2,477
{ "text": [ "severe joint pain, contorting its victims into unusual postures" ], "answer_start": [ 1660 ] }
2,642
First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, 24 January to 21 February 2020 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068164/ SHA: ce358c18aac69fc83c7b2e9a7dca4a43b0f60e2e Authors: Spiteri, Gianfranco; Fielding, James; Diercke, Michaela; Campese, Christine; Enouf, Vincent; Gaymard, Alexandre; Bella, Antonino; Sognamiglio, Paola; Sierra Moros, Maria José; Riutort, Antonio Nicolau; Demina, Yulia V.; Mahieu, Romain; Broas, Markku; Bengnér, Malin; Buda, Silke; Schilling, Julia; Filleul, Laurent; Lepoutre, Agnès; Saura, Christine; Mailles, Alexandra; Levy-Bruhl, Daniel; Coignard, Bruno; Bernard-Stoecklin, Sibylle; Behillil, Sylvie; van der Werf, Sylvie; Valette, Martine; Lina, Bruno; Riccardo, Flavia; Nicastri, Emanuele; Casas, Inmaculada; Larrauri, Amparo; Salom Castell, Magdalena; Pozo, Francisco; Maksyutov, Rinat A.; Martin, Charlotte; Van Ranst, Marc; Bossuyt, Nathalie; Siira, Lotta; Sane, Jussi; Tegmark-Wisell, Karin; Palmérus, Maria; Broberg, Eeva K.; Beauté, Julien; Jorgensen, Pernille; Bundle, Nick; Pereyaslov, Dmitriy; Adlhoch, Cornelia; Pukkila, Jukka; Pebody, Richard; Olsen, Sonja; Ciancio, Bruno Christian Date: 2020-03-05 DOI: 10.2807/1560-7917.es.2020.25.9.2000178 License: cc-by Abstract: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters’ index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. Text: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters' index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. A cluster of pneumonia of unknown origin was identified in Wuhan, China, in December 2019 [1] . On 12 January 2020, Chinese authorities shared the sequence of a novel coronavirus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from some clustered cases [2] . Since then, the disease caused by SARS-CoV-2 has been named coronavirus disease 2019 (COVID -19) . As at 21 February 2020, the virus had spread rapidly mostly within China but also to 28 other countries, including in the World Health Organization (WHO) European Region [3] [4] [5] . Here we describe the epidemiology of the first cases of COVID-19 in this region, excluding cases reported in the United Kingdom (UK), as at 21 February 2020. The study includes a comparison between cases detected among travellers from China and cases whose infection was acquired due to subsequent local transmission. On 27 January 2020, the European Centre for Disease Prevention and Control (ECDC) and the WHO Regional Office for Europe asked countries to complete a WHO standard COVID-19 case report form for all confirmed and probable cases according to WHO criteria [6] [7] [8] . The overall aim of surveillance at this time was to support the global strategy of containment of COVID-19 with rapid identification and follow-up of cases linked to affected countries in order to minimise onward transmission. The surveillance objectives were to: describe the key epidemiological and clinical characteristics of COVID-19 cases detected in Europe; inform country preparedness; and improve further case detection and management. Data collected included demographics, history of recent travel to affected areas, close contact with a probable or confirmed COVID-19 case, underlying conditions, signs and symptoms of disease at onset, type of specimens from which the virus was detected, and clinical outcome. The WHO case definition was adopted for surveillance: a confirmed case was a person with laboratory confirmation of SARS-CoV-2 infection (ECDC recommended two separate SARS-CoV-2 RT-PCR tests), irrespective of clinical signs and symptoms, whereas a probable case was a suspect case for whom testing for SARS-CoV-2 was inconclusive or positive using a pan-coronavirus assay [8] . By 31 January 2020, 47 laboratories in 31 countries, including 38 laboratories in 24 European Union and European Economic Area (EU/EEA) countries, had diagnostic capability for SARS-CoV-2 available (close to 60% of countries in the WHO European Region), with cross-border shipment arrangements in place for many of those lacking domestic testing capacity. The remaining six EU/EEA countries were expected to have diagnostic testing available by mid-February [9] . As at 09:00 on 21 February 2020, 47 confirmed cases of COVID-19 were reported in the WHO European Region and one of these cases had died [4] . Data on 38 of these cases (i.e. all except the nine reported in the UK) are included in this analysis. The first three cases detected were reported in France on 24 January 2020 and had onset of symptoms on 17, 19 and 23 January respectively [10] . The first death was reported on 15 February in France. As at 21 February, nine countries had reported cases ( Figure) : Belgium (1), Finland (1), France (12), Germany (16), Italy (3), Russia (2), Spain (2), Sweden (1) and the UK (9 -not included further). The place of infection (assessed at national level based on an incubation period presumed to be up to 14 days [11] , travel history and contact with probable or confirmed cases as per the case definition) was reported for 35 cases (missing for three cases), of whom 14 were infected in China (Hubei province: 10 cases; Shandong province: one case; province not reported for three cases). The remaining 21 cases were infected in Europe. Of these, 14 were linked to a cluster in Bavaria, Germany, and seven to a cluster in Haute-Savoie, France [12, 13] . Cases from the Bavarian cluster were reported from Germany and Spain, whereas cases from the Haute-Savoie cluster were reported from France All but two cases were hospitalised (35 of 37 where information on hospitalisation was reported), although it is likely that most were hospitalised to isolate the person rather than because of severe disease. The time from onset of symptoms to hospitalisation (and isolation) ranged between 0 and 10 days with a mean of 3.7 days (reported for 29 cases). The mean number of days to hospitalisation was 2.5 days for cases imported from China, but 4.6 days for those infected in Europe. This was mostly a result of delays in identifying the index cases of the two clusters in France and Germany. In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six took only a mean of 2 days to be hospitalised. Symptoms at the point of diagnosis were reported for 31 cases. Two cases were asymptomatic and remained so until tested negative. The asymptomatic cases were tested as part of screening following repatriation and during contact tracing respectively. Of the remaining 29, 20 reported fever, 14 reported cough and eight reported weakness. Additional symptoms reported included headaches (6 cases), sore throat (2), rhinorrhoea (2), shortness of breath (2), myalgia (1), diarrhoea (1) and nausea (1). Fever was reported as the sole symptom for nine cases. In 16 of 29 symptomatic cases, the symptoms at diagnosis were consistent with the case definition for acute respiratory infection [16] , although it is possible that cases presented additional symptoms after diagnosis and these were not reported. Data on pre-existing conditions were reported for seven cases; five had no pre-existing conditions while one was reported to be obese and one had pre-existing cardiac disease. No data on clinical signs e.g. dyspnea etc. were reported for any of the 38 cases. All hospitalised cases had a benign clinical evolution except four, two reported in Italy and two reported in France, all of whom developed viral pneumonia. All three cases who were aged 65 years or over were admitted to intensive care and required respiratory support and one French case died. The case who died was hospitalised for 21 days and required intensive care and mechanical ventilation for 19 days. The duration of hospitalisation was reported for 16 cases with a median of 13 days (range: 8-23 days). As at 21 February 2020, four cases were still hospitalised. All cases were confirmed according to specific assays targeting at least two separate genes (envelope (E) gene as a screening test and RNA-dependent RNA polymerase (RdRp) gene or nucleoprotein (N) gene for confirmation) [8, 17] . The specimen types tested were reported for 27 cases: 15 had positive nasopharyngeal swabs, nine had positive throat swabs, three cases had positive sputum, two had a positive nasal swab, one case had a positive nasopharyngeal aspirate and one a positive endotracheal aspirate. As at 09:00 on 21 February, few COVID-19 cases had been detected in Europe compared with Asia. However the situation is rapidly developing, with a large outbreak recently identified in northern Italy, with transmission in several municipalities and at least two deaths [18] . As at 5 March 2020, there are 4,250 cases including 113 deaths reported among 38 countries in the WHO European region [19] . In our analysis of early cases, we observed transmission in two broad contexts: sporadic cases among travellers from China (14 cases) and cases who acquired infection due to subsequent local transmission in Europe (21 cases). Our analysis shows that the time from symptom onset to hospitalisation/case isolation was about 3 days longer for locally acquired cases than for imported cases. People returning from affected areas are likely to have a low threshold to seek care and be tested when symptomatic, however delays in identifying the index cases of the two clusters in France and Germany meant that locally acquired cases took longer to be detected and isolated. Once the exposure is determined and contacts identified and quarantined (171 contacts in France and 200 in Germany for the clusters in Haute-Savoie and Bavaria, respectively), further cases are likely to be rapidly detected and isolated when they develop symptoms [15, 20] . In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six were hospitalised after a mean of 2 days. Locally acquired cases require significant resources for contact tracing and quarantine, and countries should be prepared to allocate considerable public health resources during the containment phase, should local clusters emerge in their population. In addition, prompt sharing of information on cases and contacts through international notification systems such as the International Health Regulations (IHR) mechanism and the European Commission's European Early Warning and Response System is essential to contain international spread of infection. All of the imported cases had a history of travel to China. This was consistent with the epidemiological situation in Asia, and supported the recommendation for testing of suspected cases with travel history to China and potentially other areas of presumed ongoing community transmission. The situation has evolved rapidly since then, however, and the number of countries reporting COVID-19 transmission increased rapidly, notably with a large outbreak in northern Italy with 3,089 cases reported as at 5 March [18, 19] . Testing of suspected cases based on geographical risk of importation needs to be complemented with additional approaches to ensure early detection of local circulation of COVID-19, including through testing of severe acute respiratory infections in hospitals irrespectively of travel history as recommended in the WHO case definition updated on 27 February 2020 [21] . The clinical presentation observed in the cases in Europe is that of an acute respiratory infection. However, of the 31 cases with information on symptoms, 20 cases presented with fever and nine cases presented only with fever and no other symptoms. These findings, which are consistent with other published case series, have prompted ECDC to include fever among several clinical signs or symptoms indicative for the suspected case definition. Three cases were aged 65 years or over. All required admission to intensive care and were tourists (imported cases). These findings could reflect the average older age of the tourist population compared with the local contacts exposed to infection in Europe and do not allow us to draw any conclusion on the proportion of severe cases that we could expect in the general population of Europe. Despite this, the finding of older individuals being at higher risk of a severe clinical course is consistent with the evidence from Chinese case series published so far although the majority of infections in China have been mild [22, 23] . This preliminary analysis is based on the first reported cases of COVID-19 cases in the WHO European Region. Given the small sample size, and limited completeness for some variables, all the results presented should be interpreted with caution. With increasing numbers of cases in Europe, data from surveillance and investigations in the region can build on the evidence from countries in Asia experiencing more widespread transmission particularly on disease spectrum and the proportion of infections with severe outcome [22] . Understanding the infection-severity is critical to help plan for the impact on the healthcare system and the wider population. Serological studies are vital to understand the proportion of cases who are asymptomatic. Hospital-based surveillance could help estimate the incidence of severe cases and identify risk factors for severity and death. Established hospital surveillance systems that are in place for influenza and other diseases in Europe may be expanded for this purpose. In addition, a number of countries in Europe are adapting and, in some cases, already using existing sentinel primary care based surveillance systems for influenza to detect community transmission of SARS-CoV-2. This approach will be used globally to help identify evidence of widespread community transmission and, should the virus spread and containment no longer be deemed feasible, to monitor intensity of disease transmission, trends and its geographical spread. Additional research is needed to complement surveillance data to build knowledge on the infectious period, modes of transmission, basic and effective reproduction numbers, and effectiveness of prevention and case management options also in settings outside of China. Such special studies are being conducted globally, including a cohort study on citizens repatriated from China to Europe, with the aim to extrapolate disease incidence and risk factors for infection in areas with community transmission. Countries together with ECDC and WHO, should use all opportunities to address these questions in a coordinated fashion at the European and global level. provided input to the outline, multiple versions of the manuscript and gave approval to the final draft.
How many cases were asymptomatic?
false
3,818
{ "text": [ "Two cases" ], "answer_start": [ 7088 ] }
1,689
Chikungunya: A Potentially Emerging Epidemic? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860491/ SHA: f7c3160bef4169d29e2a8bdd79dd6e9056d4774c Authors: Thiboutot, Michelle M.; Kannan, Senthil; Kawalekar, Omkar U.; Shedlock, Devon J.; Khan, Amir S.; Sarangan, Gopalsamy; Srikanth, Padma; Weiner, David B.; Muthumani, Karuppiah Date: 2010-04-27 DOI: 10.1371/journal.pntd.0000623 License: cc-by Abstract: Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts. Text: Chikungunya virus (CHIKV), a mosquito-borne pathogen listed by National Institute of Allergy and Infectious Diseases (NIAID) as a Category C Priority Pathogen that causes Chikungunya fever (CHIKF), has been spreading throughout Asia, Africa, and parts of Europe in recent times [1, 2, 3] . CHIKV is an arthropod-borne virus (arbovirus) and is transmitted to humans primarily by Aedes aegypti, the infamous yellow fever propagator [4, 5] . CHIKV infection is marked by severe joint pain, contorting its victims into unusual postures [6] . The disease gets its name from the Kimakonde vernacular language of Tanzania and Mozambique, and the word chikungunya means ''that which contorts or bends up'' and translates in Swahili to ''the illness of the bended walker'' [7, 8, 9] . In Africa, CHIKV is maintained in a sylvatic cycle among forest-dwelling Aedes spp. mosquitoes, wild primates, squirrels, birds, and rodents ( Figure 1 ) [10] . In Asia, the disease is vectored by Ae. aegypti and Ae. albopictus [11] . Transmission in Asia occurs in an urban cycle whereby the mosquito spreads the disease from an infected human to an uninfected human, following an epidemiological pattern similar to dengue fever [12] . The 2005-2006 epidemic of CHIKV in La Reunion islands in the Indian Ocean, spurred the discovery of a new vector species, Ae. albopictus [5] . Wrecking over one-third of the island's population, this epidemic peaked its devastation between January and February 2006, when over 46,000 cases came into light every week, including 284 deaths [5, 13] . Ae. albopictus is common in urban areas of the United States and is already flourishing in 36 states, raising grave concerns to the immunologically naive populace of the United States [14] . Accordingly, this review elaborately details the epidemiology and global expansion of CHIKV, describes its clinical features and pathogenesis and its symptoms and complications, and finally nominates a possible vaccine approach against CHIKV infection. CHIKV has been isolated into three genotypes based on phylogenetic studies. These genotypes, based on the gene sequences of an Envelope protein (E1), are Asian, East/Central/ South African, and West African [4, 11, 15] . Using phylogenetic models, Cherian et al. estimate that the Asian genotype of CHIKV emerged between 50 and 310 y ago, and the West and East African genotypes diverged between 100 and 840 y ago [15] . Since then, CHIKV has come a long way, with several mutations incorporated, and has continued to wreak epidemics in several regions. Recent activities of CHIKV include the Indian epidemic in 2005-2006, which was followed by a sudden explosion of cases in 2007. An estimated 1.3 million people across 13 states were reported to be infected in India [12, 16] , and CHIKV was also widespread in Malaysia, Sri Lanka, and Indonesia [17] . In July-August of 2007, CHIKV was reported in Italy, probably brought in by travelers from CHIKV-prone regions of India, Africa, and Indian Ocean islands such as Mauritius, Madagascar, and Seychelles. Few of the Italian isolates were found to have evolved from the Kerala isolate, which was associated with a A226V shift in E1 gene that represents a successful evolutionary adaptation in the mosquito vector similar to the ones observed in Reunion Island [2, 18, 19] . In recent times, with an increase in global travel, the risk for spreading CHIKV to non-endemic regions has heightened [1] . Several travelers have brought CHIKV home with them after visiting areas with actively infected populations [12, 20] . Such cases have been documented in European countries, Australia, Asia, and the United States [8, 21] . The United States has already reported at least twelve cases of travel-associated CHIKV, while France has reported 850 cases, and the United Kingdom 93 [8, 14] . Beyond this, CHIKV-infected travelers have also been diagnosed in Australia, Belgium, Canada, Czech Republic, French Guiana, Germany, Hong Kong, Italy, Japan, Kenya, Malaysia, Martinique, Norway, Switzerland, and Sri Lanka [21] . Some travelers were viremic, worrying public health officials about the spread of CHIKV to new areas [1, 8] . The incubation time for CHIKV is relatively short, requiring only 2-6 d with symptoms usually appearing 4-7 d post-infection [22] . Vazeille et al. detected CHIKV in the salivary glands of Ae. albopictus only 2 d after infection [5] . Upon infection, CHIKF tends to present itself in two phases. The first stage is acute, while the second stage, experienced by most but not all, is persistent, causing disabling polyarthritis. Characteristics of the acute phase include an abrupt onset of fever, arthralgia, and in some cases, maculopapular rash [6, 23] . The acute phase causes such intense joint and muscular pain that makes movement very difficult and prostrates its victims [6, 20] . Ninety-five percent of infected adults are symptomatic after infection, and of these, most become disabled for weeks to months as a result of decreased dexterity, loss of mobility, and delayed reaction. Eighteen months after disease onset, 40% of patients are found to still have anti-CHIKV IgM [6, 18, 23, 24] . The chronic stage of CHIKF is characterized by polyarthralgia that can last from weeks to years beyond the acute stage [6] . CHIKV has been shown to attack fibroblasts, explaining the involvement of muscles, joints, and skin connective tissues. The high number of nociceptive nerve endings found within the joints and muscle connective tissues can explain pain associated with CHIKF [25, 26] . More than 50% of patients who suffer from severe CHIKF are over 65 y old, and more than 33% of them die. Most adults who suffer from severe CHIKF have underlying medical conditions [6, 24, 27] . The other group that is disproportionately affected by severe CHIKV is children. Other complications associated with CHIKV, from most common to least common, include respiratory failure, cardiovascular decompensation, meningoencephalitis, severe acute hepatitis, severe cutaneous effects, other central nervous system problems, and kidney failure [6, 18, 20, 23, 24, 26, 27] . CHIKV undertakes a complex replication cycle upon host infection (Figure 2 ), which makes its genome susceptible to mutations [28, 29] . For instance, Ae. aegypti, responsible for epidemics in Kenya, Comoros, and Seychelles, carried CHIKV with an alanine in the 226 position of the E1 gene (E1-A226) [4, 18] . However, when the virus struck La Reunion Islands, a decline in population of Ae. aegypti, due to massive dichlorodiphenyltrichloroethane usage and dearth of Ae. albopictus species' www.plosntds.org population, resulted in an ecological pressure, favoring replacement of alanine at position 226 with valine (E1-A226V) [5] . This mutation allowed CHIKV's secondary vector species, Ae. albopictus, to supplement Ae. aegypti as its primary vector [5] . Within a year, the E1-A226V mutation was present in La Reunion Island, and Ae. albopictus apparently vectored the large epidemic infecting 34% of La Reunion Island's population [5] . All of the CHIKV strains isolated from Mayotte carried the E1-A226V mutation, and the mutation was also found in Madagascar in 2007 [5] . The E1-A226V mutation was not present at the beginning of the Indian Ocean Islands outbreak (before September 2005). However, more than 90% of later viral strains found there had incorporated the mutation (December-March 2006), indicating a genotype switch during the winter season [5, 18, 20] . The E1-A226V mutation also enabled an increase in infectivity of Ae. albopictus when compared to its infectivity of Ae. aegypti [4, 11, 18, 30] , and with several factors taken together, Ae. albopictus has become the new preferred and more lethal vector for CHIKV [4, 5, 11] . In fact, Tsetsarkin et al. found that a Green Fluorescent Protein tagged E1-A226V virus was 100 times more infective to Ae. albopictus than it was to Ae. aegypti [4] . In all the Indian Ocean Islands, Ae. albopictus became the main vector for CHIKV within 1-2 y after CHIKV was introduced to the region [31] . Of note is that Ae. aegypti has most likely been established in North America for over 300 y, while Ae. albopictus has been in many areas of the US, since 1985, primarily in Florida [32] and since then has expanded its range in the country. Reiskind et al. set out to determine if Ae. aegypti and Ae. albopictus mosquitoes captured in Florida were susceptible to CHIKV infection by a La Reunion isolate [32] . Each mosquito tested was highly susceptible to infection by a full-length infectious clone of the La Réunion Island isolate, CHIKV LR2006 OPY1 strain. Even though the Ae. albopictus strains were more susceptible to infection, overall ecology and differences in human biting patterns need to be studied further Characteristically, there are two rounds of translation: (+) sense genomic RNA (49S9 = 11.7 kb) acts directly as mRNA and is partially translated (59 end) to produce non-structural proteins (nsp's). These proteins are responsible for replication and formation of a complementary (2) strand, the template for further (+) strand synthesis. Subgenomic mRNA (26 S = 4.1 kb) replication occurs through the synthesis of full-length (2) intermediate RNA, which is regulated by nsp4 and p123 precursor in early infection and later by mature nsp's. Translation of the newly synthesized sub-genomic RNA results in production of structural proteins such as Capsid and protein E2-6k-E1 (from 39 end of genome). Assembly occurs at the cell surface, and the envelope is acquired as the virus buds from the cell and release and maturation almost simultaneous occurred. Replication occurs in the cytoplasm and is very rapid (,4 h) [28, 29] . doi:10.1371/journal.pntd.0000623.g002 www.plosntds.org to gain a more accurate understanding of a potential CHIKV epidemic in the US [32] . During the 7 d preceding birth, no human mother has been reported to transmit the disease vertically. However, about 50% of newborns delivered while the mother was infected with CHIKV contracted the disease from their mother, despite the method of delivery. Furthermore, there have been instances of CHIKV transmission from mother to fetus causing congenital illness and fetal death [33] . During the 2005-2006 La Reunion Island outbreaks, Ramful et al. discovered that mothers could transmit CHIKV to their progeny during the perinatal period (Day 24 to Day +1) [33, 34] , and it is associated with a high degree of morbidity. By mean Day 4 of life, all of the neonates were symptomatic for CHIKV, exhibiting common CHIKF symptoms. Six neonates were confirmed to have contracted CHIKV and developed mengoencephalitis. Of those mothers who, during the La Reunion Island epidemic, were infected long before delivery, only three fetal deaths were reported [12, 33] . Ramful et al. theorized that motherto-child transmission most likely happens transplacentally shortly before delivery [33] . A similar study by Gerardin et al. reported nineteen cases of neonatal infection associated with intrapartum maternal viremia that progressed to develop encephalitis owing to vertical transmission from infected mothers [34] . Clinical and epidemiological similarities with dengue fever make CHIKV diagnosis difficult, which may lead physicians to misdiagnose CHIKV as dengue fever; therefore, the incidence of CHIKV may actually be higher than currently believed (Table 1 ) [6, 12, 35] . The amount of time elapsed since disease onset is the most critical parameter when choosing a diagnostic test. CHIKV can be detected and isolated by culturing with mosquito cells (C6/36), Vero cells (mammalian), or in mice [26] . However, this method can take at least a week and only achieves a high sensitivity during the viremic phase, which usually only lasts up to 48 h after the bite. Five days post-infection, the viral isolation approach has a low sensitivity but is still the preferred method for detecting the CHIKV strain [12, 26, 31, 35] . RT-PCR on the other hand is a faster and more sensitive method that can be used within the first week of disease onset [26] , and it is currently the most sensitive method for detecting and quantifying viral mRNA [4, 36] . Classic serological detection, by assays such as ELISA [37] , immunofluorescence [5, 38] , complement binding, and haemagglutination inhibition [39] , constitutes the second diagnostic tool used for biological diagnosis of CHIKV infection. These proven techniques are useful for detection of Antigen in mosquitoes during epidemiological studies. These assays detect virus-specific IgM and IgG, however the sensitivity and specificity of these assays has been poorly characterized. Viral competence, or the potential of viral infection and transmission, is an important parameter that can be quantified by ELISA, viral culture, and PCR. A study by Ng et al. showed biomarkers indicative of severe CHIKV infection [40] . They found decreased levels of RANTES and increased levels of Interleukin-6 (IL-6) and Interleukin-1b (IL-1b) that could be sued for CHIKV detection in patients as indicators of CHIKV-driven cytokine storm. Couderc et al. demonstrate another cytokine, type-I IFN, as a key player in the progression to CHIKV infection [26] . Using an IFN-a/b null mouse model, they demonstrated evidence of muscles, joints, and skin as privileged CHIKV targets, which is consistent with human pathology. Although Ng et al. concluded that RANTES levels were significantly suppressed in severe CHIKF patients [40] , interestingly, an increase in levels of RANTES has been observed in dengue infection [41] . Since the symptoms of CHIKF mimic those of dengue fever, results obtained from this study strongly suggest that RANTES could be a potential distinctive biomarker that differentiates between these two clinically similar diseases. There are no approved antiviral treatments currently available for CHIKV [1, 3, 12, 42] . Currently, CHIKF is treated symptomatically, usually with non-steroidal anti-inflammatory drugs or steroids, bed rest, and fluids. Movement and mild exercise are thought to decrease stiffness and morning arthralgia, but heavy exercise may exacerbate rheumatic symptoms. Corticosteroids may be used in cases of debilitating chronic CHIKV infection. There is a debate about the appropriateness of chloroquine as treatment for unresolved, non-steroidal anti-inflammatory drugresistant arthritis [43] . A study showed that viral production was www.plosntds.org drastically reduced at 16 h post-infection after treatment with 100 mM dec-RVKR-cmk (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone), a furine inhibitor [42, 44] . Chloroquine acted by raising the pH, blocking low pH-dependent entry of virus into the cell. It is important to note that dec-RVKR-cmk or chloroquine only inhibited viral spreading from cell to cell, not CHIKV replication once it had entered the cell [43] . However, most would agree that the best weapon against CHIKV is prevention. A live CHIKV vaccine developed by the United States reached phase II clinical trial encompassing 59 healthy volunteers [45] . Eight percent of the volunteers experienced transient arthralgia, while 98% of the volunteers had seroconversion [45] . However, live CHIKV vaccines are still questionable. One cannot discount the risk of a live vaccine possibly inducing chronic rheumatism. Also, there is the question as to whether widespread use among the public could trigger mosquito transmission or lead to chronic infection or viral reversion [1] . An alternative approach would be to produce a chimeric vaccine against CHIKV. Wang et al. developed a chimeric alphavirus vaccine that is uniformly attenuated and does not cause reactogenicity in mice [3] . Three different versions of this vaccine were made using three different backbone vectors: Venezuelan equine encephalitis virus (VEEV) attenuated vaccine strain T-83, naturally attenuated eastern equine encephalitis virus (EEEV), and attenuated Sindbis virus (SINV). In short, CHIKV structural proteins were engineered into the backbones of the aforementioned vaccines to produce the chimeras [3] . These chimeras were found to stimulate a strong humoral immunity, and even at doses of 5.3-5.8 log 10 PFU, they did not trigger reactogenicity. When vaccinated mice were challenged with CHIKV, neither adult nor neonatal mice gained weight, had fever, or displayed signs of neurological illness. Upon comparison of the chimeras with the Army181/25 vaccine, the Army vaccine resulted in higher levels of viremia and replication in the joints of neonatal mice. Because the joints are known targets of CHIKV, Wang et al. noted their vaccine might avoid the negative reactogenic side effects of the Army vaccine. After being subcutaneously vaccinated with 5.3-5.8 log 10 PFU of the chimeric vaccines, mice produced strong neutralizing antibody titers. The VEEV and EEEV chimeras yielded higher neutralizing antibody titers than the SINV chimera without being more virulent. On top of this, the VEEV and EEEV CHIKV chimeras seemed to be more immunogenic than the Army vaccine despite the chimeras' lower viremia and replication in the joints of neonatal mice [3] . Tiwari et al. [46] adopted a different strategy using formalin inactivated CHIKV in combination with alhydrogel (Aluminum Hydroxide) as an adjuvant. This study clearly suggests that this vaccine elicits both humoral and cell-mediated immune responses in mice, providing its immunogenic potential. A recent study by Couderc et al. [47] showed passive immunization as a potential treatment for CHIKV infection. Using purified immunoglobulin extracted from convalescent CHIKV patients, they demonstrated effective neutralizing activity against CHIKV infection both in vitro and in vivo. This thereby establishes a potential preventive and therapeutic approach to combat CHIKV infection. Pathogenesis studies conducted with related alpha virus, like RRV, have shown the role of macrophages in persistence on infection [48] . They also demonstrated the role of RRV-specific CD8 T cells in clearing viral load in infected patients, thereby warranting similar investigations with CHIKV and the importance of investigating a cell-mediated immune response-based vaccine against CHIKV [49] . There are always certain risks associated with live attenuated or inactivated viral vaccines [50] . One way to avoid these potential problems is to construct a consensus-based DNA vaccine. DNA based vaccines have an improved safety profile as compared to live or attenuated vaccines [51, 52] . A consequence of CHIKV's rapid evolution is difficulty in constructing a vaccine that will be able to Figure 3 . Levels of CHIKV-specific IgG in mice immunized with CHIKV vaccines. Each group of C57BL/6 mice (n = 5) was immunized with 12.5 mg of pVax1 control vector or CHIKV vaccine plasmids as indicated at 0 and 2 wk. Mice were bled 2 wk after each immunization, and each group's serum pool was diluted to 1:100 and 1:500 for reaction with specific vaccine constructs. Serum was incubated for 1 h at 37uC on 96-well plates coated with 2 mg/ml of respective CHIKV peptides, and antibody was detected using anti-mouse IgG-HRP and OD was measured at 405 nm. doi:10.1371/journal.pntd.0000623.g003 www.plosntds.org effectively protect large populations from multiple strains of the virus. One of the strengths of DNA consensus vaccines is its ability to induce cross-reactive immune responses against the three distinct phylogenetic groups of CHIKV. Also DNA-based vaccines can be produced more rapidly than protein-based vaccines. Recently, Muthumani et al. constructed a vaccine that was shown to induce both humoral and cellular immunity in vivo in 3-4-wk-old female C57/BL6 mice [49] . These mice were immunized using an in vivo electroporation method to deliver the vaccine into the quadriceps muscle. The consensus construct was designed against E1, E2, and the core protein capsid. To design the construct, they aligned 21 sequences of CHIKV isolated between 1952 and 2006, using strains from differing countries, including La Reunion Island. The most common nucleotide among the sequences was chosen at each position to be used in the consensus construct, taking care not to alter the reading frame. They conducted codon and RNA optimization, added a strong Kozak sequence, and substituted signal peptide with an immunoglobulin E leader sequence to improve vaccine efficacy. After immunizing the mice, spleens were harvested along with serum and tested to determine antibody titer. After three immunizations, consensus E1, E2, and C vaccines were shown to induce T-cell immune responses leading to strong IFN-c responses and proliferation in C57/BL6 mice. Furthermore, when compared with control mice, immunized mice had higher total IgG levels as well as higher anti-E1 specific, anti-E2 specific, and anti-C specific IgG antibodies, suggesting a strong humoral immune response ( Figure 3 ) and also specificity for the antigens encoded in the vaccine constructs ( Figure 4 ). Because of its promising results and the need for a safer vaccine, this consensus DNA vaccine deserves further investigation. Determining longevity of protective effects of the vaccine and persistence of antibody and IFN-c responses could be the next step of investigation. Challenged studies of immunized mice must also be carried out. CHIKV mosquito-borne disease has caused massive outbreaks for at least half a century but is no longer confined to the www.plosntds.org developing nations. It began to encroach into the boundaries of the developing world. As a result, the NIAID has designated CHIKV as a Category C pathogen alongside the influenza and SARS-CoV viruses [3] . Realization of the potential severity of this disease is exigent; for instance, if used as a biological weapon, the world economy could be severely crippled; if enough members of the armed forces were to become infected during a military deployment, military operations could be significantly affected. Efforts to monitor the disease will only provide minimal warning in a global society, and steps to prevent the morbidity and mortality associated with pandemic are imperative [21, 31] . Despite the gravity of its infectious potency and the fear of it being a potential biological weapon, there is currently no vaccine for CHIKV infections. Live attenuated vaccine trials were carried out in 2000, but funding for the project was discontinued. Newer approaches such as DNA vaccines appear promising over conventional strategies like live attenuated or inactivated virus and thus call for further investigation. Recent advances such electroporation delivery and incorporation of adjuvants has boosted DNA vaccine efficacy [51, 53] . Despite the low antibody response to DNA vaccines, other numerous advantages have overshadowed these minor drawbacks (Table 2) , the most important one being the ability to induce both humoral and cellular immune responses [51, 54] . Judging by recent success, such as the immunogenic construct developed by Muthumani et al., DNA vaccines could play a major role in combating CHIKV [49] . Vaccines are literally a critical component of CHIKV disease control and therefore research in this area is highly encouraged. The dramatic spread of dengue viruses (DENV) throughout tropical America since 1980 via the same vectors and human hosts underscores the risk to public health in the Americas. The adverse events associated with the current live vaccine are well documented [55] . Realizing these drawbacks, earnest efforts should be taken to develop new strategies to forestall further spread and complications.
hen did Asian genotype emerge?
false
2,490
{ "text": [ "between 50 and 310 y ago," ], "answer_start": [ 3514 ] }
1,719
Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.
What are some alphavirus vectors that have been developed?
false
1,565
{ "text": [ "Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses" ], "answer_start": [ 16467 ] }
2,669
Frontiers in antiviral therapy and immunotherapy https://doi.org/10.1002/cti2.1115 SHA: facbfdfa7189ca9ff83dc30e5d241ab22e962dbf Authors: Heaton, Steven M Date: 2020 DOI: 10.1002/cti2.1115 License: cc-by Abstract: nan Text: Globally, recent decades have witnessed a growing disjunction, a 'Valley of Death' 1,2 no less, between broadening strides in fundamental biomedical research and their incommensurate reach into the clinic. Plumbing work on research funding and development pipelines through recent changes in the structure of government funding, 2 new public and private joint ventures and specialist undergraduate and postgraduate courses now aim to incorporate pathways to translation at the earliest stages. Reflecting this shift, the number of biomedical research publications targeting 'translational' concepts has increased exponentially, up 1800% between 2003 and 2014 3 and continuing to rise rapidly up to the present day. Fuelled by the availability of new research technologies, as well as changing disease, cost and other pressing issues of our time, further growth in this exciting space will undoubtedly continue. Despite recent advances in the therapeutic control of immune function and viral infection, current therapies are often challenging to develop, expensive to deploy and readily select for resistance-conferring mutants. Shaped by the hostvirus immunological 'arms race' and tempered in the forge of deep time, the biodiversity of our world is increasingly being harnessed for new biotechnologies and therapeutics. Simultaneously, a shift towards host-oriented antiviral therapies is currently underway. In this Clinical & Translational Immunology Special Feature, I illustrate a strategic vision integrating these themes to create new, effective, economical and robust antiviral therapies and immunotherapies, with both the realities and the opportunities afforded to researchers working in our changing world squarely in mind. Opening this CTI Special Feature, I outline ways these issues may be solved by creatively leveraging the so-called 'strengths' of viruses. Viral RNA polymerisation and reverse transcription enable resistance to treatment by conferring extraordinary genetic diversity. However, these exact processes ultimately restrict viral infectivity by strongly limiting virus genome sizes and their incorporation of new information. I coin this evolutionary dilemma the 'information economy paradox'. Many viruses attempt to resolve this by manipulating multifunctional or multitasking host cell proteins (MMHPs), thereby maximising host subversion and viral infectivity at minimal informational cost. 4 I argue this exposes an 'Achilles Heel' that may be safely targeted via host-oriented therapies to impose devastating informational and fitness barriers on escape mutant selection. Furthermore, since MMHPs are often conserved targets within and between virus families, MMHP-targeting therapies may exhibit both robust and broadspectrum antiviral efficacy. Achieving this through drug repurposing will break the vicious cycle of escalating therapeutic development costs and trivial escape mutant selection, both quickly and in multiple places. I also discuss alternative posttranslational and RNA-based antiviral approaches, designer vaccines, immunotherapy and the emerging field of neo-virology. 4 I anticipate international efforts in these areas over the coming decade will enable the tapping of useful new biological functions and processes, methods for controlling infection, and the deployment of symbiotic or subclinical viruses in new therapies and biotechnologies that are so crucially needed. Upon infection, pathogens stimulate expression of numerous host inflammatory factors that support recruitment and activation of immune cells. On the flip side, this same process also causes immunopathology when prolonged or deregulated. 5 In their contribution to this Special Feature, Yoshinaga and Takeuchi review endogenous RNA-binding proteins (RBPs) that post-transcriptionally control expression of crucial inflammatory factors in various tissues and their potential therapeutic applications. 6 These RBPs include tristetraprolin and AUF1, which promote degradation of AU-rich element (ARE)-containing mRNA; members of the Roquin and Regnase families, which respectively promote or effect degradation of mRNAs harbouring stem-loop structures; and the increasingly apparent role of the RNA methylation machinery in controlling inflammatory mRNA stability. These activities take place in various subcellular compartments and are differentially regulated during infection. In this way, mRNA-destabilising RBPs constitute a 'brake' on the immune system, which may ultimately be toggled therapeutically. I anticipate continued efforts in this area will lead to new methods of regaining control over inflammation in autoimmunity, selectively enhancing immunity in immunotherapy, and modulating RNA synthesis and virus replication during infection. Another mRNA under post-transcriptional regulation by Regnase-1 and Roquin is Furin, which encodes a conserved proprotein convertase crucial in human health and disease. Furin, along with other PCSK family members, is widely implicated in immune regulation, cancer and the entry, maturation or release of a broad array of evolutionarily diverse viruses including human papillomavirus (HPV), influenza (IAV), Ebola (EboV), dengue (DenV) and human immunodeficiency virus (HIV). Here, Braun and Sauter review the roles of furin in these processes, as well as the history and future of furin-targeting therapeutics. 7 They also discuss their recent work revealing how two IFN-cinducible factors exhibit broad-spectrum inhibition of IAV, measles (MV), zika (ZikV) and HIV by suppressing furin activity. 8 Over the coming decade, I expect to see an ever-finer spatiotemporal resolution of host-oriented therapies to achieve safe, effective and broad-spectrum yet costeffective therapies for clinical use. The increasing abundance of affordable, sensitive, high-throughput genome sequencing technologies has led to a recent boom in metagenomics and the cataloguing of the microbiome of our world. The MinION nanopore sequencer is one of the latest innovations in this space, enabling direct sequencing in a miniature form factor with only minimal sample preparation and a consumer-grade laptop computer. Nakagawa and colleagues here report on their latest experiments using this system, further improving its performance for use in resource-poor contexts for meningitis diagnoses. 9 While direct sequencing of viral genomic RNA is challenging, this system was recently used to directly sequence an RNA virus genome (IAV) for the first time. 10 I anticipate further improvements in the performance of such devices over the coming decade will transform virus surveillance efforts, the importance of which was underscored by the recent EboV and novel coronavirus (nCoV / COVID-19) outbreaks, enabling rapid deployment of antiviral treatments that take resistance-conferring mutations into account. Decades of basic immunology research have provided a near-complete picture of the main armaments in the human antiviral arsenal. Nevertheless, this focus on mammalian defences and pathologies has sidelined examination of the types and roles of viruses and antiviral defences that exist throughout our biosphere. One case in point is the CRISPR/Cas antiviral immune system of prokaryotes, which is now repurposed as a revolutionary gene-editing biotechnology in plants and animals. 11 Another is the ancient lineage of nucleocytosolic large DNA viruses (NCLDVs), which are emerging human pathogens that possess enormous genomes of up to several megabases in size encoding hundreds of proteins with unique and unknown functions. 12 Moreover, hundreds of human-and avian-infective viruses such as IAV strain H5N1 are known, but recent efforts indicate the true number may be in the millions and many harbour zoonotic potential. 13 It is increasingly clear that host-virus interactions have generated truly vast yet poorly understood and untapped biodiversity. Closing this Special Feature, Watanabe and Kawaoka elaborate on neo-virology, an emerging field engaged in cataloguing and characterising this biodiversity through a global consortium. 14 I predict these efforts will unlock a vast wealth of currently unexplored biodiversity, leading to biotechnologies and treatments that leverage the host-virus interactions developed throughout evolution. When biomedical innovations fall into the 'Valley of Death', patients who are therefore not reached all too often fall with them. Being entrusted with the resources and expectation to conceive, deliver and communicate dividends to society is both cherished and eagerly pursued at every stage of our careers. Nevertheless, the road to research translation is winding and is built on a foundation of basic research. Supporting industry-academia collaboration and nurturing talent and skills in the Indo-Pacific region are two of the four pillars of the National Innovation and Science Agenda. 2 These frame Australia's Medical Research and Innovation Priorities, which include antimicrobial resistance, global health and health security, drug repurposing and translational research infrastructure, 15 capturing many of the key elements of this CTI Special Feature. Establishing durable international relationships that integrate diverse expertise is essential to delivering these outcomes. To this end, NHMRC has recently taken steps under the International Engagement Strategy 16 to increase cooperation with its counterparts overseas. These include the Japan Agency for Medical Research and Development (AMED), tasked with translating the biomedical research output of that country. Given the reciprocal efforts at accelerating bilateral engagement currently underway, 17 the prospects for new areas of international cooperation and mobility have never been more exciting nor urgent. With the above in mind, all contributions to this CTI Special Feature I have selected from research presented by fellow invitees to the 2018 Awaji International Forum on Infection and Immunity (AIFII) and 2017 Consortium of Biological Sciences (ConBio) conferences in Japan. Both Australia and Japan have strong traditions in immunology and related disciplines, and I predict that the quantity, quality and importance of our bilateral cooperation will accelerate rapidly over the short to medium term. By expanding and cooperatively leveraging our respective research strengths, our efforts may yet solve the many pressing disease, cost and other sustainability issues of our time.
What does the author coin this evolutionary dilemma as?
false
4,128
{ "text": [ "'information economy paradox'." ], "answer_start": [ 2424 ] }
1,560
Relationship between hepcidin and oxidant/antioxidant status in calves with suspected neonatal septicemia https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146304/ SHA: efcd7d171bb51acf2ef0a631901900497957a3be Authors: Erkilic, E. E.; Erdogan, H. M.; Ogun, M.; Kirmizigul, A. H.; Gokce, E.; Kuru, M.; Kukurt, A. Date: 2016-11-14 DOI: 10.14202/vetworld.2016.1238-1241 License: cc-by Abstract: AIM: This study has been conducted for the purpose of determining serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves with suspected neonatal septicemia before and after treatment and the clinical significance of hepcidin in calves with suspected neonatal septicemia. MATERIALS AND METHODS: The study material consisted of 15 calves of different ages and sexes brought to the Training, Research and Application Center at the Kafkas University Faculty of Veterinary Medicine with suspected neonatal septicemia. 8.5 mL of blood was drawn from the jugular vein of each animal into coagulant tubes before and after treatment for one-off biochemical analyses and centrifuged. After this, the serum was separated. Hepcidin, TAS, TOS, and Fe levels in the serum were measured. RESULTS: While pre-treatment hepcidin levels were 58.42±3.46 ng/mL, post-treatment levels were 46.87±2.98 ng/mL (p<0.05). Pre-treatment Fe levels were 60.13±7.27 µg/dl, while post-treatment levels were 83.1±8.09 µg/dl (p<0.05). The changes in the TAS and TOS levels were also found to be statistically significant. CONCLUSION: In light of the fact that hepcidin plays a role function in the regulation of Fe as well as the fact that Fe is a significant nutritional source for many microorganisms, it was concluded that hepcidin may play a significant role in nutritional immunity and the pathogenesis of diseases. Text: Neonatal calf septicemia causes high morbidity and mortality and is one of the leading and most significant difficulties in raising cattle. Calf septicemia is the main cause of death in the neonatal period [1] . Its etiology involves bacteria (commonly Escherichia coli), viruses (rota and coronavirus), parasites, and other factors. As the disease progresses quickly and is lethal, diagnosis and treatment should be initiated as quickly as possible [2] . Hepcidin is a low molecular weight, antimicrobial peptide hormone and was first discovered in human urine [3] . It is produced by the liver as a firstline response to inflammatory reactions and high Fe concentrations [4, 5] . Hepcidin plays a fundamental role in the regulation of Fe metabolism [6] , which is a part of foundational cellular functions and thus of vital importance. On the other hand, by participating in redox reactions leading to the production of reactive oxygen species (ROSs), Fe also causes oxidative stress. Therefore, Fe has been regarded as a potentially toxic element to cells [7] . Fe also plays an important role in pathogenesis of bacterial infections as bacteria utilize Fe for survival, growth and proliferation; therefore, it is of paramount importance to control the Fe metabolism [6] . It is well known that the abundance of Fe suppresses defense system leading host vulnerable to infections. There is a significant relationship between Hepcidin, Fe metabolism, inflammation, and the immune system. The fact that hepcidin plays an active role in the regulation of Fe release from macrophages and in the control of excessive Fe absorption from the duodenum is well documented [6] . Hepcidin is a part of the natural defense mechanism, thus it limits the amount of Fe that can be utilized by pathogens [8] . In inflammatory conditions, hypoferremia is an important first-line protective mechanism in response to infections [9] . Fe also participates in redox reactions, causing the production of ROS, and thus leading to oxidative stress [7] . Free radicals play a significant role in the pathogenesis of many diseases [10] . Newborns are subject to oxidative stress during birth. It is also reported that in livestock diseases, especially enteritis and pneumonia, antioxidant capacity is efficacious [11] . This study was designed to determine the clinical significance of hepcidin in calves with suspected neonatal septicemia by evaluating serum hepcidin, total antioxidant status (TAS), total oxidant status (TOS), and Fe levels in calves suspected of neonatal septicemia before and after treatment. This study was conducted after obtaining approval from the Mehmet Akif Ersoy University Animal Experiments Local Ethics Committee (MAKU-HADYEK-Submission: 2014/77). The study consisted of 15 calves with suspected neonatal septicemia aged between 1 and 10 days old admitted to the Teaching Hospital of Veterinary Medicine. Suspected septicemia was diagnosed based on clinical (diarrhea, weakness in or absence of sucking reflex, the calf being in a supine position on the ground or being unable to stand, severe dehydration, abnormal rectal temperature [hypo-or hyperthermia], mucosal hyperemia, and full sclera) and hematological (increase in white blood cell [WBC] count) examinations; the animals were suspected to have septicemia [12, 13] . The animals were given standard treatment (antibiotic, nonsteroidal anti-inflammatory drugs, vitamin C, fluid therapy, and intestinal astringent). For determination of serum hepcidin, TAS, TOS, Fe levels, and hematological parameters; blood samples were taken before and after treatment in all cases. 8.5 mL of blood was taken from the jugular vein of each animal into coagulant tubes for biochemical analysis, and 3 mL blood was taken into ETDA tubes for hematological analysis. Samples were centrifuged at 3000 rpm for 10 min, and the serum was harvested and kept at −20°C until the analysis. Serum hepcidin (Mybiosource ® ), TAS (Rel Assay Diagnostics ® ), and TOS (Rel Assay Diagnostics ® ) were determined using commercial ELISA kits, and Fe value was measured spectrophotometrically. Hematological (WBC, lymphocyte [LYM], red blood cells [RBC], mean corpuscular volume (MCV), and hematocrit [HCT]) analysis was performed on blood counter (VG-MS4e ® , Melet Schloesıng, France). The results were evaluated using the t-test in the SPSS ® (SPSS 20, USA) statistical package program to determine the differences between values before and after treatment. Calves with suspected septicemia exhibited clinical signs of loss of appetite, fatigue, indifference to surroundings, reduced/absence of sucking reflex, cool extremities, inability to stand, diarrhea, eye sinking into their sockets, and hyperemia in the conjunctiva. The average body temperature, heart rate, and respiratory rates of the animals were 37.18±0.13°C, 104±4.33/min, and 28.86±0.75/min pre-treatment; and 38.54±0.1°C, 107.53±2.20/min and 26.40±0.36/min post-treatment, respectively. The changes in hepcidin, TAS, TOS and Fe levels in the calves with suspected septicemia before and after treatment are given in Table- 1. After treatment, serum hepcidin and TOS levels were significantly lower than before treatment in calves. On contrary, serum TAS and Fe levels were significantly higher than before treatment (Table-1 ). The treatment of calves resulted in significant changes in the hematological parameters that were examined except for RBC. The WBC count, LYM count, MCV and HCT significantly changed after treatment when compared to values obtained before treatment (Table-2 ). This study aimed to determine the clinical importance or use of hepcidin by comparing the values of serum hepcidin, TAS, TOS and Fe levels in calves with suspected neonatal septicemia before and after treatment. Clinicians rely on clinical and laboratory examinations of patients to form a working diagnosis, so hematological and serum biochemical parameters are usually used for this purpose [14] . The hematological parameters (WBC, HCT, LYM, and MCV) evaluated in this study were comparable with those reported by others in neonatal calves with diarrhea and suspected septicemia [15] [16] [17] . Treatment significantly corrected to normal values the hematological parameters that were examined with the exception of RBC. Pretreatment leukocyte count was high because of the inflammation that occurred in the organism, and that the HCT levels were high due to the dehydration that occurred due to diarrhea. Hepcidin is controlled by the presence of inflammation in the body, Fe storage, and erythropoietic activity in the bone marrow and plays a primary role in the homeostasis of Fe [4] . The increase in tissue and plasma Fe levels stimulates the synthesis of hepcidin and reduces Fe release and enteric Fe absorption from macrophages and hepatocytes [18] . Increased hepcidin concentrations during inflammation and infection reduce serum Fe levels by decreasing Fe release from macrophages and hepatocytes, and thus Fe required for microorganisms and tumor cells is restricted [19] . Serum hepcidin levels in calves with suspected septicemia were significantly high before treatment when compared to after treatment; also Fe levels were lower before treatment when compared to after treatment in this study. This situation could be related to the interaction between hepcidin and Fe and also gives credence to the role of hepcidin in the hemostasis of Fe during inflammation and infection. As in our study, Fe levels are well known to decrease in diarrheic calves when compared to healthy calves [20, 21] . Although no study exists reporting hepcidin concentration in diseased calves, studies in human subjects show that cord blood hepcidin levels might be an important indicator in diagnosing early-onset of neonatal sepsis. The cord blood hepcidin levels of neonatal infants with sepsis varied between 118.1 and 8400 ng/mL and were significantly higher than the healthy infants [22] . A similar result was reported that hepcidin concentrations in neonatal infants with sepsis were significantly higher than in healthy infants [23] . These findings along with our results add credence to the idea that hepcidin-Fe interaction may play a role in the pathogenesis of septicemia. The production of free oxygen species causes alterations in protein, lipid, and DNA during oxidative stress and leads to the development of lesions in the organs [24] . Free iron has toxic characteristics as it catalyses the production of ROSs [25] and thus causes oxidative stress [26] . The role of Fe in the development of oxidative stress may once more show the importance of hepcidin, as an important Fe regulator, with regard to enhancing antioxidant capacity through inhibiting utilization of Fe by the organism as well as the host cells. The antioxidant and oxidative system are in a constant state of balance in the organism. Any event breaking up this balance in favor of the oxidative stress molecules will cause cell damage [27, 28] . The host cells initiate the antioxidant system in case of exposure to oxidative stress [27] . Kabu et al. [16] reported TOS and TAS values in neonatal calves with diarrhea as 13.47±0.81 μmol H 2 O 2 /L and 0.51±0.02 mmol Trolox-equivalent/L, respectively, and treatment of these calves caused changes in these values of 11.21±0.26 μmol H 2 O 2 /L and 0.55±0.02 mmol Troloxequivalent/L, respectively. Studies also reported that parameters used for oxidative stress (malondialdehyde) were higher [29] and antioxidant parameters (superoxide dismutase [21] , TAS) were lower in diarrheic calves [29] . Similarly, in our study, TAS level was significantly lower and TOS level was significantly higher in diarrheic calves before treatment, and treatment caused corrections in these parameters. Decrease in TAS and increase in TOS levels demonstrated that oxidative stress was evident in the diseased calves in our study. Increased TOS and hepcidin levels before treatment are thought that associated with inflammation. After treatment increased TAS and decreased hepcidin levels support this opinion. Hepcidin may play an important part in non-specific immunity and is a key molecule that plays a role in the pathogenesis of diseases by enhancing the development of antioxidant system. However, more detailed studies are needed on the role of hepcidin in the pathogenesis of septicemia. This work was carried out in collaboration between all authors. EEE, HME and AHK: Designed the experimental procedures. EEE, EG and MK: Conducted the research work. EEE, AHK, MO and AK: Helped in laboratory analysis. All authors read and approved the final manuscript.
How does hepcidin work in the duodenum?
false
2,137
{ "text": [ "control of excessive Fe absorption" ], "answer_start": [ 3425 ] }
2,504
Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.
What does the involvement of respiratory viruses contribute to?
false
3,874
{ "text": [ "important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects" ], "answer_start": [ 4957 ] }
2,486
Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review https://doi.org/10.3390/jcm9030623 SHA: 9b0c87f808b1b66f2937d7a7acb524a756b6113b Authors: Pang, Junxiong; Wang, Min Xian; Ang, Ian Yi Han; Tan, Sharon Hui Xuan; Lewis, Ruth Frances; Chen, Jacinta I. Pei; Gutierrez, Ramona A.; Gwee, Sylvia Xiao Wei; Chua, Pearleen Ee Yong; Yang, Qian; Ng, Xian Yi; Yap, Rowena K. S.; Tan, Hao Yi; Teo, Yik Ying; Tan, Chorh Chuan; Cook, Alex R.; Yap, Jason Chin-Huat; Hsu, Li Yang Date: 2020 DOI: 10.3390/jcm9030623 License: cc-by Abstract: Rapid diagnostics, vaccines and therapeutics are important interventions for the management of the 2019 novel coronavirus (2019-nCoV) outbreak. It is timely to systematically review the potential of these interventions, including those for Middle East respiratory syndrome-Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS)-CoV, to guide policymakers globally on their prioritization of resources for research and development. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Supplementary strategies through Google Search and personal communications were used. A total of 27 studies fulfilled the criteria for review. Several laboratory protocols for confirmation of suspected 2019-nCoV cases using real-time reverse transcription polymerase chain reaction (RT-PCR) have been published. A commercial RT-PCR kit developed by the Beijing Genomic Institute is currently widely used in China and likely in Asia. However, serological assays as well as point-of-care testing kits have not been developed but are likely in the near future. Several vaccine candidates are in the pipeline. The likely earliest Phase 1 vaccine trial is a synthetic DNA-based candidate. A number of novel compounds as well as therapeutics licensed for other conditions appear to have in vitro efficacy against the 2019-nCoV. Some are being tested in clinical trials against MERS-CoV and SARS-CoV, while others have been listed for clinical trials against 2019-nCoV. However, there are currently no effective specific antivirals or drug combinations supported by high-level evidence. Text: Since mid-December 2019 and as of early February 2020, the 2019 novel coronavirus (2019-nCoV) originating from Wuhan (Hubei Province, China) has infected over 25,000 laboratory-confirmed cases across 28 countries with about 500 deaths (a case-fatality rate of about 2%). More than 90% of the cases and deaths were in China [1] . Based on the initial reported surge of cases in Wuhan, the majority were males with a median age of 55 years and linked to the Huanan Seafood Wholesale Market [2] . Most of the reported cases had similar symptoms at the onset of illness such as fever, cough, and myalgia or fatigue. Most cases developed pneumonia and some severe and even fatal respiratory diseases such as acute respiratory distress syndrome [3] . The 2019 novel coronavirus (2019-nCoV), a betacoronavirus, forms a clade within the subgenus sarbecovirus of the Orthocoronavirinae subfamily [4] . The severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are also betacoronaviruses that are zoonotic in origin and have been linked to potential fatal illness during the outbreaks in 2003 and 2012, respectively [5, 6] . Based on current evidence, pathogenicity for 2019-nCoV is about 3%, which is significantly lower than SARS-CoV (10%) and MERS-CoV (40%) [7] . However, 2019-nCoV has potentially higher transmissibility (R0: 1.4-5.5) than both SARS-CoV (R0: [2] [3] [4] [5] and MERS-CoV (R0: <1) [7] . With the possible expansion of 2019-nCoV globally [8] and the declaration of the 2019-nCoV outbreak as a Public Health Emergency of International Concern by the World Health Organization, there is an urgent need for rapid diagnostics, vaccines and therapeutics to detect, prevent and contain 2019-nCoV promptly. There is however currently a lack of understanding of what is available in the early phase of 2019-nCoV outbreak. The systematic review describes and assesses the potential rapid diagnostics, vaccines and therapeutics for 2019-nCoV, based in part on the developments for MERS-CoV and SARS-CoV. A systematic search was carried out in three major electronic databases (PubMed, Embase and Cochrane Library) to identify published studies examining the diagnosis, therapeutic drugs and vaccines for Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and the 2019 novel coronavirus (2019-nCoV), in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. There were two independent reviewers each focusing on SARS, MERS, and 2019-nCoV, respectively. A third independent reviewer was engaged to resolve any conflicting article of interest. We used the key words "SARS", "coronavirus", "MERS", "2019 Novel coronavirus", "Wuhan virus" to identify the diseases in the search strategy. The systematic searches for diagnosis, therapeutic drugs and vaccines were carried out independently and the key words "drug", "therapy", "vaccine", "diagnosis", "point of care testing" and "rapid diagnostic test" were used in conjunction with the disease key words for the respective searches. Examples of search strings can be found in Table S1 . We searched for randomized controlled trials (RCTs) and validation trials (for diagnostics test) published in English, that measured (a) the sensitivity and/or specificity of a rapid diagnostic test or a point-of-care testing kit, (b) the impact of drug therapy or (c) vaccine efficacy against either of these diseases with no date restriction applied. For the 2019-nCoV, we searched for all in vitro, animal, or human studies published in English between 1 December 2019 and 6 February 2020, on the same outcomes of interest. In addition, we reviewed the references of retrieved articles in order to identify additional studies or reports not retrieved by the initial searches. Studies that examined the mechanisms of diagnostic tests, drug therapy or vaccine efficacy against SARS, MERS and 2019-nCoV were excluded. A Google search for 2019-nCoV diagnostics (as of 6 February 2020; Table S2 ) yielded five webpage links from government and international bodies with official information and guidelines (WHO, Europe CDC, US CDC, US FDA), three webpage links on diagnostic protocols and scientific commentaries, and five webpage links on market news and press releases. Six protocols for diagnostics using reverse transcriptase polymerase chain reaction (RT-PCR) from six countries were published on WHO's website [9] . Google search for 2019-nCoV vaccines yielded 19 relevant articles. With the emergence of 2019-nCoV, real time RT-PCR remains the primary means for diagnosing the new virus strain among the many diagnostic platforms available ( [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ; Table S3 ). Among the 16 diagnostics studies selected, one study discussed the use of RT-PCR in diagnosing patients with 2019-nCoV [11] ( Table 1 ). The period and type of specimen collected for RT-PCR play an important role in the diagnosis of 2019-nCoV. It was found that the respiratory specimens were positive for the virus while serum was negative in the early period. It has also suggested that in the early days of illness, patients have high levels of virus despite the mild symptoms. Apart from the commonly used RT-PCR in diagnosing MERS-CoV, four studies identified various diagnostic methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), RT-insulated isothermal PCR (RT-iiPCR) and a one-step rRT-PCR assay based on specific TaqMan probes. RT-LAMP has similar sensitivity as real time RT-PCR. It is also highly specific and is used to detect MERS-CoV. It is comparable to the usual diagnostic tests and is rapid, simple and convenient. Likewise, RT-iiPCR and a one-step rRT-PCR assay have also shown similar sensitivity and high specificity for MER-CoV. Lastly, one study focused on the validation of the six commercial real RT-PCR kits, with high accuracy. Although real time RT-PCR is a primary method for diagnosing MERS-CoV, high levels of PCR inhibition may hinder PCR sensitivity (Table 1) . There are eleven studies that focus on SARS-CoV diagnostic testing (Table 1) . These papers described diagnostic methods to detect the virus with the majority of them using molecular testing for diagnosis. Comparison between the molecular test (i.e RT-PCR) and serological test (i.e., ELISA) showed that the molecular test has better sensitivity and specificity. Hence, enhancements to the current molecular test were conducted to improve the diagnosis. Studies looked at using nested PCR to include a pre-amplification step or incorporating N gene as an additional sensitive molecular marker to improve on the sensitivity (Table 1 ). In addition, there are seven potential rapid diagnostic kits (as of 24 January 2020; Table 2 ) available on the market for 2019-nCoV. Six of these are only for research purposes. Only one kit from Beijing Genome Institute (BGI) is approved for use in the clinical setting for rapid diagnosis. Most of the kits are for RT-PCR. There were two kits (BGI, China and Veredus, Singapore) with the capability to detect multiple pathogens using sequencing and microarray technologies, respectively. The limit of detection of the enhanced realtime PCR method was 10 2 -fold higher than the standard real-time PCR assay and 10 7fold higher than conventional PCR methods In the clinical aspect, the enhanced realtime PCR method was able to detect 6 cases of SARS-CoV positive samples that were not confirmed by any other assay [25] • The real time PCR has a threshold sensitivity of 10 genome equivalents per reaction and it has a good reproducibility with the inter-assay coefficients of variation of 1.73 to 2.72%. • 13 specimens from 6 patients were positive with viral load range from 362 to 36,240,000 genome equivalents/mL. The real-time RT-PCR reaction was more sensitive than the nested PCR reaction, as the detection limit for the nested PCR reaction was about 10 3 genome equivalents in the standard cDNA control. [34] Real-time reverse-transcription PCR (rRT-PCR); RNA-dependent RNA polymerase (RdRp); open reading frame 1a (ORF1a); Loop-mediated isothermal amplification (LAMP); enzyme-linked immunosorbent assay (ELISA); immunofluorescent assay (IFA); immunochromatographic test (ICT); nasopharyngeal aspirate (NPA). With the emergence of 2019-nCoV, there are about 15 potential vaccine candidates in the pipeline globally (Table 3 ), in which a wide range of technology (such as messenger RNA, DNA-based, nanoparticle, synthetic and modified virus-like particle) was applied. It will likely take about a year for most candidates to start phase 1 clinical trials except for those funded by Coalition for Epidemic Preparedness Innovations (CEPI). However, the kit developed by the BGI have passed emergency approval procedure of the National Medical Products Administration, and are currently used in clinical and surveillance centers of China [40] . Of the total of 570 unique studies on 2019-nCoV, SARS CoV or MERS-CoV vaccines screened, only four were eventually included in the review. Most studies on SARS and MERS vaccines were excluded as they were performed in cell or animal models ( Figure 1 ). The four studies included in this review were Phase I clinical trials on SARS or MERS vaccines (Table 4 ) [44] [45] [46] [47] . There were no studies of any population type (cell, animal, human) on the 2019-nCoV at the point of screening. The published clinical trials were mostly done in United States except for one on the SARS vaccine done in China [44] . All vaccine candidates for SARS and MERS were reported to be safe, well-tolerated and able to trigger the relevant and appropriate immune responses in the participants. In addition, we highlight six ongoing Phase I clinical trials identified in the ClinicalTrials.gov register ( [48, 49] ); Table S4 ) [50] [51] [52] . These trials are all testing the safety and immunogenicity of their respective MERS-CoV vaccine candidates but were excluded as there are no results published yet. The trials are projected to complete in December 2020 (two studies in Russia [50, 51] ) and December 2021 (in Germany [52] ). Existing literature search did not return any results on completed 2019-nCoV trials at the time of writing. Among 23 trials found from the systematic review (Table 5) , there are nine clinical trials registered under the clinical trials registry (ClinicalTrials.gov) for 2019-nCoV therapeutics [53] [54] [55] [56] [57] [58] [59] [60] [61] . Of which five studies on hydroxychloroquine, lopinavir plus ritonavir and arbidol, mesenchymal stem cells, traditional Chinese medicine and glucocorticoid therapy usage have commenced recruitment. The remaining four studies encompass investigation of antivirals, interferon atomization, darunavir and cobicistat, arbidol, and remdesivir usage for 2019-nCoV patients (Table 5) . Seroconversion measured by S1-ELISA occurred in 86% and 94% participants after 2 and 3 doses, respectively, and was maintained in 79% participants up to study end at week 60. Neutralising antibodies were detected in 50% participants at one or more time points during the study, but only 3% maintained neutralisation activity to end of study. T-cell responses were detected in 71% and 76% participants after 2 and 3 doses, respectively. There were no differences in immune responses between dose groups after 6 weeks and vaccine-induced humoral and cellular responses were respectively detected in 77% and 64% participants at week 60. [47] Molecules developed by the university scientists inhibit two coronavirus enzymes and prevent its replication. The discovered drug targets are said to be more than 95% similar to enzyme targets found on the SARS virus. Researchers note that identified drugs may not be available to address the ongoing outbreak but they hope to make it accessible for future outbreaks. [85] Besides the six completed randomized controlled trials (RCT) selected from the systematic review (Table 6) , there is only one ongoing randomized controlled trial targeted at SARS therapeutics [92] . The studies found from ClinicalTrials.gov have not been updated since 2013. While many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir or ribavirin only, there has yet to be well-designed clinical trials investigating their usage. Three completed randomized controlled trials were conducted during the SARS epidemic-3 in China, 1 in Taiwan and 2 in Hong Kong [93] [94] [95] [96] [97] . The studies respectively investigated antibiotic usage involving 190 participants, combination of western and Chinese treatment vs. Chinese treatment in 123 participants, integrative Chinese and Western treatment in 49 patients, usage of a specific Chinese medicine in four participants and early use of corticosteroid in 16 participants. Another notable study was an open non-randomized study investigating ribavirin/lopinavir/ritonavir usage in 152 participants [98] . One randomized controlled trial investigating integrative western and Chinese treatment during the SARS epidemic was excluded as it was a Chinese article [94] . There is only one ongoing randomized controlled trial targeted at MERS therapeutics [99] . It investigates the usage of Lopinavir/Ritonavir and Interferon Beta 1B. Likewise, many prospective and retrospective cohort studies conducted during the epidemic centered on usage of ribavirin with lopinavir/ritonavir/ribavirin, interferon, and convalescent plasma usage. To date, only one trial has been completed. One phase 1 clinical trial investigating the safety and tolerability of a fully human polyclonal IgG immunoglobulin (SAB-301) was found in available literature [46] . The trial conducted in the United States in 2017 demonstrated SAB-301 to be safe and well-tolerated at single doses. Another trial on MERS therapeutics was found on ClinicalTrials.gov-a phase 2/3 trial in the United States evaluating the safety, tolerability, pharmacokinetics (PK), and immunogenicity on coadministered MERS-CoV antibodies REGN3048 & REGN3051 [100]. Rapid diagnostics plays an important role in disease and outbreak management. The fast and accurate diagnosis of a specific viral infection enables prompt and accurate public health surveillance, prevention and control measures. Local transmission and clusters can be prevented or delayed by isolation of laboratory-confirmed cases and their close contacts quarantined and monitored at home. Rapid diagnostic also facilitates other specific public health interventions such as closure of high-risk facilities and areas associated with the confirmed cases for prompt infection control and environmental decontamination [11, 101] . Laboratory diagnosis can be performed by: (a) detecting the genetic material of the virus, (b) detecting the antibodies that neutralize the viral particles of interest, (c) detecting the viral epitopes of interest with antibodies (serological testing), or (d) culture and isolation of viable virus particles. The key limitations of genetic material detection are the lack of knowledge of the presence of viable virus, the potential cross-reactivity with non-specific genetic regions and the short timeframe for accurate detection during the acute infection phase. The key limitations of serological testing is the need to collect paired serum samples (in the acute and convalescent phases) from cases under investigation for confirmation to eliminate potential cross-reactivity from non-specific antibodies from past exposure and/or infection by other coronaviruses. The limitation of virus culture and isolation is the long duration and the highly specialized skills required of the technicians to process the samples. All patients recovered. Significantly shorted time from the disease onset to the symptom improvement in treatment (5.10 ± 2.83 days) compared to control group (7.62 ± 2.27 days) (p < 0.05) No significant difference in blood routine improvement, pulmonary chest shadow in chest film improvement and corticosteroid usgae between the 2 groups. However, particularly in the respect of improving clinical symptoms, elevating quality of life, promoting immune function recovery, promoting absorption of pulmonary inflammation, reducing the dosage of cortisteroid and shortening the therapeutic course, treatment with integrative chinese and western medicine treatment had obvious superiority compared with using control treatment alone. Single infusions of SAB-301 up to 50 mg/kg appear to be safe and well-tolerated in healthy participants. [46] Where the biological samples are taken from also play a role in the sensitivity of these tests. For SARS-CoV and MERS-CoV, specimens collected from the lower respiratory tract such as sputum and tracheal aspirates have higher and more prolonged levels of viral RNA because of the tropism of the virus. MERS-CoV viral loads are also higher for severe cases and have longer viral shedding compared to mild cases. Although upper respiratory tract specimens such as nasopharyngeal or oropharyngeal swabs can be used, they have potentially lower viral loads and may have higher risk of false-negatives among the mild MERS and SARS cases [102, 103] , and likely among the 2019-nCoV cases. The existing practices in detecting genetic material of coronaviruses such as SARS-CoV and MERS-CoV include (a) reverse transcription-polymerase chain reaction (RT-PCR), (b) real-time RT-PCR (rRT-PCR), (c) reverse transcription loop-mediated isothermal amplification (RT-LAMP) and (d) real-time RT-LAMP [104] . Nucleic amplification tests (NAAT) are usually preferred as in the case of MERS-CoV diagnosis as it has the highest sensitivity at the earliest time point in the acute phase of infection [102] . Chinese health authorities have recently posted the full genome of 2019-nCoV in the GenBank and in GISAID portal to facilitate in the detection of the virus [11] . Several laboratory assays have been developed to detect the novel coronavirus in Wuhan, as highlighted in WHO's interim guidance on nCoV laboratory testing of suspected cases. These include protocols from other countries such as Thailand, Japan and China [105] . The first validated diagnostic test was designed in Germany. Corman et al. had initially designed a candidate diagnostic RT-PCR assay based on the SARS or SARS-related coronavirus as it was suggested that circulating virus was SARS-like. Upon the release of the sequence, assays were selected based on the match against 2019-nCoV upon inspection of the sequence alignment. Two assays were used for the RNA dependent RNA polymerase (RdRP) gene and E gene where E gene assay acts as the first-line screening tool and RdRp gene assay as the confirmatory testing. All assays were highly sensitive and specific in that they did not cross-react with other coronavirus and also human clinical samples that contained respiratory viruses [11] . The Hong Kong University used two monoplex assays which were reactive with coronaviruses under the subgenus Sarbecovirus (consisting of 2019-nCoV, SARS-CoV and SARS-like coronavirus). Viral RNA extracted from SARS-CoV can be used as the positive control for the suggested protocol assuming that SARS has been eradicated. It is proposed that the N gene RT-PCR can be used as a screening assay while the Orf1b assay acts as a confirmatory test. However, this protocol has only been evaluated with a panel of controls with the only positive control SARS-CoV RNA. Synthetic oligonucleotide positive control or 2019-nCoV have yet to be tested [106] . The US CDC shared the protocol on the real time RT-PCR assay for the detection of the 2019-nCoV with the primers and probes designed for the universal detection of SARS-like coronavirus and the specific detection of 2019-nCoV. However, the protocol has not been validated on other platforms or chemistries apart from the protocol described. There are some limitations for the assay. Analysts engaged have to be trained and familiar with the testing procedure and result interpretation. False negative results may occur due to insufficient organisms in the specimen resulting from improper collection, transportation or handling. Also, RNA viruses may show substantial genetic variability. This could result in mismatch between the primer and probes with the target sequence which can diminish the assay performance or result in false negative results [107] . Point-of-care test kit can potentially minimize these limitations, which should be highly prioritized for research and development in the next few months. Serological testing such as ELISA, IIFT and neutralization tests are effective in determining the extent of infection, including estimating asymptomatic and attack rate. Compared to the detection of viral genome through molecular methods, serological testing detects antibodies and antigens. There would be a lag period as antibodies specifically targeting the virus would normally appear between 14 and 28 days after the illness onset [108] . Furthermore, studies suggest that low antibody titers in the second week or delayed antibody production could be associated with mortality with a high viral load. Hence, serological diagnoses are likely used when nucleic amplification tests (NAAT) are not available or accessible [102] . Vaccines can prevent and protect against infection and disease occurrence when exposed to the specific pathogen of interest, especially in vulnerable populations who are more prone to severe outcomes. In the context of the current 2019-nCoV outbreak, vaccines will help control and reduce disease transmission by creating herd immunity in addition to protecting healthy individuals from infection. This decreases the effective R0 value of the disease. Nonetheless, there are social, clinical and economic hurdles for vaccine and vaccination programmes, including (a) the willingness of the public to undergo vaccination with a novel vaccine, (b) the side effects and severe adverse reactions of vaccination, (c) the potential difference and/or low efficacy of the vaccine in populations different from the clinical trials' populations and (d) the accessibility of the vaccines to a given population (including the cost and availability of the vaccine). Vaccines against the 2019-nCoV are currently in development and none are in testing (at the time of writing). On 23 January 2020, the Coalition for Epidemic Preparedness Innovations (CEPI) announced that they will fund vaccine development programmes with Inovio, The University of Queensland and Moderna, Inc respectively, with the aim to test the experimental vaccines clinically in 16 weeks (By June 2020). The vaccine candidates will be developed by the DNA, recombinant and mRNA vaccine platforms from these organizations [109] . Based on the most recent MERS-CoV outbreak, there are already a number of vaccine candidates being developed but most are still in the preclinical testing stage. The vaccines in development include viral vector-based vaccine, DNA vaccine, subunit vaccine, virus-like particles (VLPs)-based vaccine, inactivated whole-virus (IWV) vaccine and live attenuated vaccine. The latest findings for these vaccines arebased on the review by Yong et al. (2019) in August 2019 [110] . As of the date of reporting, there is only one published clinical study on the MERS-CoV vaccine by GeneOne Life Science & Inovio Pharmaceuticals [47] . There was one SARS vaccine trial conducted by the US National Institute of Allergy and Infectious Diseases. Both Phase I clinical trials reported positive results, but only one has announced plans to proceed to Phase 2 trial [111] . Due to the close genetic relatedness of SARS-CoV (79%) with 2019-nCoV [112] , there may be potential cross-protective effect of using a safe SARS-CoV vaccine while awaiting the 2019-nCoV vaccine. However, this would require small scale phase-by-phase implementation and close monitoring of vaccinees before any large scale implementation. Apart from the timely diagnosis of cases, the achievement of favorable clinical outcomes depends on the timely treatment administered. ACE2 has been reported to be the same cell entry receptor used by 2019-nCoV to infect humans as SARS-CoV [113] . Hence, clinical similarity between the two viruses is expected, particularly in severe cases. In addition, most of those who have died from MERS-CoV, SARS-CoV and 2019-nCoV were advance in age and had underlying health conditions such as hypertension, diabetes or cardiovascular disease that compromised their immune systems [114] . Coronaviruses have error-prone RNA-dependent RNA polymerases (RdRP), which result in frequent mutations and recombination events. This results in quasispecies diversity that is closely associated with adaptive evolution and the capacity to enhance viral-cell entry to cause disease over time in a specific population at-risk [115] . Since ACE2 is abundantly present in humans in the epithelia of the lung and small intestine, coronaviruses are likely to infect the upper respiratory and gastrointestinal tract and this may influence the type of therapeutics against 2019-nCoV, similarly to SAR-CoV. However, in the years following two major coronavirus outbreaks SARS-CoV in 2003 and MERS-CoV in 2012, there remains no consensus on the optimal therapy for either disease [116, 117] . Well-designed clinical trials that provide the gold standard for assessing the therapeutic measures are scarce. No coronavirus protease inhibitors have successfully completed a preclinical development program despite large efforts exploring SARS-CoV inhibitors. The bulk of potential therapeutic strategies remain in the experimental phase, with only a handful crossing the in vitro hurdle. Stronger efforts are required in the research for treatment options for major coronaviruses given their pandemic potential. Effective treatment options are essential to maximize the restoration of affected populations to good health following infections. Clinical trials have commenced in China to identify effective treatments for 2019-nCoV based on the treatment evidence from SARS and MERS. There is currently no effective specific antiviral with high-level evidence; any specific antiviral therapy should be provided in the context of a clinical study/trial. Few treatments have shown real curative action against SARS and MERS and the literature generally describes isolated cases or small case series. Many interferons from the three classes have been tested for their antiviral activities against SARS-CoV both in vitro and in animal models. Interferon β has consistently been shown to be the most active, followed by interferon α. The use of corticosteroids with interferon alfacon-1 (synthetic interferon α) appeared to have improved oxygenation and faster resolution of chest radiograph abnormalities in observational studies with untreated controls. Interferon has been used in multiple observational studies to treat SARS-CoV and MERS-CoV patients [116, 117] . Interferons, with or without ribavirin, and lopinavir/ritonavir are most likely to be beneficial and are being trialed in China for 2019-nCoV. This drug treatment appears to be the most advanced. Timing of treatment is likely an important factor in effectiveness. A combination of ribavirin and lopinavir/ritonavir was used as a post-exposure prophylaxis in health care workers and may have reduced the risk of infection. Ribavirin alone is unlikely to have substantial antiviral activities at clinically used dosages. Hence, ribavirin with or without corticosteroids and with lopinavir and ritonavir are among the combinations employed. This was the most common agent reported in the available literature. Its efficacy has been assessed in observational studies, retrospective case series, retrospective cohort study, a prospective observational study, a prospective cohort study and randomized controlled trial ranging from seven to 229 participants [117] . Lopinavir/ritonavir (Kaletra) was the earliest protease inhibitor combination introduced for the treatment of SARS-CoV. Its efficacy was documented in several studies, causing notably lower incidence of adverse outcomes than with ribavirin alone. Combined usage with ribavirin was also associated with lower incidence of acute respiratory distress syndrome, nosocomial infection and death, amongst other favorable outcomes. Recent in vitro studies have shown another HIV protease inhibitor, nelfinavir, to have antiviral capacity against SARS-CoV, although it has yet to show favorable outcomes in animal studies [118] . Remdesivir (Gilead Sciences, GS-5734) nucleoside analogue in vitro and in vivo data support GS-5734 development as a potential pan-coronavirus antiviral based on results against several coronaviruses (CoVs), including highly pathogenic CoVs and potentially emergent BatCoVs. The use of remdesivir may be a good candidate as an investigational treatment. Improved mortality following receipt of convalescent plasma in various doses was consistently reported in several observational studies involving cases with severe acute respiratory infections (SARIs) of viral etiology. A significant reduction in the pooled odds of mortality following treatment of 0.25 compared to placebo or no therapy was observed [119] . Studies were however at moderate to high risk of bias given their small sample sizes, allocation of treatment based on the physician's discretion, and the availability of plasma. Factors like concomitant treatment may have also confounded the results. Associations between convalescent plasma and hospital length of stay, viral antibody levels, and viral load respectively were similarly inconsistent across available literature. Convalescent plasma, while promising, is likely not yet feasible, given the limited pool of potential donors and issues of scalability. Monoclonal antibody treatment is progressing. SARS-CoV enters host cells through the binding of their spike (S) protein to angiotensin converting enzyme 2 (ACE2) and CD209L [118] . Human monoclonal antibodies to the S protein have been shown to significantly reduce the severity of lung pathology in non-human primates following MERS-CoV infection [120] . Such neutralizing antibodies can be elicited by active or passive immunization using vaccines or convalescent plasma respectively. While such neutralizing antibodies can theoretically be harvested from individuals immunized with vaccines, there is uncertainty over the achievement of therapeutic levels of antibodies. Other therapeutic agents have also been reported. A known antimalarial agent, chloroquine, elicits antiviral effects against multiple viruses including HIV type 1, hepatitis B and HCoV-229E. Chloroquine is also immunomodulatory, capable of suppressing the production and release of factors which mediate the inflammatory complications of viral diseases (tumor necrosis factor and interleukin 6) [121] . It is postulated that chloroquine works by altering ACE2 glycosylation and endosomal pH. Its anti-inflammatory properties may be beneficial for the treatment of SARS. Niclosamide as a known drug used in antihelminthic treatment. The efficacy of niclosamide as an inhibitor of virus replication was proven in several assays. In both immunoblot analysis and immunofluorescence assays, niclosamide treatment was observed to completely inhibit viral antigen synthesis. Reduction of virus yield in infected cells was dose dependent. Niclosamide likely does not interfere in the early stages of virus attachment and entry into cells, nor does it function as a protease inhibitor. Mechanisms of niclosamide activity warrant further investigation [122] . Glycyrrhizin also reportedly inhibits virus adsorption and penetration in the early steps of virus replication. Glycyrrhizin was a significantly potent inhibitor with a low selectivity index when tested against several pathogenic flaviviruses. While preliminary results suggest production of nitrous oxide (which inhibits virus replication) through induction of nitrous oxide synthase, the mechanism of Glycyrrhizin against SARS-CoV remains unclear. The compound also has relatively lower toxicity compared to protease inhibitors like ribavirin [123] . Inhibitory activity was also detected in baicalin [124] , extracted from another herb used in the treatment of SARS in China and Hong Kong. Findings on these compounds are limited to in vitro studies [121] [122] [123] [124] . Due to the rapidly evolving situation of the 2019-nCoV, there will be potential limitations to the systematic review. The systematic review is likely to have publication bias as some developments have yet to be reported while for other developments there is no intention to report publicly (or in scientific platforms) due to confidentiality concerns. However, this may be limited to only a few developments for review as publicity does help in branding to some extent for the company and/or the funder. Furthermore, due to the rapid need to share the status of these developments, there may be reporting bias in some details provided by authors of the scientific articles or commentary articles in traditional media. Lastly, while it is not viable for any form of quality assessment and metaanalysis of the selected articles due to the limited data provided and the heterogeneous style of reporting by different articles, this paper has provided a comprehensive overview of the potential developments of these pharmaceutical interventions during the early phase of the outbreak. This systematic review would be useful for cross-check when the quality assessment and meta-analysis of these developments are performed as a follow-up study. Rapid diagnostics, vaccines and therapeutics are key pharmaceutical interventions to limit transmission of respiratory infectious diseases. Many potential developments on these pharmaceutical interventions for 2019-nCoV are ongoing in the containment phase of this outbreak, potentially due to better pandemic preparedness than before. However, lessons from MERS-CoV and SARS-CoV have shown that the journeys for these developments can still be challenging moving ahead. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1 : Example of full search strategy in Pubmed, Table S2 : Google Search: 2019-nCoV diagnostics, Table S3 : Summary of diagnostic assays developed for 2019-nCoV, Table S4
Why were only four studies included?
false
3,644
{ "text": [ "Most studies on SARS and MERS vaccines were excluded as they were performed in cell or animal models" ], "answer_start": [ 11532 ] }
1,595
Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3 Inflammasome https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6361828/ SHA: f02d0c1e8b0109648e578662dc250abe349a033c Authors: Chen, I-Yin; Moriyama, Miyu; Chang, Ming-Fu; Ichinohe, Takeshi Date: 2019-01-29 DOI: 10.3389/fmicb.2019.00050 License: cc-by Abstract: Nod-like receptor family, pyrin domain-containing 3 (NLRP3) regulates the secretion of proinflammatory cytokines interleukin 1 beta (IL-1β) and IL-18. We previously showed that influenza virus M2 or encephalomyocarditis virus (EMCV) 2B proteins stimulate IL-1β secretion following activation of the NLRP3 inflammasome. However, the mechanism by which severe acute respiratory syndrome coronavirus (SARS-CoV) activates the NLRP3 inflammasome remains unknown. Here, we provide direct evidence that SARS-CoV 3a protein activates the NLRP3 inflammasome in lipopolysaccharide-primed macrophages. SARS-CoV 3a was sufficient to cause the NLRP3 inflammasome activation. The ion channel activity of the 3a protein was essential for 3a-mediated IL-1β secretion. While cells uninfected or infected with a lentivirus expressing a 3a protein defective in ion channel activity expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells infected with a lentivirus expressing the 3a protein. K(+) efflux and mitochondrial reactive oxygen species were important for SARS-CoV 3a-induced NLRP3 inflammasome activation. These results highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation. Text: Severe acute respiratory syndrome coronavirus (SARS-CoV), a member of the genus Betacoronavirus within the family Coronaviridae, is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 30 kb in length. The 5 two-thirds of the genome encodes large polyprotein precursors, open reading frame (ORF) 1 and ORF1b, which are proteolytically cleaved to generate 16 non-structural proteins (Tan et al., 2005) . The 3 one-third of the genome encodes four structural proteins, spike (S), envelope (E), matrix (M) and nucleocapsid (N), and non-structural proteins, along with a set of accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b) (Perlman and Dandekar, 2005; Tan et al., 2005) . SARS-CoV is the etiological agent of SARS (Drosten et al., 2003; Fouchier et al., 2003; Ksiazek et al., 2003; Kuiken et al., 2003; Peiris et al., 2003) . At least 8,098 laboratory-confirmed cases of human infection, with a fatality rate of 9.6%, were reported to the World Health Organization from November 2002 to July 2003. High levels of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, were detected in autopsy tissues from SARS patients (He et al., 2006) . Although dysregulation of inflammatory cytokines may be involved in lung injury and the pathogenesis of SARS-CoV, the underlying molecular mechanisms are not fully understood. The innate immune systems utilizes pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (Medzhitov, 2001; Kawai and Akira, 2010) . Recognition of virus infection plays an important role in limiting virus replication at the early stages of infection. Nod-like receptor family, pyrin domain-containing 3 (NLRP3) is activated by a wide variety of stimuli, including virus infection (Bauernfeind et al., 2011) . Four models describing activation of the NLRP3 inflammasome have been proposed thus far (Hornung and Latz, 2010; Schroder et al., 2010; Tschopp and Schroder, 2010) . First, the disturbances in intracellular ionic concentrations, including K + efflux and Ca 2+ influx, play an important role (Fernandes-Alnemri et al., 2007; Petrilli et al., 2007; Arlehamn et al., 2010; Ichinohe et al., 2010; Ito et al., 2012; Murakami et al., 2012; Munoz-Planillo et al., 2013) . Second, cathepsin B and L, which are specific lysosomal cysteine proteases, are though to play a role after phagocytosis of cholesterol crystals (Duewell et al., 2010) , fibrillar peptide amyloid-beta , silica crystals, and aluminum salts . Third is the release of reactive oxygen species (ROS) or mitochondrial DNA from damaged mitochondria (Zhou et al., , 2011 Nakahira et al., 2011; Shimada et al., 2012) . Finally, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 (Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015) . Upon activation, the NLRP3 is recruited to the mitochondria via association with mitochondrial antiviral signaling (MAVS) or mitofusin 2 expressed on the outer mitochondrial membrane Subramanian et al., 2013) ; these molecules then recruit the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and pro-caspase-1 to form the NLRP3 inflammasome. This event activates the downstream molecule, caspase-1, which catalyzes the proteolytic processing of pro-IL-1β and pro-IL-18 into their active forms and stimulates their secretion (Kayagaki et al., 2015; Shi et al., 2015) . It is increasingly evident that NLRP3 detects RNA viruses by sensing the cellular damage or distress induced by viroporins (Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015) , transmembrane pore-forming proteins, encoded by certain RNA viruses; these proteins alter membrane permeability to ions by forming membrane channels (Tan et al., 2005; Chen and Ichinohe, 2015) . A recent study shows that the SARS-CoV E protein, which comprise only 76 amino acids, forms Ca 2+ -permeable ion channels and activates the NLRP3 inflammasome (Nieto-Torres et al., 2015) . Although the E and 3a proteins of SARS-CoV, which comprise 274 amino acids and contain three transmembrane domains (Zeng et al., 2004; Lu et al., 2006) , are thought to act as Na + /K + and K + channels, respectively (Wilson et al., 2004; Lu et al., 2006; Torres et al., 2007; Parthasarathy et al., 2008; Pervushin et al., 2009; Wang et al., 2011) , the role of the 3a protein in activating the NLRP3 inflammasome remains unknown. Here, we examined the role of the 3a protein in activating the NLRP3 inflammasome. Six-week-old female C57BL/6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Animal Committees of the Institute of Medical Science (The University of Tokyo). Bone marrow-derived macrophages (BMMs) were prepared as described previously (Ichinohe et al., 2009) . In brief, bone marrow was obtained from the tibia and femur by flushing with Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque). Bone marrow cells were cultured for 5 days in DMEM supplemented with 30% L929 cell supernatant containing macrophage colony-stimulating factor, 10% heat-inactivated fetal bovine serum (FBS), and L-glutamine (2 mM) at 37 • C/5% CO 2 . HEK293FT cells (a human embryonic kidney cell line) and HeLa cells (a human epithelial carcinoma cell line) were maintained in DMEM supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 µg/ml) (Nacalai Tesque). MDCK cells (Madin-Darby canine kidney cells) and HT-1080 cells (a human fibrosarcoma cell line) were grown in Eagle's minimal essential medium (E-MEM; Nacalai Tesque) supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 µg/ml) (Nacalai Tesque). Influenza A virus strain A/PR8 (H1N1) was grown at 35 • C for 2 days in the allantoic cavities of 10-day-old fertile chicken eggs (Ichinohe et al., 2009) . The viral titer was quantified in a standard plaque assay using MDCK cells (Pang et al., 2013) . Plasmids cDNAs encoding the E and M proteins of SARS-CoV Frankfurt 1 strain (Matsuyama et al., 2005) were obtained by reverse transcription and PCR of total RNA extracted from SARS-CoVinfected Vero cells, followed by PCR amplification using specific primers. pcDNA3.1D-3a-V5His was provided by Ming-Fu Chang (National Taiwan University College of Medicine, Taipei, Taiwan). To generate the plasmids pLenti6-E-V5His, pLenti6-3a-V5His, and pLenti-M-V5His, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets and then ligated into pLenti6-TOPO vectors (Invitrogen). To generate plasmids pCA7-flag-E, pCA7-flag-3a, and pCA7flag-M, pCA7-HA-E, pCA7-HA-3a, and pCA7-HA-M, cDNA fragments of E, 3a, and M were amplified from pcDNA3.1D-E-V5His, pcDNA3.1D-3a-V5His, and pcDNA3.1D-M-V5His using specific primer sets, digested with EcoR I and Not I, and subcloned into the EcoR I-Not I sites of the pCA7-flag-ASC plasmid or pCA7-HA-M2 plasmid, respectively (Ito et al., 2012) . To construct plasmids expressing the E mutant V25F, the mutated E fragments were amplified by inverse PCR with wildtype E-containing plasmids and specific primer sets. The PCR products were cleaved by Dpn I, ligated in a ligase-and T4 kinase-containing reaction and then transformed into DH5α competent cells (TOYOBO). To construct plasmids expressing the 3a mutant 3a-CS, fragments were amplified from wildtype 3a-containing plasmids using 3a-specific primer sets and transformed as described above. HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 µg pLenti6-E/3a/M-V5His, pLenti-GFP (green fluorescent protein), or pLenti-M2 using polyethylenimine (PEI) Max. At 24 h post-transfection, the cells were lysed with RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.05% sodium dodecyl sulfate (SDS), 150 mM NaCl and 1 mM EDTA). And the lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by electroblotting onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated over night with mouse anti-V5-tag (R960-25, Invitrogen), mouse anti-influenza A virus M2 (14C2, Abcam), mouse anti-GFP (GF200, Nacalai Tesque), or rabbit antitubulin (DM1A, Santa Cruz) antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories) or anti-rabbit IgG (Invitrogen). After washing 3 times with washing buffer (0.05% Tween-20/PBS), the membranes were exposed using Chemi-Lumi One Super (Nacalai Tesque), and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus (GE Healthcare). To generate lentiviruses expressing V5-tagged SARS-CoV E, 3a, and M proteins, the full-length cDNA encoding each viral protein was cloned into the pLenti6.3/V5-TOPO vector (Invitrogen) using the following primers: SARS-CoV E forward, 5 -caccatgtactcattcgtttcgga-3 , and reverse, 5 -gaccagaagatcaggaactc-3 ; SARS-CoV 3a forward, 5caccatggatttgtttatgagatt-3 , and reverse, 5 -caaaggcacgctagtagtcg-3 ; SARS-CoV M forward, 5 -caccatggcagacaacggtactat-3 , and reverse, 5 -ctgtactagcaaagcaatat-3 . Sub-confluent monolayers of HEK293FT cells seeded in a collagen-coated dish (10 cm in diameter) were transfected with 3 µg of pLenti6.3/V5-TOPO vector expressing each viral protein or EGFP together with ViraPower Packaging Mix (Invitrogen) using Lipofectamine 2000 (Invitrogen). The supernatants containing lentiviruses were harvested and filtered through a 0.45 µm filter (Millipore) at 72-96 h post-transfection (Ito et al., 2012) . The lentiviral titer was then quantified using HT-1080 cells as described previously . Bone marrow-derived macrophages were plated at a density of 8 × 10 5 in 24-well plate and infected with A/PR8 influenza virus or lentivirus at a multiplicity of infection (MOI) of 5 or 0.2 for 1 h, respectively. Then, BMMs were stimulated with 1 µg/ml of LPS and cultured for additional 23 h in complete media. Supernatants were collected at 24 h post-infection and centrifuged to remove cell debris. The amount of IL-1β in the supernatants was measured in an enzyme-linked immunosorbent assay (ELISA) using paired antibodies (eBioscience) (Ichinohe et al., 2010 . To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 µg of ER-mCherry or DsRed-Golgi (Ito et al., 2012) . At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody (M2, Sigma) followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Life Technologies). To observe the cellular distribution of NLRP3 in the E-or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 µg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 µg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA (561, MBL) and mouse anti-NLRP3 (Cryo-2; AdipoGen) antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) and Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) (Life Technologies). Fluorescent signals were observed by confocal microscopy (A1R + , Nikon). Statistical significance was tested using a two-tailed Student's t-test. P-values < 0.05 were considered statistically significant. We previously demonstrated that the influenza virus M2 protein (a proton-selective ion channel), its H37G mutant (which has lost its proton selectivity and enables the transport of other cations such as Na + and K + ), and the EMCV 2B protein (a Ca 2+ channel) stimulates NLRP3 inflammasome-mediated IL-1β secretion (Ichinohe et al., 2010; Ito et al., 2012) . In addition, the SARS-CoV E protein acts as a Ca 2+ -permeable ion channels that activates the NLRP3 inflammasome (Nieto- Torres et al., 2015) . The fact that 3a protein of SARS-CoV acts as viroporin prompted us to examine whether it also triggers inflammasome activation. Thus, we first generated lentivirus plasmids expressing V5-tagged proteins and confirmed their expression in HEK293FT cells by immunoblot analysis (Figures 1A-C) . We next transduced lipopolysaccharide (LPS)-primed BMMs with the lentiviruses expressing the SARS-CoV E, 3a, M, influenza virus M2, or EMCV 2B proteins. Consistent with previous reports (Ichinohe et al., Figure 1D) . Similarly, the lentiviruses expressing the SARS-CoV E or 3a proteins stimulated IL-1β release from LPS-primed BMMs ( Figure 1D) . Furthermore, IL-1β secretion from LPSprimed BMMs co-infected with E-and 3a-expressing lentiviruses was significantly higher than that from SARS-CoV E-expressing lentivirus-infected cells ( Figure 1E) . These data indicated that the expression of SARS-CoV viroporin 3a is sufficient to stimulate IL-1β secretion by LPS-primed BMMs. Previous studies demonstrated that the N-terminal 40 amino acids of the SARS-CoV E protein are important for ion channel formation, and that mutations N15A and V25F [located in the transmembrane domain (from amino acid residues 7-38)] prevent ion conductivity (Wilson et al., 2004; Torres et al., 2007; Verdia-Baguena et al., 2012) . In addition, the SARS-CoV 3a protein contains a cysteine-rich domain (amino acid residues 127-133) that is involved in the formation of a homodimer to generate the ion channel (Lu et al., 2006; Chan et al., 2009) . Thus, mutation of the cysteine-rich domain blocks the ion conductivity by the 3a protein (Chan et al., 2009) . To this end, we substituted amino acids Cys-127, Cys-130, and Cys-133 within the cysteine-rich domain of the SARS-CoV 3a protein with serine to generate a lentivirus expressing the ion channel activity-loss mutant, 3a-CS (Chan et al., 2009; Figure 2A) . To test whether the ion channel activity of the SARS-CoV 3a protein is required to stimulate secretion of IL-1β, we transduced LPSprimed BMMs with lentiviruses expressing the SARS-CoV E, V25F, 3a, 3a-CS, or M proteins. Consistent with a previous report (Nieto -Torres et al., 2015) , we found that the V25F mutant lentivirus failed to stimulate IL-1β release from BMMs ( Figure 2B) . Notably, the 3a-CS mutant completely abrogated IL-1β secretion (Figure 2B) , suggesting that the ion channel activity of the 3a protein is required for SARS-CoV 3a-induced IL-1β secretion. FIGURE 4 | NLRP3 inflammasome activation by SARS-CoV 3a. HeLa cells were transfected with the expression plasmid encoding NLRP3 and that encoding HA-tagged SARS-CoV 3a, 3a-CS, E, or V25F, and by with a confocal microscope. Scale bars, 10 µm. Data are representative of at least three independent experiments. Next, we determined the subcellular localization of the SARS-CoV 3a protein using confocal microscopy. When the SARS-CoV Cell-free supernatants were collected at 24 h (lentiviruses) or 6 h (ATP) post-infection or stimulation, and analyzed for IL-1β by ELISA. Data are representative of at least three independent experiments, and indicate the mean ± SD; * * P < 0.01 and * * * P < 0.001. 3a protein was expressed in HeLa cells, we observed two main distribution patterns. Consistent with previous reports (Yu et al., 2004; Yuan et al., 2005) , the 3a protein localized to the Golgi apparatus ( Figure 3A ). In addition, the 3a proteins concentrated in spot structures, which mainly localized to the endoplasmic reticulum (ER) (Figure 3B ). By contrast, the 3a-CS mutant was concentrated in the Golgi apparatus rather than in the ER and did not form spot structures (Figures 3A,B) . We next examined the intracellular localization of NLRP3. Activation of the NLRP3 inflammasome led to a redistribution from the cytosol to the perinuclear space, a process considered as a hallmark of NLRP3 activation (Zhou et al., 2011; Ito et al., 2012; Johnson et al., 2013; Moriyama et al., 2016) . Although cells expressing the ion channel activity-loss mutants 3a-CS or V25F uniformly expressed NLRP3 throughout the cytoplasm, it was redistributed to the perinuclear region in SARS-CoV 3a-or E-expressing cells (Figure 4) . Together, these data provide evidence that the ion channel activity of the SARS-CoV 3a protein is essential for triggering the NLRP3 inflammasome. Both K + Efflux and ROS Production Are Involved in the IL-1β Release Induced by the SARS-CoV 3a Protein Finally, we investigated the mechanism by which SARS-CoV 3a triggers NLRP3 inflammasome activation. A previous study showed that the 3a protein of SARS-CoV acts as a K + channel (Lu et al., 2006) . In addition, K + efflux is a well-known activator of the NLRP3 inflammasome (Mariathasan et al., 2006; Petrilli et al., 2007) . These observations prompted us to examine whether K + efflux is required for 3a-mediated IL-1β secretion. To this end, BMMs in K + -rich medium were infected with influenza A virus or lentiviruses expressing the SARS-CoV E or 3a proteins. In agreement with a previous result (Ichinohe et al., 2010) , we found that IL-1β secretion caused by influenza virus was completely blocked when the extracellular K + concentration was increased to 130 mM ( Figure 5A) . The inhibitory effect of the K + -rich medium was also observed when cells were stimulated with lentiviruses expressing the SARS-CoV E or 3a proteins ( Figure 5B ). Since mitochondrial ROS are important for NLRP3 inflammasome activation (Nakahira et al., 2011; Zhou et al., 2011) , we next stimulated BMMs with extracellular ATP or lentiviruses expressing the SARS-CoV E or 3a proteins in the presence or absence of the antioxidant, Mito-TEMPO, a scavenger that is specific for mitochondrial ROS Trnka et al., 2009) . As reported previously (Nakahira et al., 2011; Ito et al., 2012) , treatment of BMMs with Mito-TEMPO completely blocked IL-1β secretion in response to ATP ( Figure 6A) . Similarly, IL-1β release induced by the SARS-CoV E and 3a proteins was significantly inhibited by Mito-TEMPO ( Figure 6B) . These observations indicate that the SARS-CoV 3a protein disrupts intracellular ionic concentrations and causes mitochondrial damages, thereby activating the NLRP3 inflammasome. In summary, we found that the ion channel activity of SARS-CoV 3a protein is essential for activation of the NLRP3 inflammasome. In addition, both K + efflux and mitochondrial ROS production are required for SARS-CoV 3a-mediated IL-1β secretion. Thus far, several models have been proposed to explain NLRP3 inflammasome activation by RNA viruses. First, viral RNA or RNA cleavage products generated by RNase L activate the NLRP3 inflammasome via the DExD/H-box helicase, DHX33 (Allen et al., 2009; Mitoma et al., 2013; Chen et al., 2014; Chakrabarti et al., 2015) . Second, viroporins encoded by RNA viruses activates the NLRP3 inflammasome (Ichinohe et al., 2010; Ito et al., 2012; Triantafilou et al., 2013; Nieto-Torres et al., 2015) . In the case of influenza virus, the proton-selective M2 ion channel in the acidic trans-Golgi network activates the NLRP3 inflammasome (Ichinohe et al., 2010) . Interestingly, an M2 mutant in which histidine was substituted with glycine at position 37 (H37G), causing loss of proton selectivity, enables transport of other cations (i.e., Na + and K + ), thereby leading to enhanced secretion of IL-1β from LPS-primed BMMs and dendritic cells when compared with the wild-type M2 protein. In addition, the 2B proteins of EMCV, poliovirus, enterovirus 71 (EV71), and human rhinovirus (a member of the Picornaviridae family) triggers NLRP3 inflammasome activation by inducing Ca 2+ flux from the ER and Golgi compartments (Ito et al., 2012; Triantafilou et al., 2013) . Furthermore, hepatitis C virus stimulates NLRP3 inflammasome-mediated IL-1β production though its p7 viroporin (Negash et al., 2013; Farag et al., 2017) . Third, a recent study has demonstrated that the 3D protein of EV71 directly interacts with NLRP3 to facilitate the assembly of NLRP3 inflammasome complex (Wang et al., 2017) . In the case of SARS-CoV, the viroporin E forms forms Ca 2+permeable ion channels and activates the NLRP3 inflammasome (Nieto-Torres et al., 2015) . In addition, another viroporin 3a was found to induce NLRP3 inflammasome activation (Yue et al., 2018) . Although alanine substitution at Cys-133, which is required for dimer or tetramer formation (Lu et al., 2006) , still allows activation of the NLRP3 inflammasome by interacting with caspase-1 (Yue et al., 2018) , the ion channel activity-loss mutant 3a-CS (Cys-to-Ser substitution at positions Cys-127, Cys-130, and Cys-133) (Chan et al., 2009 ) completely abrogated IL-1β secretion from LPS-primed BMMs, suggesting that the 3a protein of SARS-CoV has the ability to induce the NLRP3 inflammasome activation by multiple mechanisms. Previous studies show that the 3a protein of SARS-CoV is localized to the plasma membrane (Minakshi and Padhan, 2014) and acts as a K + channel (Lu et al., 2006) , thereby (presumably) stimulating the K + efflux at the plasma membrane. Indeed, we found that IL-1β secretion caused by the 3a protein was significantly inhibited when the extracellular K + concentration increased to 130 mM. Although it remains unclear whether another viroporin 8a of SARS-CoV (Castano-Rodriguez et al., 2018) activates the NLRP3 inflammasome, these data highlights the importance of viroporins in SARS-CoV-induced NLRP3 inflammasome activation. A better understanding of the mechanism that governs the NLRP3 inflammasome will facilitate the development of more effective interventions for the treatment of infectious diseases and increase our understanding of viral pathogenesis.
What are viroporins?
false
282
{ "text": [ "transmembrane pore-forming viral proteins" ], "answer_start": [ 1545 ] }
1,631
Clara Cell 10 kDa Protein Alleviates Murine Hepatitis Virus Strain 3-Induced Fulminant Hepatitis by Inhibiting Fibrinogen-Like Protein 2 Expression https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300492/ SHA: f0c2cd2793d71f1ea11a810442a2c06d5013e899 Authors: Yu, Haijing; Liu, Yang; Wang, Hongwu; Wan, Xiaoyang; Huang, Jiaquan; Yan, Weiming; Xi, Dong; Luo, Xiaoping; Shen, Guanxin; Ning, Qin Date: 2018-12-13 DOI: 10.3389/fimmu.2018.02935 License: cc-by Abstract: Background: Fulminant hepatitis (FH) is a serious threat to human life, accompanied by massive and rapid necroinflammation. Kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for FH. Fibrinogen-like protein 2 (Fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and Fgl2 depletion represses murine hepatitis virus strain 3 (MHV-3) infection. Clara cell 10 kDa (CC10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. However, its mechanisms of action and pathogenic roles in other disease are still unclear. In this study, we aimed to determine the role of CC10 in FH and the regulation of Fgl2 by CC10. Methods: A mouse FH model was established by peritoneal injection of MHV-3. The mice received CC10 protein through tail vein injection before viral infection. Survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. The regulatory effect of CC10 on Fgl2 expression was investigated using THP-1 cells and mouse peritoneal macrophages in vitro. Results: In the mouse FH model induced by MHV-3, the survival rate increased from 0 to 12.5% in the CC10 group compared to that in the saline-only control group. Meanwhile, the levels of ALT and AST in serum were significantly decreased and liver damage was reduced. Furthermore, hepatic Fgl2, TNF-α, and IL-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of CC10 protein. In vitro, CC10 was found to significantly inhibit the expression of Fgl2 in IFN-γ-treated THP-1 cells and MHV-3-infected mouse peritoneal macrophages by western blot and real-time PCR. However, there was no direct interaction between CC10 and Fgl2 as shown by co-immunoprecipitation. Microarray investigations suggested that HMG-box transcription factor 1 (HBP1) was significantly low in CC10-treated and IFN-γ-primed THP-1 cells. HBP1-siRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in Human Umbilical Vein Endothelial cells (HUVECs). Conclusion:CC10 protects against MHV-3-induced FH via suppression of Fgl2 expression in macrophages. Such effects may be mediated by the transcription factor HBP1. Text: Fulminant hepatitis (FH) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. The underlying mechanisms and the pathogenesis of FH are not clear. However, accumulating evidence suggests that, regardless of the pathogenesis of FH, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. Accordingly, It has been shown that immune cell activation and inflammatory cytokines play an important role in FH (1) . In recent years, our laboratory has conducted extensive research on the pathogenesis of FH and found that immune cells play a key role in it. Kupffer cells, natural killer (NK) cells (2, 3) , cytotoxic T-lymphocytes (CTLs), and double negative T-cells (DNT) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. Prothrombinase Fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. This promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . Our study found that Fgl2 was highly expressed in peripheral blood mononuclear cells (PBMCs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . Gene therapy targeting Fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . Thus far, the discovery and related research involving Fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in FH. In view of the important role of Fgl2 in severe viral hepatitis, investigations concerning the regulation of Fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. Clara cell 10 kDa protein (CC10), also considered to be uteroglobin, Clara cell secretory protein, is one of members of secretoglobin superfamily. Expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . CC10 has immunomodulatory and anti-inflammatory effects. Compared to wild-type mice, CC10-knockout mice exhibited excessive airway inflammation Abbreviations: FH, fulminant hepatitis; MHV-3, murine hepatitis virus strain 3; Fgl2, Fibrinogen-like protein 2; CC10, Clara cell 10 KDa protein; ALF, acute liver failure; PFU, plaque-forming units; PBS, phosphate-buffered saline; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCA, pro-coagulant activity; HRP, horseradish peroxidase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . Reduced levels of CC10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . Previous studies and published articles show that CC10 protein can not only inhibit Th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . Moreover, CC10 inhibits the expression of an important immune regulator, osteopontin (OPN), in models of allergic rhinitis (21) . In this study, we investigated the role of CC10 in hepatitis virus strain 3 (MHV-3)-induced FH in mice and explored whether CC10 protein could regulate Fgl2 in the disease process. Female BALB/cJ mice (Shanghai Shilaike Animal Seed Center, Shanghai, China), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in Tongji Hospital with food and water. Mice were divided into two groups: CC10 group (experimental group) and phosphate-buffered saline (PBS) group (control group). This study was carried out in accordance with the recommendations of the guidelines of the National Institutes of Health and the Animal Experiment Committee of Tongji hospital. This study was reviewed and approved by the Animal Experiment Committee of Tongji hospital. The human monocyte cell line THP-1 was purchased from the Cell Institute of the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from the Biology Treasure Center of Wuhan University, China. The Chinese hamster ovary (CHO) cell line was acquired from the typical culture preservation commission cell bank, the Chinese Academy of Sciences (Shanghai, China). Human Umbilical Vein Endothelial Cells (HUVECs) and CHO cells were cultured in Dulbecco's modified Eagle's medium (DMEM), and THP-1 cells were maintained in RPMI 1,640 containing 10% heat inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin and cultured at 37 • C, 50 mL/L CO 2 and 95% humidity. Peritoneal exudative macrophages (PEMs) were obtained from BALB/cJ mice. Cells were resuspended in RPMI 1,640 supplemented with 10% FBS at 1-2 × 10 6 cells/mL in a 6-well plate and incubated for 4 h. They were then washed with RPMI 1640 medium and non-adherent cells discarded. The adherent cells were macrophages and were incubated for a further 12 h. Peritoneal exudative macrophages (PEMs) were divided into two groups. One group was supplemented with CC10 protein (150 ng/mL) and in the other group, PBS was added. After 2 h of stimulation, 1,000 plaque forming units (PFUs) of MHV-3 was added to the cells, which were then cultured for 4 h. Peritoneal exudative macrophages (PEMs) were harvested and lysed for real-time PCR and western blotting analysis. Cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method with a TUNEL apoptosis detection kit (Roche, Switzerland). Briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase K for 30 min at 37 • C. After stopping the proteinase K digestion reaction with PBS, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dUTP at a ratio of 2:29, respectively), for 2 h at 37 • C in an immunohistochemistry wet box. Following washing and blocking, each section was supplemented with reagent (converter-POD) to cover the tissues and incubated for 30 min at 37 • C in a wet box. Then, the liver tissue sections were washed with PBS, and colored with diaminobenzidine (DAB) subsequently. Hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. The expression of Fgl2 on THP-1 cells was measured by flow cytometry (BD FACS Canto II, USA). Briefly, cells (2 × 10 5 per tube) were incubated with Human TruStrain FcX (Fc Receptor Blocking solution, BioLegend, USA) for 10 min at room temperature and then incubated in the dark with mouse anti-Fgl2 antibody (1:100, Abnova,) or normal goat serum (an isotype control) at 4 • C for 40 min. Cells were washed with PBS and incubated in the dark with PE-conjugated goat anti-mouse IgG antibody (1:50, BioLegend, USA) at 4 • C for 30 min. Cells were then washed with PBS and resuspended in 300 µL PBS for study. Liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. Immunohistochemistry of liver tissues was performed using SP-9001 SPlink Detection Kits (Biotin-Streptavidin HRP Detection Systems) (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. For immunohistochemistry staining, the expression of Fgl2, fibrinogen, Fas and TNF-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse Fgl2 antibody (1:100, Proteintech, USA), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, Abcam, EngLand), polyclonal rabbit antimouse Fas antibody (1:50, Abcam, EngLand), and polyclonal rabbit anti-mouse TNF-receptor 1 antibody (1:500, Abcam, EngLand), respectively. After incubation with an horseradish peroxidase (HRP)-labeled goat IgG fraction to rabbit IgG Fc, the target protein was detected using a DAB kit (ZSGB-BIO, Beijing, China). The slides were then counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Liver tissue and cells were homogenized in RIPA lysis buffer with phenyl methane sulfonyl fluoride (PMSF) protease inhibitor. Protein lysates were separated by SDS-PAGE, and western blotting was performed using a monoclonal mouse antihuman/mouse Fgl2 (1:750, Abnova), a monoclonal mouse antihuman HBP1 (1:100, Santa Cruz, USA), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, Cell Signaling Technology, USA). Liver tissues were collected from MHV-3-infected BALB/cJ mice at 72 h, and total RNA was extracted using Trizol Reagent (Invitrogen, USA) and then reverse transcribed into cDNA by using ReverTra Ace qPCR RT kit (TOYOBO, Japan). The cDNA was then amplified by RT-PCR by using Dream Taq Green PCR Master Mix (2 ×) (Thermo Scientific, USA). Realtime quantitative PCR (qPCR) with SYBR Green Real-time PCR Master Mix (TOYOBO, Japan) was performed using a CFX96 real-time PCR detection system (Bio-Rad, USA) and mRNA levels were normalized with reference to those of the house keeping gene GAPDH. Primer sequences for qPCR amplification were as follows: mTNF-α forward, 5 ′ -TTT GAG ATC CAT GCC GTT GG-3 ′ ; mTNF-α reverse, 5 ′ -GCCA CCA CGC TCT TCT GT-3 ′ ; mIL-1β forward, 5 ′ -TGT AAT GAA AGA CGG CAC ACC-3 ′ ; mIL-1β reverse, 5 ′ -TCT TCT TTG GGT ATT GCT TGG-3 ′ . mFgl2 forward, 5 ′ -GCC AAA TGT GAG TCC CTG GAA-3 ′ ; mFgl2 reverse, 5 ′ -TTC CAC CCA AGA GCA CGT TTA AG-3 ′ ; hFgl2 forward 5 ′ -ACA GTT CAG GCT GGT GGT-3 ′ ; hFgl2 reverse, 5 ′ -GGC TTA AAG TGC TTG GGT-3 ′ ; HBP1 forward, 5 ′ -TGA AGC AGA AGC TGG GAGT-3 ′ ; HBP1 reverse, THP-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, USA) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . The CC10 group was supplemented with CC10 protein (150 ng/ml). After 2 h of stimulation, IFN-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time PCR studies. The Chinese hamster ovary (CHO) cells were cultured in 10 cm cell culture dishes with DMEM supplemented with 10% FBS until 80-90% confluence. Next, 12 µg pcDNA3.1-hFgl2 (constructed in our lab) was mixed with 12 µg pcDNA3.1-hCC10 in serumfree DMEM. The mixture was then combined with Lipofectamine 2,000 (Invitrogen, USA) and mixed gently. After incubation at 27 • C for 20 min, the solution was added to CHO cells and incubated at 37 • C in 5% CO 2 . Four to Six hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of CC10 with Fgl2. Both HUVEC and THP-1 cells express fgl2. However, in the transfection experiments, it is difficult to transfect the THP-1 cells with siRNA, so we use HUVEC instead of THP-1. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured in FIGURE 1 | CC10 protein increased survival rate and reduced liver damage in mice. (A) The survival rate of CC10 group is higher than the control group comprised of MHV-3-infected BALB/cJ mice treated with saline. CC10 protein (2 µg) or saline were injected into mice by tail vein. BALB/cJ mice then received 100 PFU of MHV-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. Then, CC10 protein (2 µg) or saline were injected into mice by tail vein following MHV-3 infection 24 h later. The survival rate was observed for 10 days (n = 24/group). Representative data from three independent experiments are shown. The survival curve was analyzed by using the Log-Rank Test. ***P < 0.001 compared with saline group. (B) Histopathology of liver tissues (H&E staining; original magnification, ×400, n = 5/group) at 72 h post-MHV-3 infection was evaluated in the two groups of MHV-3-infected BALB/cJ mice. Livers were collected from saline-treated (a) and CC10-treated (b) BALB/cJ mice at 72 h after MHV-3 infection. Arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (C) Effect of CC10 on serum ALT and AST levels (n = 6-8/group). Values represent means and standard error of three independent experiments performed in triplicate. **P < 0.01 compared with the saline group. six-well plates with DMEM supplemented with 10% FBS until 70-80% confluence. 50 pmol HBP1-siRNA was mixed with 125 µl serum-free DMEM. Two microliter Lipofectamine 2,000 was gently mixed with serum-free DMEM. After incubation at 27 • C for 5 min, the solution was added to HUVECs and incubated at 37 • C. Four hour after transfection, the medium was removed and fresh medium containing 10% FBS was added. At 48 h after transfection, cells were collected for real-time PCR and western blot analysis to evaluate the effects of HBP1 on Fgl2. At 24 h after transfection, the CC10 group was supplemented with the CC10 protein (150 ng/mL). After 4 h of stimulation, IFN-γ (10 ng/mL) was added to these cells. These cells were then cultured for 24 h before they were harvested for real-time PCR studies to evaluate the effects of CC10 on Fgl2 by HBP1. Negative control was used as a control. To detect whether there was a potential interaction between CC10 protein and Fgl2, CHO cells were transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2 for 48 h. Cells transfected with empty plasmid pcDNA3.1 (mock) were used as negative controls for CC10 gene transfection. Immunoprecipitation and immunoblotting were performed by using Pierce Co-Immunoprecipitation Kit (Pierce, USA). Total cell proteins were extracted as previously described (25) . The proteins were immunoprecipitated by mouse anti-human Fgl2 antibody (1:500, Abnova). For co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/SCGB1A1 Antibody (1:750, R&D, USA) Frontiers in Immunology | www.frontiersin.org and mouse anti-human Fgl2 antibody (1:500, Abnova). Control isotype rat IgG1 was used as a negative control for primary antibodies. The human CC10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by RT-PCR. In this study, the sequences of PCR primers for CC10 were as follows: hCC10-forward, 5 ′ -CCC TCC ACC ATG AAA CTCG-3 ′ ; hCC10-reverse, 5 ′ -TGA GAT GCT TGT GGT TTA TTG AAG-3 ′ . The PCR products were cloned into pEASY-T1 cloning vector (TransGEN, Beijing, China) and then subcloned into HindIII/XbaI site of pcDNA3.1 vector (Invitrogen, USA) to form eukaryotic expression plasmids pcDNA3.1-hCC10. Microarray analysis was used to screen changes in genome-wide gene expression patterns in THP-1 cells with or without CC10 protein. The changes in over 47,000 human gene expression patterns were assessed using Affymetrix gene microarrays (Human Genome U133 Plus 2.0) (CapitalBio Co.,Ltd., Beijing, China). Three replicates were used for microarrays analysis. Data obtained from the experiments are expressed as means ± SEM. Survival curve comparisons were performed with the Log Rank test. Multiple group analyses for data were evaluated by one-way analyses of variance. Analyses of two group results were performed using Student's t-test to evaluate the statistical significance of differences. Values of P < 0.05 indicated significance. To establish an animal model of mouse FH, MHV-3 was injected intraperitoneally to BALB/cJ mice (24 mice/group). To further study the role of CC10 in FH, recombinant mouse CC10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to MHV-3 infection. The same dose of CC10 protein or saline was then administered 24 h later. The survival rate of the CC10 and saline groups was observed for 10 days. The results showed that mice in the two groups began to die at 48 h after injection of MHV-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. In the CC10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. At the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. That is to say, the mice in the saline group died within 3 or 4 days. Three of 24 (12.5%) mice of the CC10 group recovered from fulminant viral hepatitis ( Figure 1A) . To better understand the mechanisms underlying the biological effects of the CC10 protein, liver function (ALT and AST levels in serum) and liver histology in mice of MHV-3-infected was performed. Liver tissues were harvested 72 h following MHV-3 infection, and liver histology was detected by H&E staining. These results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (Figure 1Ba ). There were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the CC10 group 72 h after MHV-3 infection (Figure 1Bb) . Serum ALT and AST levels in mice were observed 72 h after MHV-3 infection. The results showed that serum ALT and AST levels in the saline group reached a peak 72 h after MHV-3 infection, but there was no significant increase in the CC10 group compared to the levels in the control group (P < 0.01, Figure 1C) . These results suggested that CC10 protein has a role in protection against MHV-3-induced liver injury in mice. To further elucidate the mechanisms of reduced liver injury following CC10 protein injection, we investigated the cytokines TNF-α and IL-1β expression. Because these two cytokines play a crucial role in the liver damage of FH. They are characterized by an increase in apoptosis. Levels of TNF-α and IL-1β in liver tissues were markedly reduced in the CC10 group (as shown in Figure 2A) . Hepatic apoptosis (Figure 2B ) was significantly reduced in the CC10 group. We and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. Fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. We have provided liver pathology figures and liver function for MHV-3 infected mice with a fgl2 gene knockout as shown in Supplementary Figure 1 . The data was comparable with previous reports from our center and collaborators. From this current study we shown that CC10 plays a protective role in liver damage.To study the related molecules of CC10 in MHV-3-induced FH mice, we evaluated whether there was crosstalk between Fgl2 and CC10. We found that the expression of Fgl2 in the liver of mice was reduced 72 h after MHV-3 infection and treatment with CC10 protein (Figures 3A,B) . Furthermore, fibrin deposition, an indicator of liver injury associated with Fgl2 expression in FH, was also decreased in the livers of CC10-treated mice compared to that in controls (Figure 3C ). This indicates that CC10 treatment reduced liver injury after viral infection by inhibiting Fgl2 expression. We examined the effect of increasing doses of CC10 protein (0, 50, 150, and 300 ng/mL) on IFN-γ-induced Fgl2 expression in THP-1 cells. CC10 treatment showed a 10.1% decrease in THP-1 cells compared to that in control after stimulation with 10 ng/mL IFN-γ for 12 h. CC10 protein inhibited Fgl2 expression between doses of 0 ng/mL and 300 ng/mL (Figure 4A ). In particular, 150 ng/mL CC10 protein had the strongest inhibitory effect on Fgl2 expression among the doses, and we chose this dose for the following experiments. We explored the effect of different time points of stimulation with a concentration of 150 ng/mL CC10 protein. After stimulation with CC10 protein for 6, 12, and 24 h compared to the PBS control, the strongest inhibitory effect on Fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( Figure 4B ). An increasing number of studies suggest that macrophages are the primary source of Fgl2. In order to ascertain that CC10 has a direct effect on macrophages, we treated THP-1 cells with recombinant CC10 and assessed the expression of Fgl2. Unlike in controls, IFN-γ induced a significant increase in Fgl2 expression. This effect was attenuated when cells were treated with CC10 protein (Figures 4C,D) , revealing that CC10 directly reduces the levels of Fgl2 in macrophages. To further explore the possibility that CC10 protein directly acts on macrophages, we infected murine PEMs with MHV-3 in the presence of recombinant CC10 and determined Fgl2 expression. Compared to levels in the controls, MHV-3infected macrophages exhibited a significant increase in Fgl2 production, and this effect was abolished by using CC10 protein (Figures 5A,B) , indicating that CC10 directly modulates Fgl2 production in macrophages. In order to determine genes that were downregulated after stimulation by CC10 protein, we used DNA microarray analysis to screen for differentially expressed genes. THP-1 cells were cultured and PMA was added to induce differentiation into macrophages. The production of Fgl2 was stimulated by IFNγ. The experimental group was treated with CC10 protein for microarray detection of differentially expressed genes. The results showed that the most obviously downregulated genes were UBE2W, HECTD1, MIR612, ATRX, SOX4, HBP1, and Fgl2 (Supplementary Table 1) . And then these genes were tested by qPCR. However, UBE2W, HECTD1, MIR612, ATRX, and SOX4 was not differentially expressed by qPCR, while HBP1 and fgl2 were still down-regulated genes. DNA microarray analysis identified HBP1 as a down-regulated gene involved in the pathological processes of the regulation of CC10. Recently, very limited studies have explored the role of HBP1 in FH. Nevertheless, the mechanistic functions of HBP1 in FH remain largely unexplored. Therefore, we selected this gene for further study. qPCR analysis confirmed that mRNA levels of HBP1 were significantly decreased in THP-1 cells after CC10 protein stimulation compared to that in the PBS control group (Figure 6A ). We knocked down HBP1 using HBP1-siRNA. Then, transfection of HBP1-SiRNA into HUVECs was detected by qPCR and western-blotting methods. As expected, HBP1 knockdown led to significantly decreased expression of HBP1 (Figures 6B,C) . Furthermore, HBP1 knockdown impaired expression of Fgl2 (Figure 6D ), suggesting that HBP1 was able to activate Fgl2. HBP1-SiRNA was used to transfect HUVECs. Then, IFN-γ was added to induce the expression of Fgl2 followed by stimulation with CC10 protein (150 ng/ml) after 2 h. Finally, we explored the expression of Fgl2 by qPCR. The results showed that HBP1-SiRNA treatment abrogated the inhibitory effect of CC10 on Fgl2 expression in HUVECs (Figure 7) . That is to say, CC10 could suppress Fgl2 expression in macrophages. Such an effect may be mediated by the transcription factor HBP1. It is well-known that CC10 protein can suppress the immune response. In animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . Its function in FH has not been investigated yet. Here, we used a murine FH model established by MHV-3 infection to explore the effects of CC10 in this disease process. To determine the role of CC10 in the pathogenesis of FH, CC10 protein was injected into a mouse FH model established by MHV-3 infection. MHV-3-induced liver injury in CC10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. In summary, these results suggested that CC10 could reduce pathological liver damage in this FH model together with lower mortality rates followed by MHV-3 infection. MHV-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. Previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . Multiple inflammatory factors or mediators including TNF-α and IFN-γ, IL-1β and C5aR have been demonstrated to promote FH progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that CC10 substantially alleviated liver injury though survival rate improved mildly. The survival rate based on hours may be more accurate to examine the effect of CC10 on FH. It is speculated that fgl2 can mediate lethality in MHV-3-induced FH. This is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . To determine whether the tissue necrosis was mediated by Fgl2 in CC10-treated mice following infection, Fgl2 expression was observed. Results suggested that the expression of Fgl2 was significantly increased in MHV-3-induced FH mice and CC10 treatment significantly reduced the production of Fgl2 in the infected liver and serum. In addition, decreased fibrinogen deposition was also observed in the livers of CC10-treated mice. Therefore, our research results strongly clarify that the lower mortality of CC10-treated mice after MHV-3 infection is due to the lower levels of Fgl2 and decreased fibrinogen deposition. Indeed, it has been reported that Fgl2 is expressed on macrophages, and the expression of Fgl2 is believed to be induced by IFN-γ and TNF-α (22) . Cultured THP-1 cells activated by IFN-γ or IL-2 have been demonstrated, with induction of Fgl2 expression and enhanced activation of human prothrombin (23) . Therefore, in this study, we explored this cell line to investigate the modulation of CC10 on Fgl2. Surprisingly, we found that CC10 directly inhibited IFN-γ-induced Fgl2 expression in THP-1 cells. As we know, IFN-γ has proved to be the main cytokine that leads to the development and progression of FH. Also, it was shown that IFN-γ might exert its own proinflammatory biological function through enhancing Fgl2 expression. Therefore, in our study, CC10 might counter the effect of IFN-γ in the setting of FH, which substantiates its role in FH. These results demonstrated that CC10 regulates the expression of Fgl2 in macrophages. In the current study, we used co-immunoprecipitation to analyze binding between CC10 and Fgl2. In this study, we investigated possible protein-protein interactions between CC10 and Fgl2 in vitro. The Chinese hamster ovary (CHO) cells transfected with pcDNA3.1-hCC10 and pcDNA3.1-hFgl2. Cellular proteins were immunoprecipitated with anti-CC10 antibody or anti-Fgl2 antibody. Immunoblotting was performed with anti-Fgl2 and anti-CC10 antibodies. Immunoprecipitation of protein extracts from pcDNA 3.1-CC10 and pcDNA3.1-Fgl2 co-transfected CHO cells with anti-Fgl2 or anti-CC10 antibody followed by western blotting with Fgl2 and CC10 antibodies indicated that CC10 did not co-immunoprecipitate with Fgl2, showing that there is no direct relationship between CC10 and Fgl2 (data not shown). The results showed that CC10 has no direct interaction with Fgl2. From our previous study the gene of fgl2 contributed profoundly in MHV-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . Our microarray indicated a CC10 down-regulated fgl2 expression and this is further confirmed by qPCR and Western blotting in vivo (peritoneal macrophages) and in vitro (THP-1, macrophage cell line). Therefore, it is reasonable to focus on macrophages to display the effect of CC10 on fgl2 expression and eventually mice survival. We entirely agree there may be other possibilities for a protective effect of CC10 to contribute to the disease process. This is worth further studies. The potential receptor of CC10 has not been revealed yet. Our previous study have demonstrated that CC10 have effect of dendritic cells in allergic rhinitis (34) . In this research, we evaluated the effect of CC10 on macrophages functions and found Fgl2 was substantially down-regulated upon CC10 treatment, therefore, we speculate that potential CC10 receptor may be also expressed on macrophages. The potential target of CC10 on other immune cells cannot be excluded. DNA microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. By using this approach, HMGbox transcription factor 1 (HBP1) was found to be one of the most downregulated genes after CC10 treatment of THP-1 cells. HBP1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. In a recent study, it was found that HBP1 is a direct target of miR-21 and confirmed that HBP1 modulates the inhibitory function of miR-21-ASO in hepatosteatosis and carcinogenesis simultaneously (23) . HBP1 is an endogenous inhibitor of the Wnt signaling pathway in both normal and cancer cells. The tumor suppressor role of HBP1 has been reported in some malignancies, such as oral cancer and glioma (35) . However, an association between HBP1 and Fgl2 has not been investigated yet. The current study clearly demonstrated that CC10 protects against MHV-3 induced FH via suppression of Fgl2 expression. Such effects might be mediated by HBP1. However, the functional status of HBP1 in the CC10 pathway requires further research, and such studies are conducting in our laboratory. In conclusion, we demonstrated that CC10 could limit the immunopathological damage in MHV-3-induced FH mice. Our results suggest that enhancing CC10 expression by an immunotherapeutic approach might be an effective treatment for FH. HY performed all the described experiments and wrote the manuscript. YL assisted with some experiments, analyzed experimental results, and edited the manuscript. HW analyzed experimental results. XW reviewed and edited the manuscript. JH, WY, DX, XL, GS, and QN provided experimental help and design.
How does Prothrombinase Fgl2 affect the coagulation process?
false
5,296
{ "text": [ "transforming prothrombin directly into thrombin," ], "answer_start": [ 3792 ] }
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?
false
4,369
{ "text": [ "16 %" ], "answer_start": [ 50413 ] }
1,719
Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.
What is one of the issues with present vaccines?
false
1,246
{ "text": [ "low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly" ], "answer_start": [ 1987 ] }
2,642
First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, 24 January to 21 February 2020 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068164/ SHA: ce358c18aac69fc83c7b2e9a7dca4a43b0f60e2e Authors: Spiteri, Gianfranco; Fielding, James; Diercke, Michaela; Campese, Christine; Enouf, Vincent; Gaymard, Alexandre; Bella, Antonino; Sognamiglio, Paola; Sierra Moros, Maria José; Riutort, Antonio Nicolau; Demina, Yulia V.; Mahieu, Romain; Broas, Markku; Bengnér, Malin; Buda, Silke; Schilling, Julia; Filleul, Laurent; Lepoutre, Agnès; Saura, Christine; Mailles, Alexandra; Levy-Bruhl, Daniel; Coignard, Bruno; Bernard-Stoecklin, Sibylle; Behillil, Sylvie; van der Werf, Sylvie; Valette, Martine; Lina, Bruno; Riccardo, Flavia; Nicastri, Emanuele; Casas, Inmaculada; Larrauri, Amparo; Salom Castell, Magdalena; Pozo, Francisco; Maksyutov, Rinat A.; Martin, Charlotte; Van Ranst, Marc; Bossuyt, Nathalie; Siira, Lotta; Sane, Jussi; Tegmark-Wisell, Karin; Palmérus, Maria; Broberg, Eeva K.; Beauté, Julien; Jorgensen, Pernille; Bundle, Nick; Pereyaslov, Dmitriy; Adlhoch, Cornelia; Pukkila, Jukka; Pebody, Richard; Olsen, Sonja; Ciancio, Bruno Christian Date: 2020-03-05 DOI: 10.2807/1560-7917.es.2020.25.9.2000178 License: cc-by Abstract: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters’ index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. Text: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters' index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. A cluster of pneumonia of unknown origin was identified in Wuhan, China, in December 2019 [1] . On 12 January 2020, Chinese authorities shared the sequence of a novel coronavirus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from some clustered cases [2] . Since then, the disease caused by SARS-CoV-2 has been named coronavirus disease 2019 (COVID -19) . As at 21 February 2020, the virus had spread rapidly mostly within China but also to 28 other countries, including in the World Health Organization (WHO) European Region [3] [4] [5] . Here we describe the epidemiology of the first cases of COVID-19 in this region, excluding cases reported in the United Kingdom (UK), as at 21 February 2020. The study includes a comparison between cases detected among travellers from China and cases whose infection was acquired due to subsequent local transmission. On 27 January 2020, the European Centre for Disease Prevention and Control (ECDC) and the WHO Regional Office for Europe asked countries to complete a WHO standard COVID-19 case report form for all confirmed and probable cases according to WHO criteria [6] [7] [8] . The overall aim of surveillance at this time was to support the global strategy of containment of COVID-19 with rapid identification and follow-up of cases linked to affected countries in order to minimise onward transmission. The surveillance objectives were to: describe the key epidemiological and clinical characteristics of COVID-19 cases detected in Europe; inform country preparedness; and improve further case detection and management. Data collected included demographics, history of recent travel to affected areas, close contact with a probable or confirmed COVID-19 case, underlying conditions, signs and symptoms of disease at onset, type of specimens from which the virus was detected, and clinical outcome. The WHO case definition was adopted for surveillance: a confirmed case was a person with laboratory confirmation of SARS-CoV-2 infection (ECDC recommended two separate SARS-CoV-2 RT-PCR tests), irrespective of clinical signs and symptoms, whereas a probable case was a suspect case for whom testing for SARS-CoV-2 was inconclusive or positive using a pan-coronavirus assay [8] . By 31 January 2020, 47 laboratories in 31 countries, including 38 laboratories in 24 European Union and European Economic Area (EU/EEA) countries, had diagnostic capability for SARS-CoV-2 available (close to 60% of countries in the WHO European Region), with cross-border shipment arrangements in place for many of those lacking domestic testing capacity. The remaining six EU/EEA countries were expected to have diagnostic testing available by mid-February [9] . As at 09:00 on 21 February 2020, 47 confirmed cases of COVID-19 were reported in the WHO European Region and one of these cases had died [4] . Data on 38 of these cases (i.e. all except the nine reported in the UK) are included in this analysis. The first three cases detected were reported in France on 24 January 2020 and had onset of symptoms on 17, 19 and 23 January respectively [10] . The first death was reported on 15 February in France. As at 21 February, nine countries had reported cases ( Figure) : Belgium (1), Finland (1), France (12), Germany (16), Italy (3), Russia (2), Spain (2), Sweden (1) and the UK (9 -not included further). The place of infection (assessed at national level based on an incubation period presumed to be up to 14 days [11] , travel history and contact with probable or confirmed cases as per the case definition) was reported for 35 cases (missing for three cases), of whom 14 were infected in China (Hubei province: 10 cases; Shandong province: one case; province not reported for three cases). The remaining 21 cases were infected in Europe. Of these, 14 were linked to a cluster in Bavaria, Germany, and seven to a cluster in Haute-Savoie, France [12, 13] . Cases from the Bavarian cluster were reported from Germany and Spain, whereas cases from the Haute-Savoie cluster were reported from France All but two cases were hospitalised (35 of 37 where information on hospitalisation was reported), although it is likely that most were hospitalised to isolate the person rather than because of severe disease. The time from onset of symptoms to hospitalisation (and isolation) ranged between 0 and 10 days with a mean of 3.7 days (reported for 29 cases). The mean number of days to hospitalisation was 2.5 days for cases imported from China, but 4.6 days for those infected in Europe. This was mostly a result of delays in identifying the index cases of the two clusters in France and Germany. In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six took only a mean of 2 days to be hospitalised. Symptoms at the point of diagnosis were reported for 31 cases. Two cases were asymptomatic and remained so until tested negative. The asymptomatic cases were tested as part of screening following repatriation and during contact tracing respectively. Of the remaining 29, 20 reported fever, 14 reported cough and eight reported weakness. Additional symptoms reported included headaches (6 cases), sore throat (2), rhinorrhoea (2), shortness of breath (2), myalgia (1), diarrhoea (1) and nausea (1). Fever was reported as the sole symptom for nine cases. In 16 of 29 symptomatic cases, the symptoms at diagnosis were consistent with the case definition for acute respiratory infection [16] , although it is possible that cases presented additional symptoms after diagnosis and these were not reported. Data on pre-existing conditions were reported for seven cases; five had no pre-existing conditions while one was reported to be obese and one had pre-existing cardiac disease. No data on clinical signs e.g. dyspnea etc. were reported for any of the 38 cases. All hospitalised cases had a benign clinical evolution except four, two reported in Italy and two reported in France, all of whom developed viral pneumonia. All three cases who were aged 65 years or over were admitted to intensive care and required respiratory support and one French case died. The case who died was hospitalised for 21 days and required intensive care and mechanical ventilation for 19 days. The duration of hospitalisation was reported for 16 cases with a median of 13 days (range: 8-23 days). As at 21 February 2020, four cases were still hospitalised. All cases were confirmed according to specific assays targeting at least two separate genes (envelope (E) gene as a screening test and RNA-dependent RNA polymerase (RdRp) gene or nucleoprotein (N) gene for confirmation) [8, 17] . The specimen types tested were reported for 27 cases: 15 had positive nasopharyngeal swabs, nine had positive throat swabs, three cases had positive sputum, two had a positive nasal swab, one case had a positive nasopharyngeal aspirate and one a positive endotracheal aspirate. As at 09:00 on 21 February, few COVID-19 cases had been detected in Europe compared with Asia. However the situation is rapidly developing, with a large outbreak recently identified in northern Italy, with transmission in several municipalities and at least two deaths [18] . As at 5 March 2020, there are 4,250 cases including 113 deaths reported among 38 countries in the WHO European region [19] . In our analysis of early cases, we observed transmission in two broad contexts: sporadic cases among travellers from China (14 cases) and cases who acquired infection due to subsequent local transmission in Europe (21 cases). Our analysis shows that the time from symptom onset to hospitalisation/case isolation was about 3 days longer for locally acquired cases than for imported cases. People returning from affected areas are likely to have a low threshold to seek care and be tested when symptomatic, however delays in identifying the index cases of the two clusters in France and Germany meant that locally acquired cases took longer to be detected and isolated. Once the exposure is determined and contacts identified and quarantined (171 contacts in France and 200 in Germany for the clusters in Haute-Savoie and Bavaria, respectively), further cases are likely to be rapidly detected and isolated when they develop symptoms [15, 20] . In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six were hospitalised after a mean of 2 days. Locally acquired cases require significant resources for contact tracing and quarantine, and countries should be prepared to allocate considerable public health resources during the containment phase, should local clusters emerge in their population. In addition, prompt sharing of information on cases and contacts through international notification systems such as the International Health Regulations (IHR) mechanism and the European Commission's European Early Warning and Response System is essential to contain international spread of infection. All of the imported cases had a history of travel to China. This was consistent with the epidemiological situation in Asia, and supported the recommendation for testing of suspected cases with travel history to China and potentially other areas of presumed ongoing community transmission. The situation has evolved rapidly since then, however, and the number of countries reporting COVID-19 transmission increased rapidly, notably with a large outbreak in northern Italy with 3,089 cases reported as at 5 March [18, 19] . Testing of suspected cases based on geographical risk of importation needs to be complemented with additional approaches to ensure early detection of local circulation of COVID-19, including through testing of severe acute respiratory infections in hospitals irrespectively of travel history as recommended in the WHO case definition updated on 27 February 2020 [21] . The clinical presentation observed in the cases in Europe is that of an acute respiratory infection. However, of the 31 cases with information on symptoms, 20 cases presented with fever and nine cases presented only with fever and no other symptoms. These findings, which are consistent with other published case series, have prompted ECDC to include fever among several clinical signs or symptoms indicative for the suspected case definition. Three cases were aged 65 years or over. All required admission to intensive care and were tourists (imported cases). These findings could reflect the average older age of the tourist population compared with the local contacts exposed to infection in Europe and do not allow us to draw any conclusion on the proportion of severe cases that we could expect in the general population of Europe. Despite this, the finding of older individuals being at higher risk of a severe clinical course is consistent with the evidence from Chinese case series published so far although the majority of infections in China have been mild [22, 23] . This preliminary analysis is based on the first reported cases of COVID-19 cases in the WHO European Region. Given the small sample size, and limited completeness for some variables, all the results presented should be interpreted with caution. With increasing numbers of cases in Europe, data from surveillance and investigations in the region can build on the evidence from countries in Asia experiencing more widespread transmission particularly on disease spectrum and the proportion of infections with severe outcome [22] . Understanding the infection-severity is critical to help plan for the impact on the healthcare system and the wider population. Serological studies are vital to understand the proportion of cases who are asymptomatic. Hospital-based surveillance could help estimate the incidence of severe cases and identify risk factors for severity and death. Established hospital surveillance systems that are in place for influenza and other diseases in Europe may be expanded for this purpose. In addition, a number of countries in Europe are adapting and, in some cases, already using existing sentinel primary care based surveillance systems for influenza to detect community transmission of SARS-CoV-2. This approach will be used globally to help identify evidence of widespread community transmission and, should the virus spread and containment no longer be deemed feasible, to monitor intensity of disease transmission, trends and its geographical spread. Additional research is needed to complement surveillance data to build knowledge on the infectious period, modes of transmission, basic and effective reproduction numbers, and effectiveness of prevention and case management options also in settings outside of China. Such special studies are being conducted globally, including a cohort study on citizens repatriated from China to Europe, with the aim to extrapolate disease incidence and risk factors for infection in areas with community transmission. Countries together with ECDC and WHO, should use all opportunities to address these questions in a coordinated fashion at the European and global level. provided input to the outline, multiple versions of the manuscript and gave approval to the final draft.
What was the median case age?
false
3,796
{ "text": [ "42 years" ], "answer_start": [ 2026 ] }
2,459
No credible evidence supporting claims of the laboratory engineering of SARS-CoV-2 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054935/ SHA: 5a9154aee79901dd8fecd58b7bcd9b7351102d24 Authors: Liu, Shan-Lu; Saif, Linda J.; Weiss, Susan R.; Su, Lishan Date: 2020-02-26 DOI: 10.1080/22221751.2020.1733440 License: cc-by Abstract: nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 (COVID-19) (https://globalbiodefense. com/novel-coronavirus-covid-19-portal/). According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity [4, 5] . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV (RaTG13) was recently reported, which shared ∼96% homology with the SARS-CoV-2 [4] . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV [4] , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (https:// www.nature.com/articles/d41586-020-00364-2). Another claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 (>5,000 nucleotides). The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice (hence M15), due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells [10, 11] . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry [8] . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice [7] . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy (https://www.nih.gov/about-nih/who-weare/nih-director/statements/nih-lifts-funding-pausegain-function-research). The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups [5, 14] , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv (a manuscript sharing site prior to any peer review), claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author(s). Susan R. Weiss http://orcid.org/0000-0002-8155-4528
What were the results of this test?
false
3,608
{ "text": [ "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice" ], "answer_start": [ 4425 ] }
1,592
Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112080/ SHA: f01ad3545245b4f884b48aa2b69c9deb942c3e77 Authors: Aldunate, Fabián; Echeverría, Natalia; Chiodi, Daniela; López, Pablo; Sánchez-Cicerón, Adriana; Fajardo, Alvaro; Soñora, Martín; Cristina, Juan; Hernández, Nelia; Moreno, Pilar Date: 2018-08-14 DOI: 10.1155/2018/2514901 License: cc-by Abstract: Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity. Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] . Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] . The prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] . In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region. Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee). 2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol. 2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (http://www.macrogen.com). 2.4. NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates). For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] . To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1 Genotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions. To further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades. With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 . RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ). In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein. This issue limited our studies, since many of the described RASs are observed as of residue 553. Importantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed. Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] . As was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ). The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] . Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country. We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] . The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, https://www.gilead.com/-/ media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] . We did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] . The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use. Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: http:// www.hcv-trials.com/showStudy.asp?Study=86). RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV. In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] . These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population. To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions. The data used to support the findings of this study are included within the article. The authors declare that they have no conflicts of interest. Fabián Aldunate and Natalia Echeverría contributed equally to this work. Supplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)
How many RASs to NS5A inhibitors were identified?
false
3,901
{ "text": [ "2 strains out of 25 (8%)" ], "answer_start": [ 11338 ] }
2,642
First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, 24 January to 21 February 2020 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068164/ SHA: ce358c18aac69fc83c7b2e9a7dca4a43b0f60e2e Authors: Spiteri, Gianfranco; Fielding, James; Diercke, Michaela; Campese, Christine; Enouf, Vincent; Gaymard, Alexandre; Bella, Antonino; Sognamiglio, Paola; Sierra Moros, Maria José; Riutort, Antonio Nicolau; Demina, Yulia V.; Mahieu, Romain; Broas, Markku; Bengnér, Malin; Buda, Silke; Schilling, Julia; Filleul, Laurent; Lepoutre, Agnès; Saura, Christine; Mailles, Alexandra; Levy-Bruhl, Daniel; Coignard, Bruno; Bernard-Stoecklin, Sibylle; Behillil, Sylvie; van der Werf, Sylvie; Valette, Martine; Lina, Bruno; Riccardo, Flavia; Nicastri, Emanuele; Casas, Inmaculada; Larrauri, Amparo; Salom Castell, Magdalena; Pozo, Francisco; Maksyutov, Rinat A.; Martin, Charlotte; Van Ranst, Marc; Bossuyt, Nathalie; Siira, Lotta; Sane, Jussi; Tegmark-Wisell, Karin; Palmérus, Maria; Broberg, Eeva K.; Beauté, Julien; Jorgensen, Pernille; Bundle, Nick; Pereyaslov, Dmitriy; Adlhoch, Cornelia; Pukkila, Jukka; Pebody, Richard; Olsen, Sonja; Ciancio, Bruno Christian Date: 2020-03-05 DOI: 10.2807/1560-7917.es.2020.25.9.2000178 License: cc-by Abstract: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters’ index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. Text: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters' index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. A cluster of pneumonia of unknown origin was identified in Wuhan, China, in December 2019 [1] . On 12 January 2020, Chinese authorities shared the sequence of a novel coronavirus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from some clustered cases [2] . Since then, the disease caused by SARS-CoV-2 has been named coronavirus disease 2019 (COVID -19) . As at 21 February 2020, the virus had spread rapidly mostly within China but also to 28 other countries, including in the World Health Organization (WHO) European Region [3] [4] [5] . Here we describe the epidemiology of the first cases of COVID-19 in this region, excluding cases reported in the United Kingdom (UK), as at 21 February 2020. The study includes a comparison between cases detected among travellers from China and cases whose infection was acquired due to subsequent local transmission. On 27 January 2020, the European Centre for Disease Prevention and Control (ECDC) and the WHO Regional Office for Europe asked countries to complete a WHO standard COVID-19 case report form for all confirmed and probable cases according to WHO criteria [6] [7] [8] . The overall aim of surveillance at this time was to support the global strategy of containment of COVID-19 with rapid identification and follow-up of cases linked to affected countries in order to minimise onward transmission. The surveillance objectives were to: describe the key epidemiological and clinical characteristics of COVID-19 cases detected in Europe; inform country preparedness; and improve further case detection and management. Data collected included demographics, history of recent travel to affected areas, close contact with a probable or confirmed COVID-19 case, underlying conditions, signs and symptoms of disease at onset, type of specimens from which the virus was detected, and clinical outcome. The WHO case definition was adopted for surveillance: a confirmed case was a person with laboratory confirmation of SARS-CoV-2 infection (ECDC recommended two separate SARS-CoV-2 RT-PCR tests), irrespective of clinical signs and symptoms, whereas a probable case was a suspect case for whom testing for SARS-CoV-2 was inconclusive or positive using a pan-coronavirus assay [8] . By 31 January 2020, 47 laboratories in 31 countries, including 38 laboratories in 24 European Union and European Economic Area (EU/EEA) countries, had diagnostic capability for SARS-CoV-2 available (close to 60% of countries in the WHO European Region), with cross-border shipment arrangements in place for many of those lacking domestic testing capacity. The remaining six EU/EEA countries were expected to have diagnostic testing available by mid-February [9] . As at 09:00 on 21 February 2020, 47 confirmed cases of COVID-19 were reported in the WHO European Region and one of these cases had died [4] . Data on 38 of these cases (i.e. all except the nine reported in the UK) are included in this analysis. The first three cases detected were reported in France on 24 January 2020 and had onset of symptoms on 17, 19 and 23 January respectively [10] . The first death was reported on 15 February in France. As at 21 February, nine countries had reported cases ( Figure) : Belgium (1), Finland (1), France (12), Germany (16), Italy (3), Russia (2), Spain (2), Sweden (1) and the UK (9 -not included further). The place of infection (assessed at national level based on an incubation period presumed to be up to 14 days [11] , travel history and contact with probable or confirmed cases as per the case definition) was reported for 35 cases (missing for three cases), of whom 14 were infected in China (Hubei province: 10 cases; Shandong province: one case; province not reported for three cases). The remaining 21 cases were infected in Europe. Of these, 14 were linked to a cluster in Bavaria, Germany, and seven to a cluster in Haute-Savoie, France [12, 13] . Cases from the Bavarian cluster were reported from Germany and Spain, whereas cases from the Haute-Savoie cluster were reported from France All but two cases were hospitalised (35 of 37 where information on hospitalisation was reported), although it is likely that most were hospitalised to isolate the person rather than because of severe disease. The time from onset of symptoms to hospitalisation (and isolation) ranged between 0 and 10 days with a mean of 3.7 days (reported for 29 cases). The mean number of days to hospitalisation was 2.5 days for cases imported from China, but 4.6 days for those infected in Europe. This was mostly a result of delays in identifying the index cases of the two clusters in France and Germany. In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six took only a mean of 2 days to be hospitalised. Symptoms at the point of diagnosis were reported for 31 cases. Two cases were asymptomatic and remained so until tested negative. The asymptomatic cases were tested as part of screening following repatriation and during contact tracing respectively. Of the remaining 29, 20 reported fever, 14 reported cough and eight reported weakness. Additional symptoms reported included headaches (6 cases), sore throat (2), rhinorrhoea (2), shortness of breath (2), myalgia (1), diarrhoea (1) and nausea (1). Fever was reported as the sole symptom for nine cases. In 16 of 29 symptomatic cases, the symptoms at diagnosis were consistent with the case definition for acute respiratory infection [16] , although it is possible that cases presented additional symptoms after diagnosis and these were not reported. Data on pre-existing conditions were reported for seven cases; five had no pre-existing conditions while one was reported to be obese and one had pre-existing cardiac disease. No data on clinical signs e.g. dyspnea etc. were reported for any of the 38 cases. All hospitalised cases had a benign clinical evolution except four, two reported in Italy and two reported in France, all of whom developed viral pneumonia. All three cases who were aged 65 years or over were admitted to intensive care and required respiratory support and one French case died. The case who died was hospitalised for 21 days and required intensive care and mechanical ventilation for 19 days. The duration of hospitalisation was reported for 16 cases with a median of 13 days (range: 8-23 days). As at 21 February 2020, four cases were still hospitalised. All cases were confirmed according to specific assays targeting at least two separate genes (envelope (E) gene as a screening test and RNA-dependent RNA polymerase (RdRp) gene or nucleoprotein (N) gene for confirmation) [8, 17] . The specimen types tested were reported for 27 cases: 15 had positive nasopharyngeal swabs, nine had positive throat swabs, three cases had positive sputum, two had a positive nasal swab, one case had a positive nasopharyngeal aspirate and one a positive endotracheal aspirate. As at 09:00 on 21 February, few COVID-19 cases had been detected in Europe compared with Asia. However the situation is rapidly developing, with a large outbreak recently identified in northern Italy, with transmission in several municipalities and at least two deaths [18] . As at 5 March 2020, there are 4,250 cases including 113 deaths reported among 38 countries in the WHO European region [19] . In our analysis of early cases, we observed transmission in two broad contexts: sporadic cases among travellers from China (14 cases) and cases who acquired infection due to subsequent local transmission in Europe (21 cases). Our analysis shows that the time from symptom onset to hospitalisation/case isolation was about 3 days longer for locally acquired cases than for imported cases. People returning from affected areas are likely to have a low threshold to seek care and be tested when symptomatic, however delays in identifying the index cases of the two clusters in France and Germany meant that locally acquired cases took longer to be detected and isolated. Once the exposure is determined and contacts identified and quarantined (171 contacts in France and 200 in Germany for the clusters in Haute-Savoie and Bavaria, respectively), further cases are likely to be rapidly detected and isolated when they develop symptoms [15, 20] . In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six were hospitalised after a mean of 2 days. Locally acquired cases require significant resources for contact tracing and quarantine, and countries should be prepared to allocate considerable public health resources during the containment phase, should local clusters emerge in their population. In addition, prompt sharing of information on cases and contacts through international notification systems such as the International Health Regulations (IHR) mechanism and the European Commission's European Early Warning and Response System is essential to contain international spread of infection. All of the imported cases had a history of travel to China. This was consistent with the epidemiological situation in Asia, and supported the recommendation for testing of suspected cases with travel history to China and potentially other areas of presumed ongoing community transmission. The situation has evolved rapidly since then, however, and the number of countries reporting COVID-19 transmission increased rapidly, notably with a large outbreak in northern Italy with 3,089 cases reported as at 5 March [18, 19] . Testing of suspected cases based on geographical risk of importation needs to be complemented with additional approaches to ensure early detection of local circulation of COVID-19, including through testing of severe acute respiratory infections in hospitals irrespectively of travel history as recommended in the WHO case definition updated on 27 February 2020 [21] . The clinical presentation observed in the cases in Europe is that of an acute respiratory infection. However, of the 31 cases with information on symptoms, 20 cases presented with fever and nine cases presented only with fever and no other symptoms. These findings, which are consistent with other published case series, have prompted ECDC to include fever among several clinical signs or symptoms indicative for the suspected case definition. Three cases were aged 65 years or over. All required admission to intensive care and were tourists (imported cases). These findings could reflect the average older age of the tourist population compared with the local contacts exposed to infection in Europe and do not allow us to draw any conclusion on the proportion of severe cases that we could expect in the general population of Europe. Despite this, the finding of older individuals being at higher risk of a severe clinical course is consistent with the evidence from Chinese case series published so far although the majority of infections in China have been mild [22, 23] . This preliminary analysis is based on the first reported cases of COVID-19 cases in the WHO European Region. Given the small sample size, and limited completeness for some variables, all the results presented should be interpreted with caution. With increasing numbers of cases in Europe, data from surveillance and investigations in the region can build on the evidence from countries in Asia experiencing more widespread transmission particularly on disease spectrum and the proportion of infections with severe outcome [22] . Understanding the infection-severity is critical to help plan for the impact on the healthcare system and the wider population. Serological studies are vital to understand the proportion of cases who are asymptomatic. Hospital-based surveillance could help estimate the incidence of severe cases and identify risk factors for severity and death. Established hospital surveillance systems that are in place for influenza and other diseases in Europe may be expanded for this purpose. In addition, a number of countries in Europe are adapting and, in some cases, already using existing sentinel primary care based surveillance systems for influenza to detect community transmission of SARS-CoV-2. This approach will be used globally to help identify evidence of widespread community transmission and, should the virus spread and containment no longer be deemed feasible, to monitor intensity of disease transmission, trends and its geographical spread. Additional research is needed to complement surveillance data to build knowledge on the infectious period, modes of transmission, basic and effective reproduction numbers, and effectiveness of prevention and case management options also in settings outside of China. Such special studies are being conducted globally, including a cohort study on citizens repatriated from China to Europe, with the aim to extrapolate disease incidence and risk factors for infection in areas with community transmission. Countries together with ECDC and WHO, should use all opportunities to address these questions in a coordinated fashion at the European and global level. provided input to the outline, multiple versions of the manuscript and gave approval to the final draft.
What testing and detection are needed?
false
3,839
{ "text": [ "Testing of suspected cases based on geographical risk of importation needs to be complemented with additional approaches to ensure early detection of local circulation of COVID-19, including through testing of severe acute respiratory infections in hospitals irrespectively of travel history as recommended in the WHO case definition updated on 27 February 2020" ], "answer_start": [ 11773 ] }
1,719
Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.
What opportunity has the termination of smallpox vaccination provided?
false
1,636
{ "text": [ "has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns" ], "answer_start": [ 28943 ] }
776
It is Unlikely That Influenza Viruses Will Cause a Pandemic Again Like What Happened in 1918 and 1919 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019839/ Song, Liting 2014-05-07 DOI:10.3389/fpubh.2014.00039 License:cc-by Abstract: nan Text: Influenza and influenza viruses are wellknown popular topics to medical professionals and the general public. Influenza viruses had caused a pandemic globally during 1918 and 1919, and that influenza pandemic had taken away more than 20 million people's lives in the world. However, in my opinion, it is unlikely that influenza viruses will again cause a pandemic on a level (both of the morbidity rate and the mortality rate) comparable to what happened in 1918 and 1919. Influenza viruses very easily reassort, recombine, and point mutate in nature due to their segmented RNA genome structures, however, unlike highly pathogenic (virulent) viruses like rabies virus, Lassa fever virus, smallpox virus, eastern equine encephalitis virus, Ebola virus, Marburg virus, and human immunodeficiency virus 1 (HIV-1); most influenza viruses (wild types and mutants) are moderately pathogenic. The case fatality rates of some highly virulent viruses and related references are listed in Table 1 . On November 11, 1918 , the fighting of World War I was stopped, and World War I was officially ended on June 28, 1919 with the signing of the Versailles Treaty. It is estimated that around 8.5-10 million soldiers lost their lives in World War I due to battle. The war also directly caused more than 6 million civilian deaths. Millions of people suffered from hunger and malnutrition during the war. Malnutrition weakened the human immune system and made a person more vulnerable to infectious diseases like tuberculosis and influenza, therefore, hunger and malnutrition were indirectly responsible for millions of deaths in the world in that period of time. For example, about 700,000 Germans died from malnutrition-related diseases in the years of 1914-1918. During the 1918-1919 influenza pandemic, between 21 and 25 million people died of influenza worldwide. Those people were killed both directly and indirectly by influenza virus infections. Many families were too poor to buy food and coal, and to afford health care expenses when their family members were ill. Influenza virus could infect all members of a family, and this could result in no one left to feed the fires, and to prepare food for the whole family, even if they had firewood, coal, and food left in their homes. Sadly, a large number of people died of influenza virus infections along with starvation, cold, and poor living conditions (8) . In recent years, while hunger and malnutrition are not major and serious problems in some developed countries anymore, they are still very difficult to overcome in many developing countries. In these less-developed countries, there were approximately 925 million people who suffered from hunger; 125 million children were underweight; and 195 million children were stunted each year (9) . Nevertheless, in comparison to 1918 and 1919, currently, we have much better social and economic conditions and public health systems globally; and generally speaking, the majority of people in the world have better nutritional and educational statuses; better living and working conditions; therefore, better general health and immunity. Furthermore, in 1918 and 1919, physicians and nurses almost had nothing in their hands to help individuals who were infected by influenza viruses. Today, although we still do not have very effective, powerful, and practical anti-influenza drugs available, we at least have some improved, useful, and helpful anti-viral drugs like zanamivir, and effective, convenient anti-cold medicines like Tylenol or Advil. We do not have a universal vaccine to prevent all influenza virus infections, but we can make effective vaccines to a specific influenza virus strain in a short time. Actually, in the United States of America, the influenza classed mortality rate declined from 10.2/100,000 in the 1940s to 0.56/100,000 in the 1990s; and the classed mortality rates of 1957-1958 and 1968-1969 influenza pandemics were not remarkably different from the non-pandemic seasons (10) . Because of the above reasons, we can optimistically assume that even the same strain of influenza virus, which caused pandemic in 1918 and 1919, would not be able to kill millions of people and cause a pandemic comparable to the 1918-1919 pandemic again in the future. Additionally, a significant number of viruses can cause influenza-like syndromes, such as rhinovirus, parainfluenza virus, adenovirus, coronavirus, respiratory syncytial virus, Coxsackie B virus, echovirus, and metapneumovirus (11, 12) . Some of the above-mentioned viruses like adenovirus and mutated coronavirus could cause problems that are comparable to influenza viruses (13, 14) . The World Health Organization (WHO) mistakenly raised the level of influenza pandemic alert from phase 5 to the highest phase 6 on June 11, 2009 (15) . However, the truth was that most cases of H1N1 influenza A virus infections were mild, the symptomatic case fatality rate was only 0.005% in New Zealand (16) ; and in New York City, the case fatality rate was 0.0094-0.0147% for persons ≥65 years old, and for those of 0-17 years old, the case fatality rate was 0.0008-0.0012% (17) . Some researchers argued that it should not have been called an influenza pandemic in the first place if the clinical severity was considered (15, (18) (19) (20) . I believe it was unwise that we had paid too much www.frontiersin.org 23) . Not surprisingly, every year there would be some influenza patients and a few of them would die from the infections, as it is almost impossible to eliminate influenza viruses from the natural environment in many years. The severity of a viral infection is determined by both of the viral virulence (pathogenicity) and the host immunity. Some researchers' opinions on H7N9 avian influenza virus were incorrect and/or inadequate. They mainly focused on influenza viruses and worried about viral mutations, viral pathogenicity, viral adaptation, and transmission. They overestimated the negative part of socio-economic factors of the present east China: overcrowded population in the epidemic region; very busy national and international transportation and travel; a large number of live poultry markets . . . but they underestimated the currently changed, developed, and improved positive part of socio-economic factors in China. The following factors might be used to explain why that H7N9 influenza A virus epidemic was limited and controlled in China, and only a few immunocompromised patients were killed by H7N9 influenza A virus. First, China has a relatively organized and effective public health system, there are four levels of (national, provincial, prefectural-level city, and county) centers for disease control and prevention all over China (24) . Second, physicians and nurses in China were prepared and knowledgeable of influenza virus infections. Third, samples from patients with suspected influenza virus infections were collected and sent to the local and national centers for disease control and prevention promptly. H7N9 influenza A viruses were isolated and identified very quickly. Thereby, they were able to diagnose, confirm, and report three cases of H7N9 influenza patients in the early stage of the epidemic (24, 25) . Fourth, health care and public health workers were protected properly. Consequently, none of the health professionals was infected by H7N9 influenza A virus in 2013. However, a surgeon died of H7N9 influenza in Shanghai, China in January of 2014 (26) . Fifth, they detected H7N9 influenza A viruses from the samples of chickens, pigeons, and the environment of live poultry markets in Shanghai (27) ; and closed the live poultry markets of the involved epidemic region quickly. Sixth, patients were isolated and treated timely in hospitals, 74% (1251/1689) of those close contacts of H7N9 influenza patients were monitored and observed. Thus, H7N9 influenza A virus could not spread to a bigger population (24) . Last but not least, we are connected to the Internet now, and it seems that our planet is much smaller today than the earlier days when we did not have the Internet, because communication and information exchange have become so fast, easy, and convenient presently. During that avian influenza epidemic, some influenza experts in the world shared/exchanged H7N9 influenza A virus information and provided professional consultations and suggestions efficiently and rapidly. All these public health routine practices and measures resulted in that H7N9 influenza epidemic being controlled and stopped in China (24) . I have to point out that the cases of diagnosed H7N9 avian influenza A virus infection might only be the tip of the iceberg. Aside from one laboratory confirmed asymptotic case of H7N9 influenza A virus infection in Beijing (22), there were probably many undetected mild or asymptotic cases of influenza A H7N9 infection. The reason is that most people usually think a common cold is a very common and normal occurrence, and they don't take flu-like illnesses seriously. In most situations, they would just stay home and take some medicines. Only those who have very severe flu-like symptoms would see doctors, and thereby be detected and diagnosed, accordingly the real case fatality rate should be much lower than the detected 32.14% (45/140, one case from Taiwan, and one case from Hong Kong) (22, 23). Nowadays, we travel faster, and we travel more frequently and globally, and we have more complicated social activities and lifestyles, thereby increasing the chances of viral mutation; and we realize that influenza viruses are even easier to reassort, recombine, and mutate in nature than many other RNA viruses. However, we are now living in a technologically, economically, and socially much better and advanced society. I believe influenza virus infections are controllable and preventable, with the increased population health and immunity, with the WHO Global Influenza Surveillance and Response System, and with standard/routine epidemiological practices, and with new effective anti-viral agents and vaccines in production in the future. Now, I first predict that influenza viruses will unlikely again cause a pandemic on a level comparable to what happened in 1918 and 1919. Hopefully, one day we could consider a strategy to produce a universal vaccine that can prevent people from infections of all influenza virus strains, or we could produce some very effective anti-influenza virus drugs; then influenza would not be a problem anymore. We should learn lessons from the mistakes we made in the past. It is reasonable and necessary to be cautious about influenza viruses, but overreactions or catastrophic reactions should be avoided in the future. My opinion is anti-traditional; the purpose of this article is to influence public health policy, and to save some of the limited resources and money for more important diseases like heart diseases, cancer, diabetes, AIDS, hepatitises, and tuberculosis (15) . Liting Song: conception of manuscript, drafting of manuscript, critical revision of manuscript, and final approval of manuscript. The author would like to recognize the contributions of the reviewers and editors of this manuscript for their corrections and editing, and Dr. Emanuel Goldman for correcting errors related to grammar and syntax of the final manuscript.
What factors would be responsible in future for the prevention of an Influenza pandemic?
false
302
{ "text": [ "influenza virus infections are controllable and preventable, with the increased population health and immunity, with the WHO Global Influenza Surveillance and Response System, and with standard/routine epidemiological practices, and with new effective anti-viral agents and vaccines in production in the future." ], "answer_start": [ 10047 ] }
1,674
Beyond phage display: non-traditional applications of the filamentous bacteriophage as a vaccine carrier, therapeutic biologic, and bioconjugation scaffold https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523942/ SHA: f00f183d0bce0091a02349ec1eab44a76dad9bc4 Authors: Henry, Kevin A.; Arbabi-Ghahroudi, Mehdi; Scott, Jamie K. Date: 2015-08-04 DOI: 10.3389/fmicb.2015.00755 License: cc-by Abstract: For the past 25 years, phage display technology has been an invaluable tool for studies of protein–protein interactions. However, the inherent biological, biochemical, and biophysical properties of filamentous bacteriophage, as well as the ease of its genetic manipulation, also make it an attractive platform outside the traditional phage display canon. This review will focus on the unique properties of the filamentous bacteriophage and highlight its diverse applications in current research. Particular emphases are placed on: (i) the advantages of the phage as a vaccine carrier, including its high immunogenicity, relative antigenic simplicity and ability to activate a range of immune responses, (ii) the phage’s potential as a prophylactic and therapeutic agent for infectious and chronic diseases, (iii) the regularity of the virion major coat protein lattice, which enables a variety of bioconjugation and surface chemistry applications, particularly in nanomaterials, and (iv) the phage’s large population sizes and fast generation times, which make it an excellent model system for directed protein evolution. Despite their ubiquity in the biosphere, metagenomics work is just beginning to explore the ecology of filamentous and non-filamentous phage, and their role in the evolution of bacterial populations. Thus, the filamentous phage represents a robust, inexpensive, and versatile microorganism whose bioengineering applications continue to expand in new directions, although its limitations in some spheres impose obstacles to its widespread adoption and use. Text: The filamentous bacteriophage (genera Inovirus and Plectrovirus) are non-enveloped, rod-shaped viruses of Escherichia coli whose long helical capsids encapsulate a single-stranded circular DNA genome. Subsequent to the independent discovery of bacteriophage by Twort (1915) and d 'Hérelle (1917) , the first filamentous phage, f1, was isolated in Loeb (1960) and later characterized as a member of a larger group of phage (Ff, including f1, M13, and fd phage) specific for the E. coli conjugative F pilus (Hofschneider and Mueller-Jensen, 1963; Marvin and Hoffmann-Berling, 1963; Zinder et al., 1963; Salivar et al., 1964) . Soon thereafter, filamentous phage were discovered that do not use F-pili for entry (If and Ike; Meynell and Lawn, 1968; Khatoon et al., 1972) , and over time the list of known filamentous phage has expanded to over 60 members (Fauquet et al., 2005) , including temperate and Gram-positivetropic species. Work by multiple groups over the past 50 years has contributed to a relatively sophisticated understanding of filamentous phage structure, biology and life cycle (reviewed in Marvin, 1998; Rakonjac et al., 2011; Rakonjac, 2012) . In the mid-1980s, the principle of modifying the filamentous phage genome to display polypeptides as fusions to coat proteins on the virion surface was invented by Smith and colleagues (Smith, 1985; Parmley and Smith, 1988) . Based on the ideas described in Parmley and Smith (1988) , groups in California, Germany, and the UK developed phage-display platforms to create and screen libraries of peptide and folded-protein variants (Bass et al., 1990; Devlin et al., 1990; McCafferty et al., 1990; Scott and Smith, 1990; Breitling et al., 1991; Kang et al., 1991) . This technology allowed, for the first time, the ability to seamlessly connect genetic information with protein function for a large number of protein variants simultaneously, and has been widely and productively exploited in studies of proteinprotein interactions. Many excellent reviews are available on phage-display libraries and their applications (Kehoe and Kay, 2005; Bratkovic, 2010; Pande et al., 2010) . However, the phage also has a number of unique structural and biological properties that make it highly useful in areas of research that have received far less attention. Thus, the purpose of this review is to highlight recent and current work using filamentous phage in novel and nontraditional applications. Specifically, we refer to projects that rely on the filamentous phage as a key element, but whose primary purpose is not the generation or screening of phagedisplayed libraries to obtain binding polypeptide ligands. These tend to fall into four major categories of use: (i) filamentous phage as a vaccine carrier; (ii) engineered filamentous phage as a therapeutic biologic agent in infectious and chronic diseases; (iii) filamentous phage as a scaffold for bioconjugation and surface chemistry; and (iv) filamentous phage as an engine for evolving variants of displayed proteins with novel functions. A final section is dedicated to recent developments in filamentous phage ecology and phage-host interactions. Common themes shared amongst all these applications include the unique biological, immunological, and physicochemical properties of the phage, its ability to display a variety of biomolecules in modular fashion, and its relative simplicity and ease of manipulation. Nearly all applications of the filamentous phage depend on its ability to display polypeptides on the virion's surface as fusions to phage coat proteins ( Table 1) . The display mode determines the maximum tolerated size of the fused polypeptide, its copy number on the phage, and potentially, the structure of the displayed polypeptide. Display may be achieved by fusing DNA encoding a polypeptide of interest directly to the gene encoding a coat protein within the phage genome (type 8 display on pVIII, type 3 display on pIII, etc.), resulting in fully recombinant phage. Much more commonly, however, only one copy of the coat protein is modified in the presence of a second, wild-type copy (e.g., type 88 display if both recombinant and wild-type pVIII genes are on the phage genome, type 8+8 display if the Parmley and Smith (1988), McConnell et al. (1994) , Rondot et al. (2001) Hybrid (type 33 and 3+3 systems) Type 3+3 system <1 2 Smith and Scott (1993) , Smith and Petrenko (1997) pVI Hybrid (type 6+6 system) Yes <1 2 >25 kDa Hufton et al. (1999) pVII Fully recombinant (type 7 system) No ∼5 >25 kDa Kwasnikowski et al. (2005) Hybrid (type 7+7 system) Yes <1 2 Gao et al. (1999) pVIII Fully recombinant (landscape phage; type 8 system) No 2700 3 ∼5-8 residues Kishchenko et al. (1994) , Petrenko et al. (1996) Hybrid (type 88 and 8+8 systems) Type 8+8 system ∼1-300 2 >50 kDa Scott and Smith (1990) , Greenwood et al. (1991) , Smith and Fernandez (2004) pIX Fully recombinant (type 9+9 * system) Yes ∼5 >25 kDa Gao et al. (2002) Hybrid (type 9+9 system) No <1 2 Gao et al. (1999) , Shi et al. (2010) , Tornetta et al. (2010) 1 Asterisks indicate non-functional copies of the coat protein are present in the genome of the helper phage used to rescue a phagemid whose coat protein has been fused to a recombinant polypeptide. 2 The copy number depends on polypeptide size; typically <1 copy per phage particle but for pVIII peptide display can be up to ∼15% of pVIII molecules in hybrid virions. 3 The total number of pVIII molecules depends on the phage genome size; one pVIII molecule is added for every 2.3 nucleotides in the viral genome. recombinant gene 8 is on a plasmid with a phage origin of replication) resulting in a hybrid virion bearing two different types of a given coat protein. Multivalent display on some coat proteins can also be enforced using helper phage bearing nonfunctional copies of the relevant coat protein gene (e.g., type 3 * +3 display). By far the most commonly used coat proteins for display are the major coat protein, pVIII, and the minor coat protein, pIII, with the major advantage of the former being higher copy number display (up to ∼15% of recombinant pVIII molecules in a hybrid virion, at least for short peptide fusions), and of the latter being the ability to display some folded proteins at an appreciable copy number (1-5 per phage particle). While pVIII display of folded proteins on hybrid phage is possible, it typically results in a copy number of much less than 1 per virion (Sidhu et al., 2000) . For the purposes of this review, we use the term "phage display" to refer to a recombinant filamentous phage displaying a single polypeptide sequence on its surface (or more rarely, bispecific display achieved via fusion of polypeptides to two different capsid proteins), and the term "phage-displayed library" to refer to a diverse pool of recombinant filamentous phage displaying an array of polypeptide variants (e.g., antibody fragments; peptides). Such libraries are typically screened by iterative cycles of panning against an immobilized protein of interest (e.g., antigen for phage-displayed antibody libraries; antibody for phage-displayed peptide libraries) followed by amplification of the bound phage in E. coli cells. Early work with anti-phage antisera generated for species classification purposes demonstrated that the filamentous phage virion is highly immunogenic in the absence of adjuvants (Meynell and Lawn, 1968 ) and that only the major coat protein, pVIII, and the minor coat protein, pIII, are targeted by antibodies (Pratt et al., 1969; Woolford et al., 1977) . Thus, the idea of using the phage as carrier to elicit antibodies against poorly immunogenic haptens or polypeptide was a natural extension of the ability to display recombinant exogenous sequences on its surface, which was first demonstrated by de la Cruz et al. (1988) . The phage particle's low cost of production, high stability and potential for high valency display of foreign antigen (via pVIII display) also made it attractive as a vaccine carrier, especially during the early stages of development of recombinant protein technology. Building upon existing peptide-carrier technology, the first filamentous phage-based vaccine immunogens displayed short amino acid sequences derived directly from proteins of interest as recombinant fusions to pVIII or pIII (de la Cruz et al., 1988) . As library technology was developed and refined, phage-based antigens displaying peptide ligands of monoclonal antibodies (selected from random peptide libraries using the antibody, thus simulating with varying degrees of success the antibody's folded epitope on its cognate antigen; Geysen et al., 1986; Knittelfelder et al., 2009) were also generated for immunization purposes, with the goal of eliciting anti-peptide antibodies that also recognize the native protein. Some of the pioneering work in this area used peptides derived from infectious disease antigens (or peptide ligands of antibodies against these antigens; Table 2) , including malaria and human immunodeficiency virus type 1 (HIV-1). When displayed on phage, peptides encoding the repeat regions of the malarial circumsporozoite protein and merozoite surface protein 1 were immunogenic in mice and rabbits (de la Cruz et al., 1988; Greenwood et al., 1991; Willis et al., 1993; Demangel et al., 1996) , and antibodies raised against the latter cross-reacted with the full-length protein. Various peptide determinants (or mimics thereof) of HIV-1 gp120, gp41, gag, and reverse transcriptase were immunogenic when displayed on or conjugated to phage coat proteins (Minenkova et al., 1993; di Marzo Veronese et al., 1994; De Berardinis et al., 1999; Scala et al., 1999; Chen et al., 2001; van Houten et al., 2006 van Houten et al., , 2010 , and in some cases elicited antibodies that were able to weakly neutralize lab-adapted viruses (di Marzo Veronese et al., 1994; Scala et al., 1999) . The list of animal and human infections for which phage-displayed peptide immunogens have been developed as vaccine leads continues to expand and includes bacterial, fungal, viral, and parasitic pathogens ( Table 2) . While in some cases the results of these studies have been promising, antibody epitope-based peptide vaccines are no longer an area of active research for several reasons: (i) in many cases, peptides incompletely or inadequately mimic epitopes on folded proteins (Irving et al., 2010 ; see below); (ii) antibodies against a single epitope may be of limited utility, especially for highly variable pathogens (Van Regenmortel, 2012); and (iii) for pathogens for which protective immune responses are generated efficiently during natural infection, peptide vaccines offer few advantages over recombinant subunit and live vector vaccines, which have become easier to produce over time. More recently, peptide-displaying phage have been used in attempts to generate therapeutic antibody responses for chronic diseases, cancer, immunotherapy, and immunocontraception. Immunization with phage displaying Alzheimer's disease β-amyloid fibril peptides elicited anti-aggregating antibodies in mice and guinea pigs (Frenkel et al., 2000 (Frenkel et al., , 2003 Esposito et al., 2008; Tanaka et al., 2011) , possibly reduced amyloid plaque formation in mice (Frenkel et al., 2003; Solomon, 2005; Esposito et al., 2008) , and may have helped maintain cognitive abilities in a transgenic mouse model of Alzheimer's disease (Lavie et al., 2004) ; however, it remains unclear how such antibodies are proposed to cross the blood-brain barrier. Yip et al. (2001) found that antibodies raised in mice against an ERBB2/HER2 peptide could inhibit breast-cancer cell proliferation. Phage displaying peptide ligands of an anti-IgE antibody elicited antibodies that bound purified IgE molecules (Rudolf et al., 1998) , which may be useful in allergy immunotherapy. Several strategies for phage-based contraceptive vaccines have been proposed for control of animal populations. For example, immunization with phage displaying follicle-stimulating hormone peptides on pVIII elicited antibodies that impaired the fertility of mice and ewes (Abdennebi et al., 1999) . Phage displaying or chemically Rubinchik and Chow (2000) conjugated to sperm antigen peptides or peptide mimics (Samoylova et al., 2012a,b) and gonadotropin-releasing hormone (Samoylov et al., 2012) are also in development. For the most part, peptides displayed on phage elicit antibodies in experimental animals ( Table 2) , although this depends on characteristics of the peptide and the method of its display: pIII fusions tend toward lower immunogenicity than pVIII fusions (Greenwood et al., 1991) possibly due to copy number differences (pIII: 1-5 copies vs. pVIII: estimated at several hundred copies; Malik et al., 1996) . In fact, the phage is at least as immunogenic as traditional carrier proteins such as bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH; Melzer et al., 2003; Su et al., 2007) , and has comparatively few endogenous B-cell epitopes to divert the antibody response from its intended target (Henry et al., 2011) . Excepting small epitopes that can be accurately represented by a contiguous short amino acid sequence, however, it has been extremely difficult to elicit antibody responses that cross-react with native protein epitopes using peptides. The overall picture is considerably bleaker than that painted by Table 2 , since in several studies either: (i) peptide ligands selected from phage-displayed libraries were classified by the authors as mimics of discontinuous epitopes if they bore no obvious sequence homology to the native protein, which is weak evidence of non-linearity, or (ii) the evidence for cross-reactivity of antibodies elicited by immunization with phage-displayed peptides with native protein was uncompelling. Irving et al. (2010) describe at least one reason for this lack of success: it seems that peptide antigens elicit a set of topologically restricted antibodies that are largely unable to recognize discontinuous or complex epitopes on larger biomolecules. While the peptide may mimic the chemistry of a given epitope on a folded protein (allowing it to crossreact with a targeted antibody), being a smaller molecule, it cannot mimic the topology of that antibody's full epitope. Despite this, the filamentous phage remains highly useful as a carrier for peptides with relatively simple secondary structures, which may be stablilized via anchoring to the coat proteins (Henry et al., 2011) . This may be especially true of peptides with poor inherent immunogenicity, which may be increased by high-valency display and phage-associated adjuvanticity (see Immunological Mechanisms of Vaccination with Filamentous Phage below). The filamentous phage has been used to a lesser extent as a carrier for T-cell peptide epitopes, primarily as fusion proteins with pVIII ( Table 3) . Early work, showing that immunization with phage elicited T-cell help (Kölsch et al., 1971; Willis et al., 1993) , was confirmed by several subsequent studies (De Berardinis et al., 1999; Ulivieri et al., 2008) . From the perspective of vaccination against infectious disease, De Berardinis et al. (2000) showed that a cytotoxic T-cell (CTL) epitope from HIV-1 reverse transcriptase could elicit antigen-specific CTLs in vitro and in vivo without addition of exogenous helper T-cell epitopes, presumably since these are already present in the phage coat proteins (Mascolo et al., 2007) . Similarly, efficient priming of CTLs was observed against phage-displayed T-cell epitopes from Hepatitis B virus (Wan et al., 2001) and Candida albicans (Yang et al., 2005a; Wang et al., 2006 Wang et al., , 2014d , which, together with other types of immune responses, protected mice against systemic candidiasis. Vaccination with a combination of phagedisplayed peptides elicited antigen-specific CTLs that proved effective in reducing porcine cysticercosis in a randomized controlled trial (Manoutcharian et al., 2004; Morales et al., 2008) . While the correlates of vaccine-induced immune protection for infectious diseases, where they are known, are almost exclusively serum or mucosal antibodies (Plotkin, 2010) , In certain vaccine applications, the filamentous phage has been used as a carrier for larger molecules that would be immunogenic even in isolation. Initially, the major advantages to phage display of such antigens were speed, ease of purification and low cost of production (Gram et al., 1993) . E. coli F17a-G adhesin (Van Gerven et al., 2008) , hepatitis B core antigen (Bahadir et al., 2011) , and hepatitis B surface antigen (Balcioglu et al., 2014) all elicited antibody responses when displayed on pIII, although none of these studies compared the immunogenicity of the phage-displayed proteins with that of the purified protein alone. Phage displaying Schistosoma mansoni glutathione S-transferase on pIII elicited an antibody response that was both higher in titer and of different isotypes compared to immunization with the protein alone (Rao et al., 2003) . Two studies of antiidiotypic vaccines have used the phage as a carrier for antibody fragments bearing immunogenic idiotypes. Immunization with phage displaying the 1E10 idiotype scFv (mimicking a Vibrio anguillarum surface epitope) elicited antibodies that protected flounder fish from Vibrio anguillarum challenge (Xia et al., 2005) . A chemically linked phage-BCL1 tumor-specific idiotype vaccine was weakly immunogenic in mice but extended survival time in a B-cell lymphoma model (Roehnisch et al., 2013) , and was welltolerated and immunogenic in patients with multiple myeloma (Roehnisch et al., 2014) . One study of DNA vaccination with an anti-laminarin scFv found that DNA encoding a pIII-scFv fusion protein elicited stronger humoral and cell-mediated immune responses than DNA encoding the scFv alone (Cuesta et al., 2006) , suggesting that under some circumstances, endogenous phage T-cell epitopes can enhance the immunogenicity of associated proteins. Taken together, the results of these studies show that as a particulate virus-like particle, the filamentous phage likely triggers different types of immune responses than recombinant protein antigens, and provide additional T-cell help to displayed or conjugated proteins. However, the low copy number of pIII-displayed proteins, as well as potentially unwanted phage-associated adjuvanticity, can make display of recombinant proteins by phage a suboptimal vaccine choice. Although our understanding of the immune response against the filamentous phage pales in comparison to classical model antigens such as ovalbumin, recent work has begun to shed light on the immune mechanisms activated in response to phage vaccination (Figure 1) . The phage particle is immunogenic without adjuvant in all species tested to date, including mice (Willis et al., 1993) , rats (Dente et al., 1994) , rabbits (de la Cruz et al., 1988) , guinea pigs (Frenkel et al., 2000; Kim et al., 2004) , fish (Coull et al., 1996; Xia et al., 2005) , non-human primates (Chen et al., 2001) , and humans (Roehnisch et al., 2014) . Various routes of immunization have been employed, including oral administration (Delmastro et al., 1997) as well as subcutaneous (Grabowska et al., 2000) , intraperitoneal (van Houten et al., 2006) , intramuscular (Samoylova et al., 2012a) , intravenous (Vaks and Benhar, 2011) , and intradermal injection (Roehnisch et al., 2013) ; no published study has directly compared the effect of administration route on filamentous phage immunogenicity. Antibodies are generated against only three major sites on the virion: (i) the surface-exposed N-terminal ∼12 residues of the pVIII monomer lattice (Terry et al., 1997; Kneissel et al., 1999) ; (ii) the N-terminal N1 and N2 domains of pIII (van Houten et al., 2010) ; and (iii) bacterial lipopolysaccharide (LPS) embedded in the phage coat (Henry et al., 2011) . In mice, serum antibody titers against the phage typically reach 1:10 5 -1:10 6 after 2-3 immunizations, and are maintained for at least 1 year postimmunization (Frenkel et al., 2000) . Primary antibody responses against the phage appear to be composed of a mixture of IgM and IgG2b isotypes in C57BL/6 mice, while secondary antibody responses are composed primarily of IgG1 and IgG2b isotypes, with a lesser contribution of IgG2c and IgG3 isotypes (Hashiguchi et al., 2010) . Deletion of the surface-exposed N1 and N2 domains of pIII produces a truncated form of this protein that does not elicit antibodies, but also results in a non-infective phage particle with lower overall immunogenicity (van Houten et al., 2010) . FIGURE 1 | Types of immune responses elicited in response to immunization with filamentous bacteriophage. As a virus-like particle, the filamentous phage engages multiple arms of the immune system, beginning with cellular effectors of innate immunity (macrophages, neutrophils, and possibly natural killer cells), which are recruited to tumor sites by phage displaying tumor-targeting moieties. The phage likely activates T-cell independent antibody responses, either via phage-associated TLR ligands or cross-linking by the pVIII lattice. After processing by antigen-presenting cells, phage-derived peptides are presented on MHC class II and cross-presented on MHC class I, resulting in activation of short-lived CTLs and an array of helper T-cell types, which help prime memory CTL and high-affinity B-cell responses. Frontiers in Microbiology | www.frontiersin.org Although serum anti-phage antibody titers appear to be at least partially T-cell dependent (Kölsch et al., 1971; Willis et al., 1993; De Berardinis et al., 1999; van Houten et al., 2010) , many circulating pVIII-specific B cells in the blood are devoid of somatic mutation even after repeated biweekly immunizations, suggesting that under these conditions, the phage activates T-cell-independent B-cell responses in addition to highaffinity T-cell-dependent responses (Murira, 2014) . Filamentous phage particles can be processed by antigen-presenting cells and presented on MHC class II molecules (Gaubin et al., 2003; Ulivieri et al., 2008) and can activate T H 1, T H 2, and T H 17 helper T cells (Yang et al., 2005a; Wang et al., 2014d) . Anti-phage T H 2 responses were enhanced through display of CTLA-4 peptides fused to pIII (Kajihara et al., 2000) . Phage proteins can also be cross-presented on MHC class I molecules (Wan et al., 2005) and can prime two waves of CTL responses, consisting first of short-lived CTLs and later of long-lived memory CTLs that require CD4 + T-cell help (Del Pozzo et al., 2010) . The latter CTLs mediate a delayed-type hypersensitivity reaction (Fang et al., 2005; Del Pozzo et al., 2010) . The phage particle is self-adjuvanting through multiple mechanisms. Host cell wall-derived LPS enhances the virion's immunogenicity, and its removal by polymyxin B chromatography reduces antibody titers against phage coat proteins (Grabowska et al., 2000) . The phage's singlestranded DNA genome contains CpG motifs and may also have an adjuvant effect. The antibody response against the phage is entirely dependent on MyD88 signaling and is modulated by stimulation of several Toll-like receptors (Hashiguchi et al., 2010) , indicating that innate immunity plays an important but largely uncharacterized role in the activation of anti-phage adaptive immune responses. Biodistribution studies of the phage after intravenous injection show that it is cleared from the blood within hours through the reticuloendothelial system (Molenaar et al., 2002) , particularly of the liver and spleen, where it is retained for days (Zou et al., 2004) , potentially activating marginal-zone B-cell responses. Thus, the filamentous phage is not only a highly immunogenic carrier, but by virtue of activating a range of innate and adaptive immune responses, serves as an excellent model virus-like particle antigen. Long before the identification of filamentous phage, other types of bacteriophage were already being used for antibacterial therapy in the former Soviet Union and Eastern Europe (reviewed in Sulakvelidze et al., 2001) . The filamentous phage, with its nonlytic life cycle, has less obvious clinical uses, despite the fact that the host specificity of Inovirus and Plectrovirus includes many pathogens of medical importance, including Salmonella, E. coli, Shigella, Pseudomonas, Clostridium, and Mycoplasma species. In an effort to enhance their bactericidal activity, genetically modified filamentous phage have been used as a "Trojan horse" to introduce various antibacterial agents into cells. M13 and Pf3 phage engineered to express either BglII restriction endonuclease (Hagens and Blasi, 2003; Hagens et al., 2004) , lambda phage S holin (Hagens and Blasi, 2003) or a lethal catabolite gene activator protein (Moradpour et al., 2009) effectively killed E. coli and Pseudomonas aeruginosa cells, respectively, with no concomitant release of LPS (Hagens and Blasi, 2003; Hagens et al., 2004) . Unfortunately, the rapid emergence of resistant bacteria with modified F pili represents a major and possibly insurmountable obstacle to this approach. However, there are some indications that filamentous phage can exert useful but more subtle effects upon their bacterial hosts that may not result in the development of resistance to infection. Several studies have reported increased antibiotic sensitivity in bacterial populations simultaneously infected with either wild type filamentous phage (Hagens et al., 2006) or phage engineered to repress the cellular SOS response (Lu and Collins, 2009) . Filamentous phage f1 infection inhibited early stage, but not mature, biofilm formation in E. coli (May et al., 2011) . Thus, unmodified filamentous phage may be of future interest as elements of combination therapeutics against certain drug-resistant infections. More advanced therapeutic applications of the filamentous phage emerge when it is modified to express a targeting moiety specific for pathogenic cells and/or proteins for the treatment of infectious diseases, cancer and autoimmunity (Figure 2) . The first work in this area showed as proof-of-concept that phage encoding a GFP expression cassette and displaying a HER2specific scFv on all copies of pIII were internalized into breast tumor cells, resulting in GFP expression (Poul and Marks, 1999) . M13 or fd phage displaying either a targeting peptide or antibody fragment and tethered to chloramphenicol by a labile crosslinker were more potent inhibitors of Staphylococcus aureus growth than high-concentration free chloramphenicol (Yacoby et al., 2006; Vaks and Benhar, 2011) . M13 phage loaded with doxorubicin and displaying a targeting peptide on pIII specifically killed prostate cancer cells in vitro (Ghosh et al., 2012a) . Tumorspecific peptide:pVIII fusion proteins selected from "landscape" phage (Romanov et al., 2001; Abbineni et al., 2010; Fagbohun et al., 2012 Fagbohun et al., , 2013 Lang et al., 2014; Wang et al., 2014a) were able to target and deliver siRNA-, paclitaxel-, and doxorubicincontaining liposomes to tumor cells (Jayanna et al., 2010a; Wang et al., 2010a Wang et al., ,b,c, 2014b Bedi et al., 2011 Bedi et al., , 2013 Bedi et al., , 2014 ; they were non-toxic and increased tumor remission rates in mouse models (Jayanna et al., 2010b; Wang et al., 2014b,c) . Using the B16-OVA tumor model, Eriksson et al. (2007) showed that phage displaying peptides and/or Fabs specific for tumor antigens delayed tumor growth and improved survival, owing in large part to activation of tumor-associated macrophages and recruitment of neutrophils to the tumor site (Eriksson et al., 2009) . Phage displaying an scFv against β-amyloid fibrils showed promise as a diagnostic (Frenkel and Solomon, 2002) and therapeutic (Solomon, 2008) reagent for Alzheimer's disease and Parkinson's disease due to the unanticipated ability of the phage to penetrate into brain tissue (Ksendzovsky et al., 2012) . Similarly, phage displaying an immunodominant peptide epitope derived from myelin oligodendrocyte glycoprotein depleted pathogenic demyelinating antibodies in brain tissue in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis (Rakover et al., 2010) . The advantages of the filamentous phage in this context over traditional antibody-drug or protein-peptide conjugates are (i) its ability to carry very high amounts of drug or peptide, and (ii) its ability to access anatomical compartments that cannot generally be reached by systemic administration of a protein. Unlike most therapeutic biologics, the filamentous phage's production in bacteria complicates its use in humans in several ways. First and foremost, crude preparations of filamentous phage typically contain very high levels of contaminating LPS, in the range of ∼10 2 -10 4 endotoxin units (EU)/mL (Boratynski et al., 2004; Branston et al., 2015) , which have the potential to cause severe adverse reactions. LPS is not completely removed by polyethylene glycol precipitation or cesium chloride density gradient centrifugation (Smith and Gingrich, 2005; Branston et al., 2015) , but its levels can be reduced dramatically using additional purification steps such as size exclusion chromatography (Boratynski et al., 2004; Zakharova et al., 2005) , polymyxin B chromatography (Grabowska et al., 2000) , and treatment with detergents such as Triton X-100 or Triton X-114 (Roehnisch et al., 2014; Branston et al., 2015) . These strategies routinely achieve endotoxin levels of <1 EU/mL as measured by the limulus amebocyte lysate (LAL) assay, well below the FDA limit for parenteral administration of 5 EU/kg body weight/dose, although concerns remain regarding the presence of residual virion-associated LPS which may be undetectable. A second and perhaps unavoidable consequence of the filamentous phage's bacterial production is inherent heterogeneity of particle size and the spectrum of host cellderived virion-associated and soluble contaminants, which may be cause for safety concerns and restrict its use to high-risk groups. Many types of bacteriophage and engineered phage variants, including filamentous phage, have been proposed for prophylactic use ex vivo in food safety, either in the production pipeline (reviewed in Dalmasso et al., 2014) or for detection of foodborne pathogens post-production (reviewed in Schmelcher and Loessner, 2014) . Filamentous phage displaying a tetracysteine tag on pIII were used to detect E. coli cells through staining with biarsenical dye . M13 phage functionalized with metallic silver were highly bactericidal against E. coli and Staphylococcus epidermidis . Biosensors based on surface plasmon resonance (Nanduri et al., 2007) , piezoelectric transducers (Olsen et al., 2006) , linear dichroism (Pacheco-Gomez et al., 2012) , and magnetoelastic sensor technology (Lakshmanan et al., 2007; Huang et al., 2009) were devised using filamentous phage displaying scFv or conjugated to whole IgG against E. coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus anthracis with limits of detection on the order of 10 2 -10 6 bacterial cells/mL. Proof of concept has been demonstrated for use of such phage-based biosensors to detect bacterial contamination of live produce (Li et al., 2010b) and eggs (Chai et al., 2012) . The filamentous phage particle is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (Henry et al., 2011) . The regularity of the phage pVIII lattice and its diversity of chemically addressable groups make it an ideal scaffold for bioconjugation (Figure 3) . The most commonly used approach is functionalization of amine groups with NHS esters (van Houten et al., 2006 (van Houten et al., , 2010 Yacoby et al., 2006) , although this can result in unwanted acylation of pIII and any displayed biomolecules. Carboxyl groups and tyrosine residues can also be functionalized using carbodiimide coupling and diazonium coupling, respectively (Li et al., 2010a) . Carrico et al. (2012) developed methods to specifically label pVIII N-termini without modification of exposed lysine residues through a two-step transamination-oxime formation reaction. Specific modification of phage coat proteins is even more easily accomplished using genetically modified phage displaying peptides (Ng et al., 2012) or enzymes (Chen et al., 2007; Hess et al., 2012) , but this can be cumbersome and is less general in application. For more than a decade, interest in the filamentous phage as a building block for nanomaterials has been growing because of its unique physicochemical properties, with emerging applications in magnetics, optics, and electronics. It has long been known that above a certain concentration threshold, phage can form ordered crystalline suspensions (Welsh et al., 1996) . Lee et al. (2002) engineered M13 phage to display a ZnS-binding peptide on pIII and showed that, in the presence of ZnS nanoparticles, they selfassemble into highly ordered film biomaterials that can be aligned using magnetic fields. Taking advantage of the ability to display substrate-specific peptides at known locations on the phage filament Hess et al., 2012) , this pioneering FIGURE 3 | Chemically addressable groups of the filamentous bacteriophage major coat protein lattice. The filamentous phage virion is made up of ∼2,500-4,000 overlapping copies of the 50-residue major coat protein, pVIII, arranged in a shingle-type lattice. Each monomer has an array of chemically addressable groups available for bioorthogonal conjugation, including two primary amine groups (shown in red), three carboxyl groups (show in blue) and two hydroxyl groups (show in green). The 12 N-terminal residues generally exposed to the immune system for antibody binding are in bold underline. Figure adapted from structural data of Marvin, 1990 , freely available in PDB and SCOPe databases. work became the basis for construction of two-and threedimensional nanomaterials with more advanced architectures, including semiconducting nanowires (Mao et al., 2003 (Mao et al., , 2004 , nanoparticles , and nanocomposites (Oh et al., 2012; Chen et al., 2014) . Using hybrid M13 phage displaying Co 3 O 4 -and gold-binding peptides on pVIII as a scaffold to assemble nanowires on polyelectrolyte multilayers, Nam et al. (2006) produced a thin, flexible lithium ion battery, which could be stamped onto platinum microband current collectors (Nam et al., 2008) . The electrochemical properties of such batteries were further improved through pIII-display of single-walled carbon nanotube-binding peptides (Lee et al., 2009) , offering an approach for sustainable production of nanostructured electrodes from poorly conductive starting materials. Phagebased nanomaterials have found applications in cancer imaging (Ghosh et al., 2012b; Yi et al., 2012) , photocatalytic water splitting (Nam et al., 2010a; Neltner et al., 2010) , light harvesting (Nam et al., 2010b; Chen et al., 2013) , photoresponsive technologies (Murugesan et al., 2013) , neural electrodes (Kim et al., 2014) , and piezoelectric energy generation (Murugesan et al., 2013) . Thus, the unique physicochemical properties of the phage, in combination with modular display of peptides and proteins with known binding specificity, have spawned wholly novel materials with diverse applications. It is worth noting that the unusual biophysical properties of the filamentous phage can also be exploited in the study of structures of other macromolecules. Magnetic alignment of high-concentration filamentous phage in solution can partially order DNA, RNA, proteins, and other biomolecules for measurement of dipolar coupling interactions (Hansen et al., 1998 (Hansen et al., , 2000 Dahlke Ojennus et al., 1999) in NMR spectroscopy. Because of their large population sizes, short generation times, small genome sizes and ease of manipulation, various filamentous and non-filamentous bacteriophages have been used as models of experimental evolution (reviewed in Husimi, 1989; Wichman and Brown, 2010; Kawecki et al., 2012; Hall et al., 2013) . The filamentous phage has additional practical uses in protein engineering and directed protein evolution, due to its unique tolerance of genetic modifications that allow biomolecules to be displayed on the virion surface. First and foremost among these applications is in vitro affinity maturation of antibody fragments displayed on pIII. Libraries of variant Fabs and single chain antibodies can be generated via random or sitedirected mutagenesis and selected on the basis of improved or altered binding, roughly mimicking the somatic evolution strategy of the immune system (Marks et al., 1992; Bradbury et al., 2011) . However, other in vitro display systems, such as yeast display, have important advantages over the filamentous phage for affinity maturation (although each display technology has complementary strengths; Koide and Koide, 2012) , and regardless of the display method, selection of "improved" variants can be slow and cumbersome. Iterative methods have been developed to combine computationally designed mutations (Lippow et al., 2007) and circumvent the screening of combinatorial libraries, but these have had limited success to date. Recently, Esvelt et al. (2011) developed a novel strategy for directed evolution of filamentous phage-displayed proteins, called phage-assisted continuous evolution (PACE), which allows multiple rounds of evolution per day with little experimental intervention. The authors engineered M13 phage to encode an exogenous protein (the subject for directed evolution), whose functional activity triggers gene III expression from an accessory plasmid; variants of the exogenous protein arise by random mutagenesis during phage replication, the rate of which can be increased by inducible expression of error-prone DNA polymerases. By supplying limiting amounts of receptive E. coli cells to the engineered phage variants, Esvelt et al. (2011) elegantly linked phage infectivity and production of offspring with the presence of a desired protein phenotype. Carlson et al. (2014) later showed that PACE selection stringency could be modulated by providing small amounts of pIII independently of protein phenotype, and undesirable protein functions negatively selected by linking them to expression of a truncated pIII variant that impairs infectivity in a dominant negative fashion. PACE is currently limited to protein functions that can be linked in some way to the expression of a gene III reporter, such as protein-protein interaction, recombination, DNA or RNA binding, and enzymatic catalysis (Meyer and Ellington, 2011) . This approach represents a promising avenue for both basic research in molecular evolution (Dickinson et al., 2013) and synthetic biology, including antibody engineering. Filamentous bacteriophage have been recovered from diverse environmental sources, including soil (Murugaiyan et al., 2011) , coastal fresh water (Xue et al., 2012) , alpine lakes (Hofer and Sommaruga, 2001) and deep sea bacteria (Jian et al., 2012) , but not, perhaps surprisingly, the human gut (Kim et al., 2011) . The environmental "phageome" in soil and water represent the largest source of replicating DNA on the planet, and is estimated to contain upward of 10 30 viral particles (Ashelford et al., 2003; Chibani-Chennoufi et al., 2004; Suttle, 2005) . The few studies attempting to investigate filamentous phage environmental ecology using classical environmental microbiology techniques (typically direct observation by electron microscopy) found that filamentous phage made up anywhere from 0 to 100% of all viral particles (Demuth et al., 1993; Pina et al., 1998; Hofer and Sommaruga, 2001) . There was some evidence of seasonal fluctuation of filamentous phage populations in tandem with the relative abundance of free-living heterotrophic bacteria (Hofer and Sommaruga, 2001) . Environmental metagenomics efforts are just beginning to unravel the composition of viral ecosystems. The existing data suggest that filamentous phage comprise minor constituents of viral communities in freshwater (Roux et al., 2012) and reclaimed and potable water (Rosario et al., 2009) but have much higher frequencies in wastewater and sewage (Cantalupo et al., 2011; Alhamlan et al., 2013) , with the caveat that biases inherent to the methodologies for ascertaining these data (purification of viral particles, sequencing biases) have not been not well validated. There are no data describing the population dynamics of filamentous phage and their host species in the natural environment. At the individual virus-bacterium level, it is clear that filamentous phage can modulate host phenotype, including the virulence of important human and crop pathogens. This can occur either through direct effects of phage replication on cell growth and physiology, or, more typically, by horizontal transfer of genetic material contained within episomes and/or chromosomally integrated prophage. Temperate filamentous phage may also play a role in genome evolution (reviewed in Canchaya et al., 2003) . Perhaps the best-studied example of virulence modulation by filamentous phage is that of Vibrio cholerae, whose full virulence requires lysogenic conversion by the cholera toxin-encoding CTXφ phage (Waldor and Mekalanos, 1996) . Integration of CTXφ phage occurs at specific sites in the genome; these sequences are introduced through the combined action of another filamentous phage, fs2φ, and a satellite filamentous phage, TLC-Knφ1 (Hassan et al., 2010) . Thus, filamentous phage species interact and coevolve with each other in addition to their hosts. Infection by filamentous phage has been implicated in the virulence of Yersinia pestis (Derbise et al., 2007) , Neisseria meningitidis (Bille et al., 2005 (Bille et al., , 2008 , Vibrio parahaemolyticus (Iida et al., 2001) , E. coli 018:K1:H7 (Gonzalez et al., 2002) , Xanthomonas campestris (Kamiunten and Wakimoto, 1982) , and P. aeruginosa (Webb et al., 2004) , although in most of these cases, the specific mechanisms modulating virulence are unclear. Phage infection can both enhance or repress virulence depending on the characteristics of the phage, the host bacterium, and the environmental milieu, as is the case for the bacterial wilt pathogen Ralstonia solanacearum (Yamada, 2013) . Since infection results in downregulation of the pili used for viral entry, filamentous phage treatment has been proposed as a hypothetical means of inhibiting bacterial conjugation and horizontal gene transfer, so as to prevent the spread of antibiotic resistance genes (Lin et al., 2011) . Finally, the filamentous phage may also play a future role in the preservation of biodiversity of other organisms in at-risk ecosystems. Engineered phage have been proposed for use in bioremediation, either displaying antibody fragments of desired specificity for filtration of toxins and environmental contaminants (Petrenko and Makowski, 1993) , or as biodegradable polymers displaying peptides selected for their ability to aggregate pollutants, such as oil sands tailings (Curtis et al., 2011 (Curtis et al., , 2013 . Engineered phage displaying peptides that specifically bind inorganic materials have also been proposed for use in more advanced and less intrusive mineral separation technologies (Curtis et al., 2009 ). The filamentous phage represents a highly versatile organism whose uses extend far beyond traditional phage display and affinity selection of antibodies and polypeptides of desired specificity. Its high immunogenicity and ability to display a variety of surface antigens make the phage an excellent particulate vaccine carrier, although its bacterial production and preparation heterogeneity likely limits its applications in human vaccines at present, despite being apparently safe and well-tolerated in animals and people. Unanticipated characteristics of the phage particle, such as crossing of the blood-brain barrier and formation of highly ordered liquid crystalline phases, have opened up entirely new avenues of research in therapeutics for chronic disease and the design of nanomaterials. Our comparatively detailed understanding of the interactions of model filamentous phage with their bacterial hosts has allowed researchers to harness the phage life cycle to direct protein evolution in the lab. Hopefully, deeper knowledge of phage-host interactions at an ecological level may produce novel strategies to control bacterial pathogenesis. While novel applications of the filamentous phage continue to be developed, the phage is likely to retain its position as a workhorse for therapeutic antibody discovery for many years to come, even with the advent of competing technologies. KH and JS conceived and wrote the manuscript. MA-G read the manuscript and commented on the text.
What is the structure of a filamentous phage particle?
false
1,761
{ "text": [ "is enclosed by a rod-like protein capsid, ∼1000 nm long and 5 nm wide, made up almost entirely of overlapping pVIII monomers, each of which lies ∼27 angstroms from its nearest neighbor and exposes two amine groups as well as at least three carboxyl groups (Henry et al., 2011)" ], "answer_start": [ 33493 ] }
1,546
First Complete Genome Sequence of a French Bovine coronavirus Strain https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477389/ SHA: eef0ecf5b8e7b179dadaef967e65f2ab68f021e1 Authors: Kin, Nathalie; Guerard, Pauline; Diancourt, Laure; Caro, Valérie; Vabret, Astrid; Ar Gouilh, Meriadeg Date: 2017-05-25 DOI: 10.1128/genomea.00319-17 License: cc-by Abstract: We sequenced the first Bovine coronavirus (BCoV) complete genome sequence from France. This BCoV was directly sequenced from a fecal sample collected from a calf in Normandy in 2014. Text: B ovine coronavirus (BCoV) belongs to the Nidovirales order, the Coronaviridae family, the Coronavirinae subfamily, and the Betacoronavirus (https://talk.ictvonline.org/ ICTV/proposals/2008.085-122V.v4.Coronaviridae.pdf). Its genome is a single-stranded, linear, and nonsegmented RNA of around 31 kb. BCoV is responsible for respiratory and enteric diseases in cattle, particularly during winter (1, 2) . To date, the 19 complete BCoV genome sequences available in GenBank databases (consulted on 17 January 2017) originated from the United States or Asia. Here, we report the first complete genome sequence of a BCoV detected in France. The BCoV/FRA-EPI/CAEN/2014/13 strain was obtained from a fecal sample collected from a 1-week-old calf in Normandy in 2014. The presence of BCoV in the fecal sample was assessed using an in-house reverse transcription-PCR (RT-PCR) targeting the M gene (3). A cDNA library was synthesized using SuperScript III (Invitrogen, Carlsbad, CA, USA) and hexamers. The complete genome sequencing of overlapping PCR products was carried out in both directions, using original primers and Sanger's dideoxy sequencing. Sequencing reactions were performed as previously described (3). Sequences were assembled and annotated using the Geneious software (version 5.1.6). We obtained a sequence counting 30,847 nucleotides. The orf1ab, HE, S, ns5, E, M, and N genes of the obtained BCoV were submitted to a Blastn analysis. According to these analyses, the orf1ab (20kb nucleotides, located at the 5= side of the genome) gene is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F strain from the United Arab Emirates (accession no. KF906251), with a nucleotide identity of 99.19%. Conversely, the NS2, HE, S, ns5, and M genes are closely related to the BCoV Bubalus/Italy/179/07-11 strain (accession no. EU019216), with nucleotide identities of 99.88%, 99.45%, 99.02%, 98.79%, and 99.28%, respectively. The E gene is closely related to the Chinese Bovine coronavirus strain BCV-AKS-01 (accession no. KU886219), with a nucleotide identity of 100%. Finally, the highest Blastn score for the N gene was found with the American enteric BCoV-ENT (accession no. AF391541), associated with a nucleotide identity of 100%. Multiple-sequence alignment, including 20 BCoVs and 10 clade A betacoronaviruses closely related to BCoV from North America, two DcCoVs from the United Arab Emirates, and two Human coronavirus OC43 (HCoV-OC43) strains from France, was performed using the Muscle algorithm implemented in MEGA7 (4, 5) . The phylogenetic analysis on the orf1ab confirms that BCoV/FRA-EPI/CAEN/2014/13 is closely related to the Dromedary camel coronavirus (DcCoV) HKU23-23-362F. The orf1ab gene of these two viruses together clustered separately from that of BCoV and BCoV-like viruses from North America and Asia. This finding also confirms the results from our previous analysis on partial genomes in which nsp12, S, and N genes of American and Asian BCoVs group together in a cluster tentatively named C 1 . The nsp12 and N coding regions of BCoVs from France and DcCoVs from the United Arab Emirates clustered together in C 2 . The DcCoV S gene individualized from both HCoV-OC43 and BCoV S genes. Potential recombination events could be at the origin of DcCoV. Accession number(s). The complete genome sequence sequence of the BCoV/FRA-EPI/CAEN/2014/13 isolate has been deposited in GenBank under the accession number KX982264.
How many nucleotides does bovine coronavirus contain?
false
919
{ "text": [ "30,847 nucleotides" ], "answer_start": [ 1873 ] }
1,719
Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.
What is the result of rAd5 trials?
false
1,530
{ "text": [ "clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy," ], "answer_start": [ 10212 ] }
2,683
Estimating the number of infections and the impact of non- pharmaceutical interventions on COVID-19 in 11 European countries 30 March 2020 Imperial College COVID-19 Response Team Seth Flaxmani Swapnil Mishra*, Axel Gandy*, H JulietteT Unwin, Helen Coupland, Thomas A Mellan, Harrison Zhu, Tresnia Berah, Jeffrey W Eaton, Pablo N P Guzman, Nora Schmit, Lucia Cilloni, Kylie E C Ainslie, Marc Baguelin, Isobel Blake, Adhiratha Boonyasiri, Olivia Boyd, Lorenzo Cattarino, Constanze Ciavarella, Laura Cooper, Zulma Cucunuba’, Gina Cuomo—Dannenburg, Amy Dighe, Bimandra Djaafara, Ilaria Dorigatti, Sabine van Elsland, Rich FitzJohn, Han Fu, Katy Gaythorpe, Lily Geidelberg, Nicholas Grassly, Wi|| Green, Timothy Hallett, Arran Hamlet, Wes Hinsley, Ben Jeffrey, David Jorgensen, Edward Knock, Daniel Laydon, Gemma Nedjati—Gilani, Pierre Nouvellet, Kris Parag, Igor Siveroni, Hayley Thompson, Robert Verity, Erik Volz, Caroline Walters, Haowei Wang, Yuanrong Wang, Oliver Watson, Peter Winskill, Xiaoyue Xi, Charles Whittaker, Patrick GT Walker, Azra Ghani, Christl A. Donnelly, Steven Riley, Lucy C Okell, Michaela A C Vollmer, NeilM.Ferguson1and Samir Bhatt*1 Department of Infectious Disease Epidemiology, Imperial College London Department of Mathematics, Imperial College London WHO Collaborating Centre for Infectious Disease Modelling MRC Centre for Global Infectious Disease Analysis Abdul LatifJameeI Institute for Disease and Emergency Analytics, Imperial College London Department of Statistics, University of Oxford *Contributed equally 1Correspondence: nei|[email protected], [email protected] Summary Following the emergence of a novel coronavirus (SARS-CoV-Z) and its spread outside of China, Europe is now experiencing large epidemics. In response, many European countries have implemented unprecedented non-pharmaceutical interventions including case isolation, the closure of schools and universities, banning of mass gatherings and/or public events, and most recently, widescale social distancing including local and national Iockdowns. In this report, we use a semi-mechanistic Bayesian hierarchical model to attempt to infer the impact of these interventions across 11 European countries. Our methods assume that changes in the reproductive number— a measure of transmission - are an immediate response to these interventions being implemented rather than broader gradual changes in behaviour. Our model estimates these changes by calculating backwards from the deaths observed over time to estimate transmission that occurred several weeks prior, allowing for the time lag between infection and death. One of the key assumptions of the model is that each intervention has the same effect on the reproduction number across countries and over time. This allows us to leverage a greater amount of data across Europe to estimate these effects. It also means that our results are driven strongly by the data from countries with more advanced epidemics, and earlier interventions, such as Italy and Spain. We find that the slowing growth in daily reported deaths in Italy is consistent with a significant impact of interventions implemented several weeks earlier. In Italy, we estimate that the effective reproduction number, Rt, dropped to close to 1 around the time of Iockdown (11th March), although with a high level of uncertainty. Overall, we estimate that countries have managed to reduce their reproduction number. Our estimates have wide credible intervals and contain 1 for countries that have implemented a|| interventions considered in our analysis. This means that the reproduction number may be above or below this value. With current interventions remaining in place to at least the end of March, we estimate that interventions across all 11 countries will have averted 59,000 deaths up to 31 March [95% credible interval 21,000-120,000]. Many more deaths will be averted through ensuring that interventions remain in place until transmission drops to low levels. We estimate that, across all 11 countries between 7 and 43 million individuals have been infected with SARS-CoV-Z up to 28th March, representing between 1.88% and 11.43% ofthe population. The proportion of the population infected to date — the attack rate - is estimated to be highest in Spain followed by Italy and lowest in Germany and Norway, reflecting the relative stages of the epidemics. Given the lag of 2-3 weeks between when transmission changes occur and when their impact can be observed in trends in mortality, for most of the countries considered here it remains too early to be certain that recent interventions have been effective. If interventions in countries at earlier stages of their epidemic, such as Germany or the UK, are more or less effective than they were in the countries with advanced epidemics, on which our estimates are largely based, or if interventions have improved or worsened over time, then our estimates of the reproduction number and deaths averted would change accordingly. It is therefore critical that the current interventions remain in place and trends in cases and deaths are closely monitored in the coming days and weeks to provide reassurance that transmission of SARS-Cov-Z is slowing. SUGGESTED CITATION Seth Flaxman, Swapnil Mishra, Axel Gandy et 0/. Estimating the number of infections and the impact of non— pharmaceutical interventions on COVID—19 in 11 European countries. Imperial College London (2020), doi: https://doi.org/10.25561/77731 1 Introduction Following the emergence of a novel coronavirus (SARS-CoV-Z) in Wuhan, China in December 2019 and its global spread, large epidemics of the disease, caused by the virus designated COVID-19, have emerged in Europe. In response to the rising numbers of cases and deaths, and to maintain the capacity of health systems to treat as many severe cases as possible, European countries, like those in other continents, have implemented or are in the process of implementing measures to control their epidemics. These large-scale non-pharmaceutical interventions vary between countries but include social distancing (such as banning large gatherings and advising individuals not to socialize outside their households), border closures, school closures, measures to isolate symptomatic individuals and their contacts, and large-scale lockdowns of populations with all but essential internal travel banned. Understanding firstly, whether these interventions are having the desired impact of controlling the epidemic and secondly, which interventions are necessary to maintain control, is critical given their large economic and social costs. The key aim ofthese interventions is to reduce the effective reproduction number, Rt, ofthe infection, a fundamental epidemiological quantity representing the average number of infections, at time t, per infected case over the course of their infection. Ith is maintained at less than 1, the incidence of new infections decreases, ultimately resulting in control of the epidemic. If Rt is greater than 1, then infections will increase (dependent on how much greater than 1 the reproduction number is) until the epidemic peaks and eventually declines due to acquisition of herd immunity. In China, strict movement restrictions and other measures including case isolation and quarantine began to be introduced from 23rd January, which achieved a downward trend in the number of confirmed new cases during February, resulting in zero new confirmed indigenous cases in Wuhan by March 19th. Studies have estimated how Rt changed during this time in different areas ofChina from around 2-4 during the uncontrolled epidemic down to below 1, with an estimated 7-9 fold decrease in the number of daily contacts per person.1'2 Control measures such as social distancing, intensive testing, and contact tracing in other countries such as Singapore and South Korea have successfully reduced case incidence in recent weeks, although there is a riskthe virus will spread again once control measures are relaxed.3'4 The epidemic began slightly laterin Europe, from January or later in different regions.5 Countries have implemented different combinations of control measures and the level of adherence to government recommendations on social distancing is likely to vary between countries, in part due to different levels of enforcement. Estimating reproduction numbers for SARS-CoV-Z presents challenges due to the high proportion of infections not detected by health systems”7 and regular changes in testing policies, resulting in different proportions of infections being detected over time and between countries. Most countries so far only have the capacity to test a small proportion of suspected cases and tests are reserved for severely ill patients or for high-risk groups (e.g. contacts of cases). Looking at case data, therefore, gives a systematically biased view of trends. An alternative way to estimate the course of the epidemic is to back-calculate infections from observed deaths. Reported deaths are likely to be more reliable, although the early focus of most surveillance systems on cases with reported travel histories to China may mean that some early deaths will have been missed. Whilst the recent trends in deaths will therefore be informative, there is a time lag in observing the effect of interventions on deaths since there is a 2-3-week period between infection, onset of symptoms and outcome. In this report, we fit a novel Bayesian mechanistic model of the infection cycle to observed deaths in 11 European countries, inferring plausible upper and lower bounds (Bayesian credible intervals) of the total populations infected (attack rates), case detection probabilities, and the reproduction number over time (Rt). We fit the model jointly to COVID-19 data from all these countries to assess whether there is evidence that interventions have so far been successful at reducing Rt below 1, with the strong assumption that particular interventions are achieving a similar impact in different countries and that the efficacy of those interventions remains constant over time. The model is informed more strongly by countries with larger numbers of deaths and which implemented interventions earlier, therefore estimates of recent Rt in countries with more recent interventions are contingent on similar intervention impacts. Data in the coming weeks will enable estimation of country-specific Rt with greater precision. Model and data details are presented in the appendix, validation and sensitivity are also presented in the appendix, and general limitations presented below in the conclusions. 2 Results The timing of interventions should be taken in the context of when an individual country’s epidemic started to grow along with the speed with which control measures were implemented. Italy was the first to begin intervention measures, and other countries followed soon afterwards (Figure 1). Most interventions began around 12th-14th March. We analyzed data on deaths up to 28th March, giving a 2-3-week window over which to estimate the effect of interventions. Currently, most countries in our study have implemented all major non-pharmaceutical interventions. For each country, we model the number of infections, the number of deaths, and Rt, the effective reproduction number over time, with Rt changing only when an intervention is introduced (Figure 2- 12). Rt is the average number of secondary infections per infected individual, assuming that the interventions that are in place at time t stay in place throughout their entire infectious period. Every country has its own individual starting reproduction number Rt before interventions take place. Specific interventions are assumed to have the same relative impact on Rt in each country when they were introduced there and are informed by mortality data across all countries. Figure l: Intervention timings for the 11 European countries included in the analysis. For further details see Appendix 8.6. 2.1 Estimated true numbers of infections and current attack rates In all countries, we estimate there are orders of magnitude fewer infections detected (Figure 2) than true infections, mostly likely due to mild and asymptomatic infections as well as limited testing capacity. In Italy, our results suggest that, cumulatively, 5.9 [1.9-15.2] million people have been infected as of March 28th, giving an attack rate of 9.8% [3.2%-25%] of the population (Table 1). Spain has recently seen a large increase in the number of deaths, and given its smaller population, our model estimates that a higher proportion of the population, 15.0% (7.0 [18-19] million people) have been infected to date. Germany is estimated to have one of the lowest attack rates at 0.7% with 600,000 [240,000-1,500,000] people infected. Imperial College COVID-19 Response Team Table l: Posterior model estimates of percentage of total population infected as of 28th March 2020. Country % of total population infected (mean [95% credible intervall) Austria 1.1% [0.36%-3.1%] Belgium 3.7% [1.3%-9.7%] Denmark 1.1% [0.40%-3.1%] France 3.0% [1.1%-7.4%] Germany 0.72% [0.28%-1.8%] Italy 9.8% [3.2%-26%] Norway 0.41% [0.09%-1.2%] Spain 15% [3.7%-41%] Sweden 3.1% [0.85%-8.4%] Switzerland 3.2% [1.3%-7.6%] United Kingdom 2.7% [1.2%-5.4%] 2.2 Reproduction numbers and impact of interventions Averaged across all countries, we estimate initial reproduction numbers of around 3.87 [3.01-4.66], which is in line with other estimates.1'8 These estimates are informed by our choice of serial interval distribution and the initial growth rate of observed deaths. A shorter assumed serial interval results in lower starting reproduction numbers (Appendix 8.4.2, Appendix 8.4.6). The initial reproduction numbers are also uncertain due to (a) importation being the dominant source of new infections early in the epidemic, rather than local transmission (b) possible under-ascertainment in deaths particularly before testing became widespread. We estimate large changes in Rt in response to the combined non-pharmaceutical interventions. Our results, which are driven largely by countries with advanced epidemics and larger numbers of deaths (e.g. Italy, Spain), suggest that these interventions have together had a substantial impact on transmission, as measured by changes in the estimated reproduction number Rt. Across all countries we find current estimates of Rt to range from a posterior mean of 0.97 [0.14-2.14] for Norway to a posterior mean of2.64 [1.40-4.18] for Sweden, with an average of 1.43 across the 11 country posterior means, a 64% reduction compared to the pre-intervention values. We note that these estimates are contingent on intervention impact being the same in different countries and at different times. In all countries but Sweden, under the same assumptions, we estimate that the current reproduction number includes 1 in the uncertainty range. The estimated reproduction number for Sweden is higher, not because the mortality trends are significantly different from any other country, but as an artefact of our model, which assumes a smaller reduction in Rt because no full lockdown has been ordered so far. Overall, we cannot yet conclude whether current interventions are sufficient to drive Rt below 1 (posterior probability of being less than 1.0 is 44% on average across the countries). We are also unable to conclude whether interventions may be different between countries or over time. There remains a high level of uncertainty in these estimates. It is too early to detect substantial intervention impact in many countries at earlier stages of their epidemic (e.g. Germany, UK, Norway). Many interventions have occurred only recently, and their effects have not yet been fully observed due to the time lag between infection and death. This uncertainty will reduce as more data become available. For all countries, our model fits observed deaths data well (Bayesian goodness of fit tests). We also found that our model can reliably forecast daily deaths 3 days into the future, by withholding the latest 3 days of data and comparing model predictions to observed deaths (Appendix 8.3). The close spacing of interventions in time made it statistically impossible to determine which had the greatest effect (Figure 1, Figure 4). However, when doing a sensitivity analysis (Appendix 8.4.3) with uninformative prior distributions (where interventions can increase deaths) we find similar impact of Imperial College COVID-19 Response Team interventions, which shows that our choice of prior distribution is not driving the effects we see in the main analysis. Figure 2: Country-level estimates of infections, deaths and Rt. Left: daily number of infections, brown bars are reported infections, blue bands are predicted infections, dark blue 50% credible interval (CI), light blue 95% CI. The number of daily infections estimated by our model drops immediately after an intervention, as we assume that all infected people become immediately less infectious through the intervention. Afterwards, if the Rt is above 1, the number of infections will starts growing again. Middle: daily number of deaths, brown bars are reported deaths, blue bands are predicted deaths, CI as in left plot. Right: time-varying reproduction number Rt, dark green 50% CI, light green 95% CI. Icons are interventions shown at the time they occurred. Imperial College COVID-19 Response Team Table 2: Totalforecasted deaths since the beginning of the epidemic up to 31 March in our model and in a counterfactual model (assuming no intervention had taken place). Estimated averted deaths over this time period as a result of the interventions. Numbers in brackets are 95% credible intervals. 2.3 Estimated impact of interventions on deaths Table 2 shows total forecasted deaths since the beginning of the epidemic up to and including 31 March under ourfitted model and under the counterfactual model, which predicts what would have happened if no interventions were implemented (and R, = R0 i.e. the initial reproduction number estimated before interventions). Again, the assumption in these predictions is that intervention impact is the same across countries and time. The model without interventions was unable to capture recent trends in deaths in several countries, where the rate of increase had clearly slowed (Figure 3). Trends were confirmed statistically by Bayesian leave-one-out cross-validation and the widely applicable information criterion assessments —WA|C). By comparing the deaths predicted under the model with no interventions to the deaths predicted in our intervention model, we calculated the total deaths averted up to the end of March. We find that, across 11 countries, since the beginning of the epidemic, 59,000 [21,000-120,000] deaths have been averted due to interventions. In Italy and Spain, where the epidemic is advanced, 38,000 [13,000- 84,000] and 16,000 [5,400-35,000] deaths have been averted, respectively. Even in the UK, which is much earlier in its epidemic, we predict 370 [73-1,000] deaths have been averted. These numbers give only the deaths averted that would have occurred up to 31 March. lfwe were to include the deaths of currently infected individuals in both models, which might happen after 31 March, then the deaths averted would be substantially higher. Figure 3: Daily number of confirmed deaths, predictions (up to 28 March) and forecasts (after) for (a) Italy and (b) Spain from our model with interventions (blue) and from the no interventions counterfactual model (pink); credible intervals are shown one week into the future. Other countries are shown in Appendix 8.6. 03/0 25% 50% 753% 100% (no effect on transmissibility) (ends transmissibility Relative % reduction in R. Figure 4: Our model includes five covariates for governmental interventions, adjusting for whether the intervention was the first one undertaken by the government in response to COVID-19 (red) or was subsequent to other interventions (green). Mean relative percentage reduction in Rt is shown with 95% posterior credible intervals. If 100% reduction is achieved, Rt = 0 and there is no more transmission of COVID-19. No effects are significantly different from any others, probably due to the fact that many interventions occurred on the same day or within days of each other as shown in Figure l. 3 Discussion During this early phase of control measures against the novel coronavirus in Europe, we analyze trends in numbers of deaths to assess the extent to which transmission is being reduced. Representing the COVlD-19 infection process using a semi-mechanistic, joint, Bayesian hierarchical model, we can reproduce trends observed in the data on deaths and can forecast accurately over short time horizons. We estimate that there have been many more infections than are currently reported. The high level of under-ascertainment of infections that we estimate here is likely due to the focus on testing in hospital settings rather than in the community. Despite this, only a small minority of individuals in each country have been infected, with an attack rate on average of 4.9% [l.9%-ll%] with considerable variation between countries (Table 1). Our estimates imply that the populations in Europe are not close to herd immunity ("50-75% if R0 is 2-4). Further, with Rt values dropping substantially, the rate of acquisition of herd immunity will slow down rapidly. This implies that the virus will be able to spread rapidly should interventions be lifted. Such estimates of the attack rate to date urgently need to be validated by newly developed antibody tests in representative population surveys, once these become available. We estimate that major non-pharmaceutical interventions have had a substantial impact on the time- varying reproduction numbers in countries where there has been time to observe intervention effects on trends in deaths (Italy, Spain). lfadherence in those countries has changed since that initial period, then our forecast of future deaths will be affected accordingly: increasing adherence over time will have resulted in fewer deaths and decreasing adherence in more deaths. Similarly, our estimates of the impact ofinterventions in other countries should be viewed with caution if the same interventions have achieved different levels of adherence than was initially the case in Italy and Spain. Due to the implementation of interventions in rapid succession in many countries, there are not enough data to estimate the individual effect size of each intervention, and we discourage attributing associations to individual intervention. In some cases, such as Norway, where all interventions were implemented at once, these individual effects are by definition unidentifiable. Despite this, while individual impacts cannot be determined, their estimated joint impact is strongly empirically justified (see Appendix 8.4 for sensitivity analysis). While the growth in daily deaths has decreased, due to the lag between infections and deaths, continued rises in daily deaths are to be expected for some time. To understand the impact of interventions, we fit a counterfactual model without the interventions and compare this to the actual model. Consider Italy and the UK - two countries at very different stages in their epidemics. For the UK, where interventions are very recent, much of the intervention strength is borrowed from countries with older epidemics. The results suggest that interventions will have a large impact on infections and deaths despite counts of both rising. For Italy, where far more time has passed since the interventions have been implemented, it is clear that the model without interventions does not fit well to the data, and cannot explain the sub-linear (on the logarithmic scale) reduction in deaths (see Figure 10). The counterfactual model for Italy suggests that despite mounting pressure on health systems, interventions have averted a health care catastrophe where the number of new deaths would have been 3.7 times higher (38,000 deaths averted) than currently observed. Even in the UK, much earlier in its epidemic, the recent interventions are forecasted to avert 370 total deaths up to 31 of March. 4 Conclusion and Limitations Modern understanding of infectious disease with a global publicized response has meant that nationwide interventions could be implemented with widespread adherence and support. Given observed infection fatality ratios and the epidemiology of COVlD-19, major non-pharmaceutical interventions have had a substantial impact in reducing transmission in countries with more advanced epidemics. It is too early to be sure whether similar reductions will be seen in countries at earlier stages of their epidemic. While we cannot determine which set of interventions have been most successful, taken together, we can already see changes in the trends of new deaths. When forecasting 3 days and looking over the whole epidemic the number of deaths averted is substantial. We note that substantial innovation is taking place, and new more effective interventions or refinements of current interventions, alongside behavioral changes will further contribute to reductions in infections. We cannot say for certain that the current measures have controlled the epidemic in Europe; however, if current trends continue, there is reason for optimism. Our approach is semi-mechanistic. We propose a plausible structure for the infection process and then estimate parameters empirically. However, many parameters had to be given strong prior distributions or had to be fixed. For these assumptions, we have provided relevant citations to previous studies. As more data become available and better estimates arise, we will update these in weekly reports. Our choice of serial interval distribution strongly influences the prior distribution for starting R0. Our infection fatality ratio, and infection-to-onset-to-death distributions strongly influence the rate of death and hence the estimated number of true underlying cases. We also assume that the effect of interventions is the same in all countries, which may not be fully realistic. This assumption implies that countries with early interventions and more deaths since these interventions (e.g. Italy, Spain) strongly influence estimates of intervention impact in countries at earlier stages of their epidemic with fewer deaths (e.g. Germany, UK). We have tried to create consistent definitions of all interventions and document details of this in Appendix 8.6. However, invariably there will be differences from country to country in the strength of their intervention — for example, most countries have banned gatherings of more than 2 people when implementing a lockdown, whereas in Sweden the government only banned gatherings of more than 10 people. These differences can skew impacts in countries with very little data. We believe that our uncertainty to some degree can cover these differences, and as more data become available, coefficients should become more reliable. However, despite these strong assumptions, there is sufficient signal in the data to estimate changes in R, (see the sensitivity analysis reported in Appendix 8.4.3) and this signal will stand to increase with time. In our Bayesian hierarchical framework, we robustly quantify the uncertainty in our parameter estimates and posterior predictions. This can be seen in the very wide credible intervals in more recent days, where little or no death data are available to inform the estimates. Furthermore, we predict intervention impact at country-level, but different trends may be in place in different parts of each country. For example, the epidemic in northern Italy was subject to controls earlier than the rest of the country. 5 Data Our model utilizes daily real-time death data from the ECDC (European Centre of Disease Control), where we catalogue case data for 11 European countries currently experiencing the epidemic: Austria, Belgium, Denmark, France, Germany, Italy, Norway, Spain, Sweden, Switzerland and the United Kingdom. The ECDC provides information on confirmed cases and deaths attributable to COVID-19. However, the case data are highly unrepresentative of the incidence of infections due to underreporting as well as systematic and country-specific changes in testing. We, therefore, use only deaths attributable to COVID-19 in our model; we do not use the ECDC case estimates at all. While the observed deaths still have some degree of unreliability, again due to changes in reporting and testing, we believe the data are ofsufficient fidelity to model. For population counts, we use UNPOP age-stratified counts.10 We also catalogue data on the nature and type of major non-pharmaceutical interventions. We looked at the government webpages from each country as well as their official public health division/information webpages to identify the latest advice/laws being issued by the government and public health authorities. We collected the following: School closure ordered: This intervention refers to nationwide extraordinary school closures which in most cases refer to both primary and secondary schools closing (for most countries this also includes the closure of otherforms of higher education or the advice to teach remotely). In the case of Denmark and Sweden, we allowed partial school closures of only secondary schools. The date of the school closure is taken to be the effective date when the schools started to be closed (ifthis was on a Monday, the date used was the one of the previous Saturdays as pupils and students effectively stayed at home from that date onwards). Case-based measures: This intervention comprises strong recommendations or laws to the general public and primary care about self—isolation when showing COVID-19-like symptoms. These also include nationwide testing programs where individuals can be tested and subsequently self—isolated. Our definition is restricted to nationwide government advice to all individuals (e.g. UK) or to all primary care and excludes regional only advice. These do not include containment phase interventions such as isolation if travelling back from an epidemic country such as China. Public events banned: This refers to banning all public events of more than 100 participants such as sports events. Social distancing encouraged: As one of the first interventions against the spread of the COVID-19 pandemic, many governments have published advice on social distancing including the recommendation to work from home wherever possible, reducing use ofpublictransport and all other non-essential contact. The dates used are those when social distancing has officially been recommended by the government; the advice may include maintaining a recommended physical distance from others. Lockdown decreed: There are several different scenarios that the media refers to as lockdown. As an overall definition, we consider regulations/legislations regarding strict face-to-face social interaction: including the banning of any non-essential public gatherings, closure of educational and public/cultural institutions, ordering people to stay home apart from exercise and essential tasks. We include special cases where these are not explicitly mentioned on government websites but are enforced by the police (e.g. France). The dates used are the effective dates when these legislations have been implemented. We note that lockdown encompasses other interventions previously implemented. First intervention: As Figure 1 shows, European governments have escalated interventions rapidly, and in some examples (Norway/Denmark) have implemented these interventions all on a single day. Therefore, given the temporal autocorrelation inherent in government intervention, we include a binary covariate for the first intervention, which can be interpreted as a government decision to take major action to control COVID-19. A full list of the timing of these interventions and the sources we have used can be found in Appendix 8.6. 6 Methods Summary A Visual summary of our model is presented in Figure 5 (details in Appendix 8.1 and 8.2). Replication code is available at https://github.com/|mperia|CollegeLondon/covid19model/releases/tag/vl.0 We fit our model to observed deaths according to ECDC data from 11 European countries. The modelled deaths are informed by an infection-to-onset distribution (time from infection to the onset of symptoms), an onset-to-death distribution (time from the onset of symptoms to death), and the population-averaged infection fatality ratio (adjusted for the age structure and contact patterns of each country, see Appendix). Given these distributions and ratios, modelled deaths are a function of the number of infections. The modelled number of infections is informed by the serial interval distribution (the average time from infection of one person to the time at which they infect another) and the time-varying reproduction number. Finally, the time-varying reproduction number is a function of the initial reproduction number before interventions and the effect sizes from interventions. Figure 5: Summary of model components. Following the hierarchy from bottom to top gives us a full framework to see how interventions affect infections, which can result in deaths. We use Bayesian inference to ensure our modelled deaths can reproduce the observed deaths as closely as possible. From bottom to top in Figure 5, there is an implicit lag in time that means the effect of very recent interventions manifest weakly in current deaths (and get stronger as time progresses). To maximise the ability to observe intervention impact on deaths, we fit our model jointly for all 11 European countries, which results in a large data set. Our model jointly estimates the effect sizes of interventions. We have evaluated the effect ofour Bayesian prior distribution choices and evaluate our Bayesian posterior calibration to ensure our results are statistically robust (Appendix 8.4). 7 Acknowledgements Initial research on covariates in Appendix 8.6 was crowdsourced; we thank a number of people across the world for help with this. This work was supported by Centre funding from the UK Medical Research Council under a concordat with the UK Department for International Development, the NIHR Health Protection Research Unit in Modelling Methodology and CommunityJameel. 8 Appendix: Model Specifics, Validation and Sensitivity Analysis 8.1 Death model We observe daily deaths Dam for days t E 1, ...,n and countries m E 1, ...,p. These daily deaths are modelled using a positive real-Valued function dam = E(Dam) that represents the expected number of deaths attributed to COVID-19. Dam is assumed to follow a negative binomial distribution with The expected number of deaths (1 in a given country on a given day is a function of the number of infections C occurring in previous days. At the beginning of the epidemic, the observed deaths in a country can be dominated by deaths that result from infection that are not locally acquired. To avoid biasing our model by this, we only include observed deaths from the day after a country has cumulatively observed 10 deaths in our model. To mechanistically link ourfunction for deaths to infected cases, we use a previously estimated COVID- 19 infection-fatality-ratio ifr (probability of death given infection)9 together with a distribution oftimes from infection to death TE. The ifr is derived from estimates presented in Verity et al11 which assumed homogeneous attack rates across age-groups. To better match estimates of attack rates by age generated using more detailed information on country and age-specific mixing patterns, we scale these estimates (the unadjusted ifr, referred to here as ifr’) in the following way as in previous work.4 Let Ca be the number of infections generated in age-group a, Na the underlying size of the population in that age group and AR“ 2 Ca/Na the age-group-specific attack rate. The adjusted ifr is then given by: ifra = fififié, where AR50_59 is the predicted attack-rate in the 50-59 year age-group after incorporating country-specific patterns of contact and mixing. This age-group was chosen as the reference as it had the lowest predicted level of underreporting in previous analyses of data from the Chinese epidemic“. We obtained country-specific estimates of attack rate by age, AR“, for the 11 European countries in our analysis from a previous study which incorporates information on contact between individuals of different ages in countries across Europe.12 We then obtained overall ifr estimates for each country adjusting for both demography and age-specific attack rates. Using estimated epidemiological information from previous studies,“'11 we assume TE to be the sum of two independent random times: the incubation period (infection to onset of symptoms or infection- to-onset) distribution and the time between onset of symptoms and death (onset-to-death). The infection-to-onset distribution is Gamma distributed with mean 5.1 days and coefficient of variation 0.86. The onset-to-death distribution is also Gamma distributed with a mean of 18.8 days and a coefficient of va riation 0.45. ifrm is population averaged over the age structure of a given country. The infection-to-death distribution is therefore given by: um ~ ifrm ~ (Gamma(5.1,0.86) + Gamma(18.8,0.45)) Figure 6 shows the infection-to-death distribution and the resulting survival function that integrates to the infection fatality ratio. Figure 6: Left, infection-to-death distribution (mean 23.9 days). Right, survival probability of infected individuals per day given the infection fatality ratio (1%) and the infection-to-death distribution on the left. Using the probability of death distribution, the expected number of deaths dam, on a given day t, for country, m, is given by the following discrete sum: The number of deaths today is the sum of the past infections weighted by their probability of death, where the probability of death depends on the number of days since infection. 8.2 Infection model The true number of infected individuals, C, is modelled using a discrete renewal process. This approach has been used in numerous previous studies13'16 and has a strong theoretical basis in stochastic individual-based counting processes such as Hawkes process and the Bellman-Harris process.”18 The renewal model is related to the Susceptible-Infected-Recovered model, except the renewal is not expressed in differential form. To model the number ofinfections over time we need to specify a serial interval distribution g with density g(T), (the time between when a person gets infected and when they subsequently infect another other people), which we choose to be Gamma distributed: g ~ Gamma (6.50.62). The serial interval distribution is shown below in Figure 7 and is assumed to be the same for all countries. Figure 7: Serial interval distribution g with a mean of 6.5 days. Given the serial interval distribution, the number of infections Eamon a given day t, and country, m, is given by the following discrete convolution function: _ t—1 Cam — Ram ZT=0 Cr,mgt—‘r r where, similarto the probability ofdeath function, the daily serial interval is discretized by fs+0.5 1.5 gs = T=s—0.Sg(T)dT fors = 2,3, and 91 = fT=Og(T)dT. Infections today depend on the number of infections in the previous days, weighted by the discretized serial interval distribution. This weighting is then scaled by the country-specific time-Varying reproduction number, Ram, that models the average number of secondary infections at a given time. The functional form for the time-Varying reproduction number was chosen to be as simple as possible to minimize the impact of strong prior assumptions: we use a piecewise constant function that scales Ram from a baseline prior R0,m and is driven by known major non-pharmaceutical interventions occurring in different countries and times. We included 6 interventions, one of which is constructed from the other 5 interventions, which are timings of school and university closures (k=l), self—isolating if ill (k=2), banning of public events (k=3), any government intervention in place (k=4), implementing a partial or complete lockdown (k=5) and encouraging social distancing and isolation (k=6). We denote the indicator variable for intervention k E 1,2,3,4,5,6 by IkI’m, which is 1 if intervention k is in place in country m at time t and 0 otherwise. The covariate ”any government intervention” (k=4) indicates if any of the other 5 interventions are in effect,i.e.14’t’m equals 1 at time t if any of the interventions k E 1,2,3,4,5 are in effect in country m at time t and equals 0 otherwise. Covariate 4 has the interpretation of indicating the onset of major government intervention. The effect of each intervention is assumed to be multiplicative. Ram is therefore a function ofthe intervention indicators Ik’t’m in place at time t in country m: Ram : R0,m eXp(— 212:1 O(Rheum)- The exponential form was used to ensure positivity of the reproduction number, with R0,m constrained to be positive as it appears outside the exponential. The impact of each intervention on Ram is characterised by a set of parameters 0(1, ...,OL6, with independent prior distributions chosen to be ock ~ Gamma(. 5,1). The impacts ock are shared between all m countries and therefore they are informed by all available data. The prior distribution for R0 was chosen to be R0,m ~ Normal(2.4, IKI) with K ~ Normal(0,0.5), Once again, K is the same among all countries to share information. We assume that seeding of new infections begins 30 days before the day after a country has cumulatively observed 10 deaths. From this date, we seed our model with 6 sequential days of infections drawn from cl’m,...,66’m~EXponential(T), where T~Exponential(0.03). These seed infections are inferred in our Bayesian posterior distribution. We estimated parameters jointly for all 11 countries in a single hierarchical model. Fitting was done in the probabilistic programming language Stan,19 using an adaptive Hamiltonian Monte Carlo (HMC) sampler. We ran 8 chains for 4000 iterations with 2000 iterations of warmup and a thinning factor 4 to obtain 2000 posterior samples. Posterior convergence was assessed using the Rhat statistic and by diagnosing divergent transitions of the HMC sampler. Prior-posterior calibrations were also performed (see below). 8.3 Validation We validate accuracy of point estimates of our model using cross-Validation. In our cross-validation scheme, we leave out 3 days of known death data (non-cumulative) and fit our model. We forecast what the model predicts for these three days. We present the individual forecasts for each day, as well as the average forecast for those three days. The cross-validation results are shown in the Figure 8. Figure 8: Cross-Validation results for 3-day and 3-day aggregatedforecasts Figure 8 provides strong empirical justification for our model specification and mechanism. Our accurate forecast over a three-day time horizon suggests that our fitted estimates for Rt are appropriate and plausible. Along with from point estimates we all evaluate our posterior credible intervals using the Rhat statistic. The Rhat statistic measures whether our Markov Chain Monte Carlo (MCMC) chains have converged to the equilibrium distribution (the correct posterior distribution). Figure 9 shows the Rhat statistics for all of our parameters Figure 9: Rhat statistics - values close to 1 indicate MCMC convergence. Figure 9 indicates that our MCMC have converged. In fitting we also ensured that the MCMC sampler experienced no divergent transitions - suggesting non pathological posterior topologies. 8.4 SensitivityAnalysis 8.4.1 Forecasting on log-linear scale to assess signal in the data As we have highlighted throughout in this report, the lag between deaths and infections means that it ta kes time for information to propagate backwa rds from deaths to infections, and ultimately to Rt. A conclusion of this report is the prediction of a slowing of Rt in response to major interventions. To gain intuition that this is data driven and not simply a consequence of highly constrained model assumptions, we show death forecasts on a log-linear scale. On this scale a line which curves below a linear trend is indicative of slowing in the growth of the epidemic. Figure 10 to Figure 12 show these forecasts for Italy, Spain and the UK. They show this slowing down in the daily number of deaths. Our model suggests that Italy, a country that has the highest death toll of COVID-19, will see a slowing in the increase in daily deaths over the coming week compared to the early stages of the epidemic. We investigated the sensitivity of our estimates of starting and final Rt to our assumed serial interval distribution. For this we considered several scenarios, in which we changed the serial interval distribution mean, from a value of 6.5 days, to have values of 5, 6, 7 and 8 days. In Figure 13, we show our estimates of R0, the starting reproduction number before interventions, for each of these scenarios. The relative ordering of the Rt=0 in the countries is consistent in all settings. However, as expected, the scale of Rt=0 is considerably affected by this change — a longer serial interval results in a higher estimated Rt=0. This is because to reach the currently observed size of the epidemics, a longer assumed serial interval is compensated by a higher estimated R0. Additionally, in Figure 14, we show our estimates of Rt at the most recent model time point, again for each ofthese scenarios. The serial interval mean can influence Rt substantially, however, the posterior credible intervals of Rt are broadly overlapping. Figure 13: Initial reproduction number R0 for different serial interval (SI) distributions (means between 5 and 8 days). We use 6.5 days in our main analysis. Figure 14: Rt on 28 March 2020 estimated for all countries, with serial interval (SI) distribution means between 5 and 8 days. We use 6.5 days in our main analysis. 8.4.3 Uninformative prior sensitivity on or We ran our model using implausible uninformative prior distributions on the intervention effects, allowing the effect of an intervention to increase or decrease Rt. To avoid collinearity, we ran 6 separate models, with effects summarized below (compare with the main analysis in Figure 4). In this series of univariate analyses, we find (Figure 15) that all effects on their own serve to decrease Rt. This gives us confidence that our choice of prior distribution is not driving the effects we see in the main analysis. Lockdown has a very large effect, most likely due to the fact that it occurs after other interventions in our dataset. The relatively large effect sizes for the other interventions are most likely due to the coincidence of the interventions in time, such that one intervention is a proxy for a few others. Figure 15: Effects of different interventions when used as the only covariate in the model. 8.4.4 To assess prior assumptions on our piecewise constant functional form for Rt we test using a nonparametric function with a Gaussian process prior distribution. We fit a model with a Gaussian process prior distribution to data from Italy where there is the largest signal in death data. We find that the Gaussian process has a very similartrend to the piecewise constant model and reverts to the mean in regions of no data. The correspondence of a completely nonparametric function and our piecewise constant function suggests a suitable parametric specification of Rt. Nonparametric fitting of Rf using a Gaussian process: 8.4.5 Leave country out analysis Due to the different lengths of each European countries’ epidemic, some countries, such as Italy have much more data than others (such as the UK). To ensure that we are not leveraging too much information from any one country we perform a ”leave one country out” sensitivity analysis, where we rerun the model without a different country each time. Figure 16 and Figure 17 are examples for results for the UK, leaving out Italy and Spain. In general, for all countries, we observed no significant dependence on any one country. Figure 16: Model results for the UK, when not using data from Italy for fitting the model. See the Figure 17: Model results for the UK, when not using data from Spain for fitting the model. See caption of Figure 2 for an explanation of the plots. 8.4.6 Starting reproduction numbers vs theoretical predictions To validate our starting reproduction numbers, we compare our fitted values to those theoretically expected from a simpler model assuming exponential growth rate, and a serial interval distribution mean. We fit a linear model with a Poisson likelihood and log link function and extracting the daily growth rate r. For well-known theoretical results from the renewal equation, given a serial interval distribution g(r) with mean m and standard deviation 5, given a = mZ/S2 and b = m/SZ, and a subsequently R0 = (1 + %) .Figure 18 shows theoretically derived R0 along with our fitted estimates of Rt=0 from our Bayesian hierarchical model. As shown in Figure 18 there is large correspondence between our estimated starting reproduction number and the basic reproduction number implied by the growth rate r. R0 (red) vs R(FO) (black) Figure 18: Our estimated R0 (black) versus theoretically derived Ru(red) from a log-linear regression fit. 8.5 Counterfactual analysis — interventions vs no interventions Figure 19: Daily number of confirmed deaths, predictions (up to 28 March) and forecasts (after) for all countries except Italy and Spain from our model with interventions (blue) and from the no interventions counterfactual model (pink); credible intervals are shown one week into the future. DOI: https://doi.org/10.25561/77731 Page 28 of 35 30 March 2020 Imperial College COVID-19 Response Team 8.6 Data sources and Timeline of Interventions Figure 1 and Table 3 display the interventions by the 11 countries in our study and the dates these interventions became effective. Table 3: Timeline of Interventions. Country Type Event Date effective School closure ordered Nationwide school closures.20 14/3/2020 Public events banned Banning of gatherings of more than 5 people.21 10/3/2020 Banning all access to public spaces and gatherings Lockdown of more than 5 people. Advice to maintain 1m ordered distance.22 16/3/2020 Social distancing encouraged Recommendation to maintain a distance of 1m.22 16/3/2020 Case-based Austria measures Implemented at lockdown.22 16/3/2020 School closure ordered Nationwide school closures.23 14/3/2020 Public events All recreational activities cancelled regardless of banned size.23 12/3/2020 Citizens are required to stay at home except for Lockdown work and essential journeys. Going outdoors only ordered with household members or 1 friend.24 18/3/2020 Public transport recommended only for essential Social distancing journeys, work from home encouraged, all public encouraged places e.g. restaurants closed.23 14/3/2020 Case-based Everyone should stay at home if experiencing a Belgium measures cough or fever.25 10/3/2020 School closure Secondary schools shut and universities (primary ordered schools also shut on 16th).26 13/3/2020 Public events Bans of events >100 people, closed cultural banned institutions, leisure facilities etc.27 12/3/2020 Lockdown Bans of gatherings of >10 people in public and all ordered public places were shut.27 18/3/2020 Limited use of public transport. All cultural Social distancing institutions shut and recommend keeping encouraged appropriate distance.28 13/3/2020 Case-based Everyone should stay at home if experiencing a Denmark measures cough or fever.29 12/3/2020 School closure ordered Nationwide school closures.30 14/3/2020 Public events banned Bans of events >100 people.31 13/3/2020 Lockdown Everybody has to stay at home. Need a self- ordered authorisation form to leave home.32 17/3/2020 Social distancing encouraged Advice at the time of lockdown.32 16/3/2020 Case-based France measures Advice at the time of lockdown.32 16/03/2020 School closure ordered Nationwide school closures.33 14/3/2020 Public events No gatherings of >1000 people. Otherwise banned regional restrictions only until lockdown.34 22/3/2020 Lockdown Gatherings of > 2 people banned, 1.5 m ordered distance.35 22/3/2020 Social distancing Avoid social interaction wherever possible encouraged recommended by Merkel.36 12/3/2020 Advice for everyone experiencing symptoms to Case-based contact a health care agency to get tested and Germany measures then self—isolate.37 6/3/2020 School closure ordered Nationwide school closures.38 5/3/2020 Public events banned The government bans all public events.39 9/3/2020 Lockdown The government closes all public places. People ordered have to stay at home except for essential travel.40 11/3/2020 A distance of more than 1m has to be kept and Social distancing any other form of alternative aggregation is to be encouraged excluded.40 9/3/2020 Case-based Advice to self—isolate if experiencing symptoms Italy measures and quarantine if tested positive.41 9/3/2020 Norwegian Directorate of Health closes all School closure educational institutions. Including childcare ordered facilities and all schools.42 13/3/2020 Public events The Directorate of Health bans all non-necessary banned social contact.42 12/3/2020 Lockdown Only people living together are allowed outside ordered together. Everyone has to keep a 2m distance.43 24/3/2020 Social distancing The Directorate of Health advises against all encouraged travelling and non-necessary social contacts.42 16/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a Norway measures cough or fever symptoms.44 15/3/2020 ordered Nationwide school closures.45 13/3/2020 Public events banned Banning of all public events by lockdown.46 14/3/2020 Lockdown ordered Nationwide lockdown.43 14/3/2020 Social distancing Advice on social distancing and working remotely encouraged from home.47 9/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a Spain measures cough or fever symptoms.47 17/3/2020 School closure ordered Colleges and upper secondary schools shut.48 18/3/2020 Public events banned The government bans events >500 people.49 12/3/2020 Lockdown ordered No lockdown occurred. NA People even with mild symptoms are told to limit Social distancing social contact, encouragement to work from encouraged home.50 16/3/2020 Case-based Advice to self—isolate if experiencing a cough or Sweden measures fever symptoms.51 10/3/2020 School closure ordered No in person teaching until 4th of April.52 14/3/2020 Public events banned The government bans events >100 people.52 13/3/2020 Lockdown ordered Gatherings of more than 5 people are banned.53 2020-03-20 Advice on keeping distance. All businesses where Social distancing this cannot be realised have been closed in all encouraged states (kantons).54 16/3/2020 Case-based Advice to self—isolate if experiencing a cough or Switzerland measures fever symptoms.55 2/3/2020 Nationwide school closure. Childminders, School closure nurseries and sixth forms are told to follow the ordered guidance.56 21/3/2020 Public events banned Implemented with lockdown.57 24/3/2020 Gatherings of more than 2 people not from the Lockdown same household are banned and police ordered enforceable.57 24/3/2020 Social distancing Advice to avoid pubs, clubs, theatres and other encouraged public institutions.58 16/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a UK measures cough or fever symptoms.59 12/3/2020 9 References 1. Li, R. et al. Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV2). Science (2020) doi:10.1126/science.abb3221. 2. Zhang, J. et al. Patterns of human social contact and contact with animals in Shanghai, China. 5cLRep.9,1—11(2019) 3. Worldometers.info. Hong Kong: coronavirus cases. https://www.wo rldometers.info/co ronavirus/country/china-hong-kong-sar/. 4. Ferguson, N. et al. Impact of non-pharmaceutical interventions (NPIs) to reduce COVID-19 mortality and healthcare demand (Report 9). https://www.imperial.ac.uk/mrc-global-infectious- disease-analysis/news--wuhan-coronavirus/. 5. Cereda, D. et al. The early phase of the COVID-19 outbreak in Lombardy, Italy. arXiv (2020). 6. Zhao, A. J. et al. Title: Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 Brief Title : Antibody responses in COVID-19 patients. (2020). 7. Jombart, T. et al. Inferring the number of COVID-19 cases from recently reported deaths. medRXiV 2020.03.10.20033761(2020)doi:10.1101/2020.03.10.20033761. 8. Zhang, J. et al. Age profile of susceptibility, mixing, and social distancing shape the dynamics of the novel coronavirus disease 2019 outbreak in China. (2020) doi:10.1101/2020.03.19.20039107. 9. Lourenco, J. et al. Fundamental principles of epidemic spread highlight the immediate need for large-scale serological surveys to assess the stage of the SARS-CoV-2 epidemic. doi:10.1101/2020.03.24.20042291 10. United Nations, Department of Economic and Social Affairs, Population Division. World Population Prospects 2019: Data Booket. ST/ESA/SER.A/424. (2019). 11. Verity, R. et al. Estimates ofthe severity of COVID-19 disease. Lancet Infect Dis in press, (2020). 12. Walker, P. G. T. et al. Report 12: The Global Impact of COVID-19 and Strategies for Mitigation and Suppression. 13. Fraser, C. Estimating Individual and Household Reproduction Numbers in an Emerging Epidemic. PL05 ONE 2, e758 (2007). 14. Cori, A., Ferguson, N. M., Fraser, C. & Cauchemez, S. A New Framework and Software to Estimate Time-Varying Reproduction Numbers During Epidemics. Am. J. Epidemiol. 178, 1505—1512 (20131 15. Nouvellet, P. et al. A simple approach to measure transmissibility and forecast incidence. Epidemics 22, 29—35 (2018). 16. Cauchemez, 8., Valleron, A. J., Boelle, P. Y., Flahault, A. & Ferguson, N. M. Estimating the impact of school closure on influenza transmission from Sentinel data. Nature 452, 750—754 (2008). 17. Bellman, R. & Harris, T. On Age-Dependent Binary Branching Processes. Ann. Math. 55, 280— 295(19521 18. Bellman, R. & Harris, T. E. On the Theory of Age-Dependent Stochastic Branching Processes. Proc. Natl. Acad. Sci. 34, 601—604 (1948). 19. Stan Development Team. 2018. The Stan Core Library, Version 2.18.0. http://mc-stan.org. 20. Bundesministerium. Coronavirus (COVID-19): Status quo — Schulen, Hochschulen, Universitaten und Forschungsinstitutionen. https://www.bmbwf.gv.at/Ministerium/Informationspflicht/corona/corona_status.html. 21. Henley, J. Coronavirus: EU states enact tough measures to stem spread. The Guardian https://www.theguardian.com/world/2020/mar/10/coronavirus-several-eu-states-ban-mass-events- after-italian-lockdown (2020). 22. Bundesministerium. Coronavirus - Aktuelle MaBnahmen. https://www.sozialministerium.at/Informationen-zum-Coronavirus/Coronavirus—Aktuelle- MaBnahmen.html (2020). 23. Federal Public Service. Coronavirus : Phase 2 maintained, transition to the federal phase and additional measures. https://www.info-coronavirus.be/en/2020/03/12/phase-2-maintained- transition-to-the-federal-phase-and-additional-measures/ (2020). 24. Belgium.be. Coronavirus: reinforced measures | Belgium.be. https://www.belgium.be/en/news/2020/coronavirus_reinforced_measures (2020). 25. Federal Public Service. Protect yourself and protect the others. https://www.info- coronavirus.be/en/2020/03/10/protect-yourself—and-protect-the-others/ (2020). 26. Wikipedia. 2020 coronavirus pandemic in Denmark. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_Denmark. 27. Stephensen, Emma K|inker; Hansen, T. S. Danmark lukker ned: Her er regeringens nye tiltag. TV2 https://nyheder.tv2.dk/samfund/2020-03-11-danmark-lukker-ned-her-er-regeringens-nye-tiltag (20201 28. Politi. Nye tiltag mod covid-19. Politi https://politi.dk/coronavirus-i-danmark/seneste-nyt-fra- myndighederne/nye-tiltag-mod-covid-19 (2020). 29. Styrelsen for Patientsikkerhed. Indberetning om covid-19zlnformation om mulighed for p\aabud til enkeltpersoner (coronavirus/covid-19). https://stps.dk/da/ansvar-og- retningslinjer/vejledning/indberetning-om-covid-19/#. 30. Wikipedia. 2020 coronavirus pandemic in France. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_France. 31. The Local. France bans gatherings of more than 100 people as coronavirus death toll rises - The Local. The Local https://www.thelocal.fr/20200313/france-bans-gatherings-of—over-100-people- to-fight-coronavirus-pandemic (2020). 32. Henley, Jon; Willsher, Kim; Kassam, A. Coronavirus: France imposes lockdown as EU calls for 30-day travel ban. The Guardian https://www.theguardian.com/world/2020/mar/16/coronavirus- spain-takes-over-private-healthcare-amid-more-european-lockdowns (2020). 33. Wikipedia. 2020 coronavirus pandemic in Germany. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_Germany. 34. BMI. Coronavirus: Fragen und Antworten. Bundesministerium des Innern,fur Bau und Heimat https://web.archive.org/web/20200317073042/https://www.bmi.bund.de/SharedDocs/faqs/DE/the men/bevoelkerungsschutz/coronavirus/coronavirus-faqs.htmI#doc13738352bodyText7. 35. BBC News. Coronavirus: Germany tightens curbs and bans meetings of more than two. BBC News https://www.bbc.co.uk/news/world-europe-51999080 (2020). 36. Bundesregierung. Kanzlerin trifft Regierungschefs der Lander Sozialkontakte vermeiden, Ausbreitung verlangsamen. https://www.bundesregierung.de/breg-de/themen/coronavirus/mpk- 1730186(2020) 37. Robert Koch Institut. Antworten auf haufig gestellte Fragen zum Coronavirus SARS-CoV-2. Robert Koch Institut https://web.archive.org/web/20200312004624/https://www.rki.de/SharedDocs/FAQ/NCOV2019/F AQ_Liste.html (2020). 38. Ministero della Salute. Governo annuncia sospensione dell’attivita didattica dal 5 a|15 marzo. Ministero della Salute http://www.salute.gov.it/portale/nuovocoronavirus/dettaglioVideoNuovoCoronavirus.jsp?lingua=ita liano&menu=multimedia&p=video&id=2052 (2020). 39. CNN. Italy prohibits travel and cancels all public events in its northern region. CNN https://edition.cnn.com/2020/03/08/europe/italy-coronavirus-lockdown-europe-intl/index.html (2020). 40. Attualita. Coronavirus: stop a pub, cinema, teatro e discoteche anche a Roma. Ecco cosa prevede il nuovo decreto. Roma Today https://www.romatoday.it/attualita/coronavirus-pub-cinema- teatri-locali-chiusi-nuovo-decreto.html (2020). 41. Gazzetta Ufficiale. DECRETO DEL PRESIDENTE DEL CONSIGLIO DEl MINISTRI. Gazzetta Ufflclale https://www.gazzettaufficiale.it/eli/id/2020/03/08/20A01522/sg (2020). 42. Helsedirektoratet. The Norwegian Directorate of Health has issued a decision to close schools and other educational institutions. Helsedirektoratet https://www.helsedirektoratet.no/nyheter/the- norwegian-directorate-of—health-has-issued-a-decision-to-close-schools-and-other-educationa|- institutions (2020). 43. Krostensen, Mette; Hellem-Hansen, Viktoria L.; Tandstad, B. Folkehelseinstituttet mener 23.000 kan vaere smittet. NRK https://www.nrk.no/norge/folkehelseinstituttet-mener-23.000-kan- vaere-smittet-1.14958149 (2020). 44. Norweigen Government. The Government is establishing clear quarantine and isolation rules. regjeringen.no https://www.regjeringen.no/en/aktuelt/the-government-is-establishing-clear- quarantine-and-isolation-rules/id2693647/ (2020). 45. Wikipedia. 2020 coronavirus pandemic in Spain. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_Spain. 46. Gabinete de Prensa. El Gobierno anuncia nuevas medidas para evitar la extension del nuevo coronavirus COVID-19. Gobierno de Espana https://www.mscbs.gob.es/gabinete/notasPrensa.do?id=4807 (2020). 47. Gabinete de Prensa. El Consejo Interterritorial del SNS acuerda medidas concretas para zonas con transmision comunitaria significativa de coronavirus. Gobierno de Espana https://www.mscbs.gob.es/gabinete/notasPrensa.do?id=4806 (2020). 48. Folkhalsomyndigheten. Larosaten och gymnasieskolor uppmanas nu att bedriva distansundervisning. Folkhdlsomyndigheten https://www.folkhalsomyndigheten.se/nyheter-och- press/nyhetsarkiv/2020/mars/larosaten-och-gymnasieskolor-uppmanas-nu-att-bedriva- distansundervisning(2020). 49. The Local. Sweden bans large events to halt coronavirus spread. The Local https://www.theloca|.se/20200311/sweden-to-ban-large-public-gatherings-over-coronavirus (2020). 50. Radosevich. Stockholmers urged to work from home as COVID-19 community spread confirmed. Sveriges Radio https://sverigesradio.se/sida/artikel.aspx?programid=2054&artikel=7430511(2020). 51. Folkhalsomyndigheten. Flera tecken p\aa samhallsspridning av covid-19 i Sverige. Folkhdlsomyndigheten https://www.folkhalsomyndigheten.se/nyheter-och- press/nyhetsarkiv/2020/mars/flera-tecken-pa-samhallsspridning-av-covid-19-i-sverige/ (2020). 52. Bundesamt fur Gesendheit BAG. Bundesrat verscharft Massnahmen gegen das Coronavirus zum Schutz der Gesundheit und unterstUtzt betroffene Branchen. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das-bag/aktuell/medienmitteilungen.msg-id-78437.html (20201 53. Bundesamt fur Gesundheit BAG. Coronavirus: Bundesrat verbietet Ansammlungen von mehr als fUnf Personen. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das- bag/aktuell/medienmitteilungen.msg-id-78513.html (2020). 54. Bundesamt fur Gesundheit BAG. Coronavirus: Bundesrat erklart die «ausserordentliche Lage» und verscharft die Massnahmen. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das-bag/aktuell/medienmitteilungen.msg-id-78454.html (20201 55. Bundesamt fur Gesundheit BAG. Neue Hygiene- und Verhaltensregeln zum Schutz gegen das neue Coronavirus. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das- bag/a ktuell/medienmitteilungen.msg-id-78304.html (2020). 56. UK Government, D. for E. Schools, colleges and early years settings to close. UK Government https://www.gov.uk/government/news/schools-colleges-and-early-years-settings-to-close (2020). 57. UK Government. PM address to the nation on coronavirus: 23 March 2020. UK Government https://www.gov.uk/government/speeches/pm-address-to-the-nation-on-coronavirus-23-march- 2020(20201 58. Boycott-Owen, Mason; Bowman, Verity; Kelly-Linden, Jordan; Gartner, A. G. H. S. T. Coronavirus: Boris Johnson puts UK in lockdown as death tolls reaches 55. The Telegraph https://www.telegraph.co.uk/global-health/science-and-disease/coronavirus-news-uk-latest- update-covid-19-death-toll-cases/ (2020). 59. BBC News. Coronavirus: People with fever or ’continuous’ cough told to self—isolate. BBC News https://www.bbc.co.uk/news/uk-51857856 (2020).
What is a way to measure virus transmission?
false
799
{ "text": [ "reproductive number" ], "answer_start": [ 2265 ] }
2,585
Spread from the Sink to the Patient: In Situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377511/ SHA: 615071c8c959f24857b1bad521cc432b59719bfb Authors: Kotay, Shireen; Chai, Weidong; Guilford, William; Barry, Katie; Mathers, Amy J. Date: 2017-03-31 DOI: 10.1128/aem.03327-16 License: cc-by Abstract: There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients. Text: D espite early reports (1) (2) (3) (4) (5) , the premise that hand-wash sink traps can act as reservoirs of bacteria that cause nosocomial infections has been frequently overlooked. There has recently been an alarming increase in sink-related outbreaks worldwide, with many reports establishing an observational link (6) (7) (8) (9) (10) (11) (12) (13) . A sink often operates as an open conduit to wastewater in a patient care area that is often in the same room as the patient. Health care establishments often invest in desperate interventions to deal with nosocomial outbreaks. The preferred method for addressing most of the environmentrelated transmission is to employ enhanced cleaning using chemical and physical agents (14, 15) . Unfortunately, routine approaches are inefficient in completely eliminating drug-resistant Gammaproteobacteria in an inaccessible microbiologically active area such as a sink trap (6, (16) (17) (18) (19) (20) . The wet, humid, and relatively protected environment in a sink trap favors the formation of rich stable microbial communities (16, 21, 22) . These communities will be exposed to liquids and waste that are discarded in a sink and may include antimicrobials, discarded beverages, soap, presumably pathogenic bacteria from health care workers' hands, and other items. In short, sink traps could serve as a breeding ground for opportunistic and highly antimicrobial-resistant bacteria that cannot be easily cleaned or removed (23) (24) (25) (26) (27) (28) . There are many reports of a genetic association between pathogens found in sink traps and those found in patients (29, 30) . However, surprisingly little work has been done to understand the microscale transmission dynamics. It was previously demonstrated using a suspension of fluorescent particles (Glo Germ; Glo Germ Co., Moab, UT) that material injected into the P-trap gets dispersed around a hand-washing sink (6) . This result however has not been replicated hitherto in the follow-up studies. Dispersion has never been investigated with living organisms. Ultimately, many details remain unaddressed surrounding the spread of Enterobacteriaceae in sink-trap wastewater systems: (i) can organisms grow retrograde from the P-trap water to the sink strainer, (ii) can organisms spread from one sink to another along the internal surfaces of pipes with shared drainage systems, and (iii) which portion of a colonized drain pipe results in dispersion into the sink bowl during a hand-washing event? We aim to better understand the dispersion dynamics of Gammaproteobacteria living in the wastewater of a sink strainer and P-trap into an area where patients and health care workers could be exposed. To study this dynamic, we used a surrogate organism that could be easily tracked while remaining in the Enterobacteriaceae family, where some of the most concerning threats in antimicrobial resistance are developing (30) . Growth and colonization of GFP-expressing E. coli in the P-trap. In the first 14 days following the installation of the P-trap with established green fluorescent protein (GFP)-expressing Escherichia coli and just water running from the faucet, GFPexpressing E. coli was not detected in the tailpipe beyond 1.5 in. above the liquid level in the P-trap. GFP-expressing E. coli, however, was found to be viable in the P-trap without any nutrients added. A nutrient regimen was then instituted to understand the influence of nutrients on mobility and upward growth. The addition of tryptic soy broth (TSB) promoted GFP-expressing E. coli growth as early as day 1, with growth observed in the tailpipe 2 in. above the liquid surface in the P-trap (Table 1) . On day 7, the strainer (ϳ8 in. above the liquid in the P-trap) was found to be colonized with GFP-expressing E. coli. This translates to an average growth rate of 1 in./day along the length of the tailpipe with the addition of nutrients and without faucet operation. GFP-expressing E. coli was not detected in the faucet water. Sink-to-sink transmission of bacteria. In these experiments, a flanking sink (sink 5) was the only P-trap inoculated with GFP-expressing E. coli and therefore was the sole source for transmission to the connected sinks. Starting with a lower inoculum concentration (10 3 CFU/ml) in sink 5, on day 7, GFP-expressing E. coli was detected in the sink 2 and sink 3 P-traps (Fig. 1a) . With inoculum concentrations of 10 6 CFU/ml and Ͼ10 10 CFU/ml in sink 5, all of the sink P-traps in the sink gallery with the exception of sink 1 were found to be colonized with GFP-expressing E. coli after 7 days (Fig. 1b and c) . Faucet water and aerators tested negative for GFP-expressing E. coli. Irrespective of the starting inoculum concentration, on day 7 the highest level of colonization was recorded in the sink 3 P-trap. After day 7, when the nutrient regimen (described previously) was followed for an additional 7 days in each of the sinks in the sink gallery with an inoculum concentration of Ͼ10 10 CFU/ml, GFP-expressing E. coli was detected in the strainers of sinks 2 and 3 on day 14. This finding validated the upward growth and growth rate in the tailpipe when nutrients were added. Nonfluorescent colonies were occasionally observed in the P-trap water samples collected from the sinks, which were subsequently identified to be Pseudomonas sp. or Stenotrophomonas maltophilia, and fluorescent colonies were confirmed to be E. coli. Dispersion of microspheres from sinks. In the first dispersion experiment, when fluorescent microspheres were inoculated into the offset drain tailpiece only 4 in. below the strainer, no microspheres were detected on the polyester sheets placed on the counter space. However, when the sink bowl was coated with the microspheres, polyester sheets overlaid on the counter space captured the dispersed microspheres caused by the faucet operation. Dispersion was observed on almost all zones of the sink counter space (Fig. 2) . Relatively higher levels of dispersion were observed along the major and minor axes of the elliptical sink bowl (zones 2, 5, 6, 9, 11, and 12) . Anterior corners of the sink counter space (zones 4 and 7), which were most distant from the impact of water in the sink bowl, received the lowest dispersion. Dispersion of GFP-expressing E. coli from sinks. Initially the P-trap alone was inoculated with GFP-expressing E. coli and carefully installed, keeping the tailpipe and strainer free of GFP-expressing E. coli before operating the faucets. No fluorescent CFU were observed on the plates placed on the counter or attached to the bowl surface after faucet operation. Similarly, no fluorescent CFU were detected when GFPexpressing E. coli was inoculated into the offset drain tailpiece only 4 in. below the strainer. Interestingly, when there was conspicuous water backup over the strainer as a result of a higher water flow rate from the faucet than the drainage rate from the P-trap, dispersal was detected on the plates attached to the bowl surface. The dispersion pattern recorded when the sink bowl was coated with GFPexpressing E. coli was comparable to the pattern recorded when the sink bowl was coated with fluorescent microspheres (Fig. 2) . Dispersion was significantly higher along the axes (zones 6, 9, 11, and 12) and lower at the corners of the sink counter space (zones 4, 7, and 10). In contrast, dispersion of GFP-expressing E. coli caused by the faucet operation was much more extensive when the strainer was allowed to be colonized with GFPexpressing E. coli prior to the dispersion experiment. In addition to the sink counter space, we measured dispersion to the sink bowl, faucet, faucet handles, splatter shields, and the extended counter surface. Dispersion of GFP-expressing E. coli was highest on the plates attached to the sink bowl (Fig. 3b) . Further, dispersion was greater along the Ϫ" and "ϩ" denote the absence and presence of GFP-expressing E. coli, respectively. minor axis of the sink bowl (Fig. 3b , zones B3, B4, and B10) than along the major axis of the sink bowl, associated with a shorter distance from the strike point of the faucet water to the bowl along this axis. The next highest CFU count from the dispersal was recorded on the counter area near the faucets (Fig. 3a , zones 12 and 11). A similar pattern of higher dispersion near the faucets and lower dispersion at the corners of the counter space (Fig. 3a , zones 4, 7, and 10) was also observed using microspheres. Dispersion was also recorded in other zones of the counter space, on the Plexiglas splatter shields, faucets, faucet handles, and extended surface (Fig. 3c ). There were no GFP-expressing E. coli CFU recorded on plates placed beyond 30 in. from the strainer, demarcating the range of dispersion under these experimental conditions. Table 2 gives a summary of the total distribution loads recorded using fluorescent microspheres and GFP-expressing E. coli across each experiment. The loads of dispersion on the sink counter were comparable when the sink bowl was coated with microspheres or GFP-expressing E. coli before the faucet operation. Although the dispersion load on the sink counter was lower when the sink strainer was colonized, it is interesting to note that the sink bowl received the highest dispersion. To mimic dispersion in a hospital setting, we first investigated whether GFPexpressing E. coli would establish consistent colonization in a sink trap as many other Gammaproteobacteria implicated in nosocomial outbreaks have done (6, 28) . Many recent reports demonstrate that P-traps become colonized with highly consequential Gammaproteobacteria, which then results in nosocomial transmission (29, 31, 32) . The retained water in a sink P-trap is present to provide a water barrier to prevent off-gassing of sewer smell, but it may inadvertently provide favorable conditions for pathogenic and opportunistic antibiotic-resistant microorganisms to survive and develop resilient biofilms (3, 33) . However, the mechanism of dispersal of the bacteria in the P-trap to patients or the surrounding health care area had not been fully elucidated. We began with the hypothesis that the bacteria originate from the P-trap via droplet creation when the water from the faucet hits the P-trap water, thus contaminating the sink bowl and the surrounding area. The finding supporting this theory had been previously reported using Glo Germ particles (6) . However, in the present study with careful attention to avoid strainer and tail piece contamination, the dispersal directly from the sink P-trap with either microspheres or GFP-expressing E. coli could not be reproduced as previously reported (6) . Rather this work demonstrates a different, more staged mode of transmission from a P-trap reservoir to the sink and surrounding environment. GFP-expressing E. coli in the P-trap alone was sustained for 14 days but did not grow or mobilize up the tailpipe to the strainer with just intermittent water exposure. However, when nutrients were subsequently added to the system, the organisms rapidly grew up the tailpipe to the strainer at approximately an inch per day. In a real-world setting, motility of bacteria inside the tailpipe is restricted to relatively sporadic and brief wetting events in which swimming is an opportunity to colonize new surfaces. It is assumed that once established, the biofilm promotes the upward growth of GFP-expressing E. coli in the tailpipe at an accelerated rate. The nutrient regimen that promoted colonization in our model reflects our and others' observations of items commonly disposed of in hospital sinks (intravenous fluids, feeding supplements, and leftover beverages) (5, 32) . Transmission of bacteria between sinks via a common pipe was a key finding in this study as this highlights the concept that premise plumbing may be a more continuous system with shared microbiology than a single isolated sink. The sink gallery used in this study provided a unique in situ advantage to investigate sink-to-sink transmission of bacteria through common drains. The two possible mechanisms for P-trap strainers becoming colonized are seeding of organisms from above and retrograde spread of organisms along common pipes in a hospital wastewater infrastructure. Here we demonstrate that it is possible for GFP-expressing E. coli to contaminate adjacent P-traps with just time and water given a standard U.S. code piping rise of 0.25 ft/ft. Sink-to-sink or retrograde transmission may explain the recurrence of pathogen colonization following intervention strategies like disinfection or replacement of plumbing (23) . Sink 3 was lowest on the slope in the drain line (see Fig. 4 ) with arguably the most opportunity for reflux and retrograde wetting. Sink 1, on the other hand, was farthest away from the source (sink 5), and its P-trap had the greatest incline in the drain line connecting the sinks, which could perhaps contribute to the reasons there was no GFP-expressing E. coli colonization detected in it after 7 days. There has been more investigation about microbiologic dynamics of infectious viral particles such as those of severe acute respiratory syndrome (SARS) and Ebola viruses through premise plumbing systems (34) (35) (36) . However, the microbiology, sustainability, and dynamics might be very different, although the backflow and inoculation issues could have some parallels when comparing viruses to bacteria. As Enterobacteriaceae can either multiply or remain viable for long periods of time in biofilms coating the interior of P-traps and the connected plumbing, it may not be sustainable to target any intervention limited to a single isolated sink as a source of a particular pathogen. Data from different dispersal experiments suggest that although P-traps can act as the source or the reservoir of pathogens, the physical presence of the organism in the sink bowl or colonization of the strainer is necessary for the dispersal to occur. Colonization of strainers or drains reported in earlier studies (7, 10, 13, 24, 37) was perhaps a result of ascending biofilm growth from the P-trap to the strainer or introduction through contaminated fluids. Many of the studies used swab samples, which likely sampled the strainer rather than P-trap water (17, 20) . Once the strainer was colonized, the water from the faucet resulted in GFP-expressing E. coli dispersion in the bowl and to the surrounding surfaces of up to 30 in. The range of dispersal (6) . Greater dispersal near the faucet may be attributed to the specific designs of the sink bowl and faucet in this study, which determine the contact angle of water impact. This is an important finding since many sinks in hospitals are similar in design, with faucet handles representing a high-touch surface for the sink users (38) . It can also be concluded from the dispersion experiments that secondary and successive dispersals would likely increase the degree and the scope of dispersion. There are several limitations to this work. First the use of similar sink bowls across these sinks only examines the dispersion pattern of this particular sink design. Similarly the sink-to-sink transmission may not be applicable to all wastewater plumbing systems as the fixtures on the pipe are very close together, unlike most layouts in health care settings. However, we speculate that transmission could occur on larger systems over greater time scales, especially if heavy nutrient and contamination loads were also included. GFP-expressing E. coli is a laboratory surrogate, and the putative biofilms established in the short time frame of our experiments are unlikely to be as complex or stable as biofilms developed in a hospital wastewater system over many years. However, to address the monomicrobial dominance of the GFP-expressing E. coli added to the system, we kept the system open, and other environmental organisms were able to cocolonize in an attempt to mimic the hospital system. Another limitation was the need to add nutrients to the drain to ensure rapid and robust colonization. We are not clear how widespread the practice of disposing of dextrose-containing intravenous fluids or leftover beverages in the hand-wash sinks is; however, we have observed this practice, and anecdotally it appears to be relatively common in the United States. We also did not completely characterize the droplet sizes, nor do we demonstrate air sampling to understand if the dispersion is only droplet or if there are also aerosols that contain GFP-expressing E. coli. This would require additional testing and is planned as future work. In summary, this work for the first time better models the mechanisms of spread of multidrug-resistant pathogens arising from the sink drain and infecting patients. Droplet dispersion from the P-trap does not happen directly. Rather it is a multistage process: dispersal originates from the strainer and/or the bowl after growth of the biofilm up from the microbial reservoir of the P-trap. We also demonstrate sink-to-sink transmission via a common sanitary pipe. This work could have implications for patient safety, infection control, and interventions as well as the design of future hospital plumbing systems to eliminate this mode of transmission to vulnerable hospitalized patients. Sink gallery design. A dedicated sink gallery was set up to simulate hospital hand-washing sinks. The gallery was comprised of five sink modules assembled next to each other (Fig. 4) . The five hand-wash sink stations were identical in bowl designs and dimensions and were modeled from the most common intensive care unit hand-washing sink type in the acute care hospital at the University of Virginia Medical Center. Partitions made of 24-in.-high Plexiglas sheet were installed between the sinks to prevent splatter and cross contamination. Each sink module was built with Corian integrated sink/countertops without an overflow and fitted with an 8-in. centerset 2-handle gooseneck faucet (Elkay, Oak Brook, IL). The drain line (Dearborn Brass-Oatey, Cleveland, OH). All of the fixtures were made of brass with chrome plating. Each of the sink P-traps was connected to a 3-in. common cast-iron pipe sloping into a T-joint leading into the building sanitary line located behind sink 3 (Fig. 4) . Inoculation, growth, and establishment of GFP-expressing E. coli in sink P-traps. For the GFP-expressing E. coli strain (ATCC 25922GFP), the green fluorescent protein (GFP) gene is contained on a plasmid that also contains an ampicillin resistance gene. A single isolated colony of GFP-expressing E. coli grown from a Ϫ80°C stock was inoculated into 5 ml tryptic soy broth (TSB) (Becton, Dickinson and Company, Sparks, MD) containing 100 g/ml ampicillin (ATCC medium 2855). The inoculum concentration and method varied for each experiment. For establishment of GFP-expressing E. coli in sink P-traps, new autoclaved P-traps were filled with 100 ml 0.1ϫ TSB and inoculated with ϳ10 3 CFU/ml GFPexpressing E. coli. Following inoculation, both ends of the P-traps were covered with perforated Parafilm (Bemis, Inc., Oshkosh, WI) and allowed to incubate at room temperature (22 Ϯ 2°C) for 14 days to facilitate adherent bacterial growth. The medium in the P-trap was decanted and replaced with fresh 0.1ϫ TSB every 48 h. An aliquot of decanted medium and a swab sample from the inner surface of the P-trap were plated on tryptic soy agar (Becton, Dickinson and Company, Sparks, MD) plates containing 100 g/ml ampicillin (TSA) to monitor the growth of GFP-expressing E. coli in the P-traps. TSA plates were incubated overnight at 37°C, and CFU fluorescing under UV light were enumerated. All preparatory culturing of GFP-expressing E. coli took place in a separate room from the sink gallery to avoid unintentional contamination. Installation of P-traps colonized with GFP-expressing E.coli. After the 14-day incubation, P-traps were fastened into the plumbing of the sinks (Fig. 5a) . The remainder of the drain line was either autoclaved (strainer, tailpipe, and trap arms) prior to installation or surface disinfected (sink bowl, countertop, and faucets) with Caviwipes-1 (Meterx Research, Romulus, MI), maintaining at least 1 min of contact time. After the P-trap was installed, a daily regimen was followed in which 25 ml of TSB followed by 25 ml of 0.9% NaCl solution (saline) were added in the ratio 1:3 via the strainer (Fig. 5b) to mimic the potential nutrient exposure in the hospital. Sampling and enumeration of GFP-expressing E. coli. To monitor the growth of GFP-expressing E. coli in the plumbing, sampling ports were drilled along the length of the tailpiece (between the P-trap and the strainer) and the trap arm (between the P-trap and the common line). These holes were fitted with size 00 silicone stoppers (Cole-Parmer, Vernon Hills, IL) (Fig. 5a) . Sterile cotton swabs (Covidien, Mansfield, MA) presoaked in saline were inserted through these sampling ports, and samples were collected by turning the swab in a circular motion on the inner surface (ϳ20 cm 2 ) of the tailpipes. Sample swabs were pulse-vortexed in 3 ml saline, and serial dilutions were plated on TSA. The strainer, faucet aerator, and bowl surface were sampled with presoaked swabs and processed as described earlier. Sink-to-sink transmission of bacteria. To investigate sink-to-sink transmission of bacteria, a distal sink (sink 5) (Fig. 4) was fitted with a P-trap inoculated with GFP-expressing E. coli. The effects of different inoculum concentrations of GFP-expressing E. coli-10 3 , 10 6 , and Ͼ10 10 CFU/ml (colonized for 14 days)-were investigated. Identification to the species level of fluorescent and nonfluorescent colonies identified from mixed pipe cultures was performed using a matrix-assisted laser desorption-ionization (MALDI)-time of flight (MALDI-TOF) mass spectrometer (Vitek-MS; bioMérieux, Durham, NC). The wastewater paths of sinks 1 to 4 were either autoclaved (strainer, tailpipe, P-traps, and trap arms) prior to installation or surface disinfected (sink bowl, countertop, and faucets) with Caviwipes-1 (Meterx Research, Romulus, MI). Faucets on each of the five sinks were turned on simultaneously for 1 min, supplying water at a flow rate of 8 liters/min, once every 24 h for 7 days. No additional feed to any of the sinks was added during this period of 7 days. On days 0 and 7, P-traps on each of the five sinks were unfastened, and swab samples from the P-trap were collected and processed as described earlier. Dispersion measured using fluorescent microspheres. Fluoresbrite YO carboxylate microspheres (Polysciences, Inc.) which had a 1-m diameter and maximum excitation and emission of 529 nm and 546 nm, respectively, were used as a tracer in the preliminary experiments to understand droplet dispersion from the hand-wash sinks. To test whether microspheres could be dispersed from below the sink strainer, a 1-ml suspension of microspheres (ϳ10 10 particles) was injected through a strainer attached to a Hert 4.5-in. offset drain tailpiece typically used for wheelchair-accessible sinks (American Standard, model 7723018.002) (Fig. 5c) . The vertical distance between the strainer and microsphere suspension injected into the tailpipe was ϳ4 in. Counter space around the sink bowl was thoroughly wiped with alcohol wipes (Covidien Webcol 6818; Kendall), and polyester sheets precut to appropriate shapes were placed on the counter to cover the entire sink counter and labeled according to position (Fig. 6a) . The faucet was turned on for 5 min at a water flow rate of 1.8 to 3.0 liters/min. Polyester sheets were harvested and immediately analyzed using a ChemiDoc MP system (Bio-Rad Laboratories, Inc.) with an exposure time of 5 s. Fluorescent microspheres were enumerated from the digital micrographs using the Image Lab Software (Bio-Rad Laboratories, Inc.). To test whether microspheres could be dispersed from the surface of the sink bowl, the sink bowl was evenly coated with a 20-ml microsphere suspension (ϳ10 10 particles/ml) using a disposable swab (Sage Products, Inc., Cary, IL), and the dispersion experiment was repeated following the protocol described above. To ascertain there was no nonspecific background fluorescence in the sink and/or the water from the faucet, a control using the same protocol but without the fluorescent microspheres was performed before each experiment. Dispersion measured using GFP-expressing E.coli. Dispersion using GFP-expressing E. coli was investigated in three experiments. To test whether live organisms in the P-trap could be dispersed by running water, ϳ10 10 CFU/ml GFP-expressing E. coli in saline was added to an autoclaved P-trap and fitted into the drain line that was preautoclaved (strainer, tailpipe, and trap arms). Similarly, to test whether live organisms could be dispersed from the tailpieces of wheelchair-accessible sinks, a suspension of ϳ10 10 CFU/ml GFP-expressing E. coli was added with a syringe through the strainer into the Hert 4.5-ft offset drain tailpiece (Fig. 5c ). Just as in the microsphere dispersion experiment, the vertical distance between the strainer and GFP-expressing E. coli suspension injected into the tailpipe was ϳ4 in. We next tested whether live organisms from the surface of the sink bowl could be dispersed by running water. The sink bowl surface was evenly coated with an approximately 20-ml suspension of 10 10 CFU/ml GFP-expressing E. coli. Finally, to mimic all of these conditions, a P-trap colonized with GFP-expressing E. coli (for 14 days) was installed, and a nutrient regimen (Fig. 5b) was followed for 14 days to intentionally promote the GFP-expressing E. coli colonization in the attached tailpipe and strainer. On day 15, the dispersion experiment was performed. Before each of the GFP-expressing E. coli dispersion experiments, the counter space was thoroughly disinfected with Caviwipes-1. TSA plates were then positioned on the sink counter surrounding the bowl and an extension platform (Fig. 6b) . Additional plates were attached to the sink bowl, faucets, Plexiglas partitions, and faucet handles using adhesive tape. TSA plates were also placed 3 m away from the sink as negative controls. The faucet was turned on for 5 min with a water flow rate of 1.8 to 3.0 liters/min. Lids of the TSA plates were removed only during faucet operation. Swab samples from the faucet aerators before and after operation were collected and plated on TSA. Prior to each dispersion experiment, 50 ml water from the faucet was also collected, and aliquots were plated to assess for the presence of GFP-expressing E. coli in source water and ensure cross contamination of GFP-expressing E. coli had not occurred. A control dispersion experiment was also performed using the same protocol prior to GFP-expressing E. coli inoculation in each case. Dispersion per defined area (CFU per square centimeter) was deduced by dividing the CFU counts in the TSA plate by the surface area of the TSA plate.
Which type of bacteria are implicated in carrying genes of drug resistance?
false
544
{ "text": [ "Gammaproteobacteria" ], "answer_start": [ 507 ] }
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?
false
2,453
{ "text": [ "he clean area\nincludes the administrative office and the diagnostic room." ], "answer_start": [ 11603 ] }
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?
false
2,775
{ "text": [ "to decrease R 0" ], "answer_start": [ 15929 ] }
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?
false
1,219
{ "text": [ "December 2019" ], "answer_start": [ 974 ] }
1,676
Viruses Causing Gastroenteritis: The Known, The New and Those Beyond https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776197/ SHA: f7b30ee89775bc82607cc6bc87feb5934b47625f Authors: Oude Munnink, Bas B.; van der Hoek, Lia Date: 2016-02-19 DOI: 10.3390/v8020042 License: cc-by Abstract: The list of recently discovered gastrointestinal viruses is expanding rapidly. Whether these agents are actually involved in a disease such as diarrhea is the essential question, yet difficult to answer. In this review a summary of all viruses found in diarrhea is presented, together with the current knowledge about their connection to disease. Text: The gastrointestinal tract is a vulnerable organ for infections as there is constant contact with the outside, mainly via the oral route. Inflammation of the stomach and the intestines (gastroenteritis) can cause nausea, vomiting and diarrhea. Gastroenteritis is responsible for two to three million deaths each year, making it one of the most common causes of mortality [1] . Mainly children in developing countries, but also immuno-compromised individuals in developed countries, suffer from diarrhea. While bacterial and parasitic gastrointestinal infections are declining as a result of proper disposal of sewage and safe drinking water, viral gastroenteritis is not declining in developing countries [2] . In the developed world, viruses are already the most common pathogens causing diarrhea [3] . Although viruses infecting humans had already been described since 1901 [4] and viruses were suspected to play a role in diarrhea, it lasted until 1972, when the first virus causing gastroenteritis (norovirus) was identified in an outbreak of diarrhea in Norwalk (California, United States) [5] . Shortly after the discovery of norovirus several other viruses causing gastroenteritis were discovered: rotavirus in epithelial cells of children with gastroenteritis [6] , astrovirus in infantile diarrhea cases [7] , enteric adenoviruses in the feces of children with acute diarrhea [8] , and sapovirus during an outbreak of gastroenteritis in an orphanage in Sapporo, Japan [9] . All these viruses spread via the fecal-oral route through person-to-person transmission and are described in more detail below. Noroviruses are part of the family Caliciviridae and outbreaks of norovirus gastroenteritis have been reported in cruise ships, health care settings, schools, and in the military, but norovirus is also responsible for around 60% of all sporadic diarrhea cases (diarrhea cases where an enteropathogen could be found), reviewed in the literature [10, 11] . The pathogenesis of norovirus infection has been tested in vivo. Filtrated norovirus was given to healthy volunteers after which most of them developed diarrhea [12] . Culturing of the virus, however, has been a problem since its discovery, yet one study has recently described the cultivation of norovirus in B cells, and has revealed that co-factors, such as histo-blood antigen expressing enteric bacteria, are probably needed before enteric viruses can be cultured in vitro [13] . Sapoviruses are also members of the Caliciviridae. There are five human genogroups of sapovirus described [14] which account for 2.2%-12.7% of all gastroenteritis cases around the globe [14, 15] . Sapovirus outbreaks occur throughout the year and can be foodborne [16] . For sapoviruses it has been described that the virus was not found before onset of an outbreak, and that it was found in 95% of the patients during an outbreak, while it declined to 50% after an outbreak, indicating that the virus introduces disease in a naturally infected host [17] . Rotavirus infection is the most common cause of viral gastroenteritis among children; however, parents of infected children also often become ill and as a result rotavirus is the second most common cause of gastroenteritis in adults [18] . Studies in human volunteers have shown that infection with rotavirus causes diarrhea, results in shedding of the virus and a rise in antibody anti-virus titer after infection [19] . Additionally, astroviruses infections are common, accounting for about 10% of all sporadic diarrhea cases [20] . Astrovirus has been isolated from diseased people, filtrated and administered to healthy individuals after which in some of the volunteers diarrheal disease was observed and astrovirus was shed in their stools [21] . The virus can replicate in human embryonic kidney cells and was detected by electron microscopy (EM) [21] . Adenoviruses are responsible for around 1.5%-5.4% of the diarrhea cases in children under the age of 2 years, reviewed in the literature [22] . Of the 57 identified adenovirus types [23] , only adenoviruses type 40 and 41 are associated with diarrhea [24] . Next to these two types, adenovirus type 52 can also cause gastroenteritis [25] , although it has been argued whether type 52 is actually a separate type since there is not sufficient distance to adenovirus type 41 [26] . Adenoviruses can generally be propagated in cell lines; however, enteric adenovirus 40/41 are difficult to culture, reviewed in the literature [27] . In the 1980s and 1990s some viral agents were identified for which the direct association with disease is less clear. Aichi viruses are members of the Picornaviridae identified in fecal samples of patients with gastroenteritis [28] . Aichi virus infection has been shown to elicit an immune response [29] . Since their discovery, two case-control studies were performed, but, although both studies only found Aichi virus in stools of diarrheic patients, the prevalence of Aichi virus (0.5% and 1.8%) was too low to find a significant association with diarrhea [30, 31] . In immuno-compromised hosts the virus is found in higher quantities and is not associated with diarrhea [32] . Toroviruses, part of the Coronaviridae, were first identified in 1984 in stools of children and adults with gastroenteritis [33] . Torovirus infection is associated with diarrhea [34] and is more frequently observed in immuno-compromised patients and in nosocomial infected individuals [34] . Retrospective analysis of nosocomial viral gastroenteritis in a pediatric hospital revealed that in 67% of the cases torovirus could be detected [35] . However, only a limited number of studies report the detection of torovirus and therefore the true pathogenesis and prevalence of this virus remains elusive. Picobirnaviruses belong to the Picobirnaviridae and were first detected in the feces of children with gastroenteritis [36] . Since the initial discovery, the virus has been detected in fecal samples of several animal species, and it has been shown that the viruses are genetically highly diverse without a clear species clustering, reviewed in the literature [37] . This high sequence diversity has also been observed within particular outbreaks of gastroenteritis [38, 39] , limiting the likelihood that picobirnaviruses are actually causing outbreaks, as no distinct single source of infection can be identified. In 1907 the first tissue culture system was developed which was regarded as the golden standard for virus detection for a long time, reviewed in the literature [40] . In the 1930's serology and electron microscopy were introduced which boosted the discovery of new viruses. During these years, these methods developed fruitfully but viruses infecting the gastrointestinal tract were especially difficult to culture. Throughout the last several decades, several DNA-based techniques have been developed for virus discovery that boosted the identification of novel viruses in stool samples. The four most used methods are: 1. Universal primer-PCR [41] ; 2. Random priming-based PCR [42] ; 3. Virus Discovery cDNA, Amplified Fragment Length Polymorphism (VIDISCA) [43] ; and 4. Sequence-Independent Single Primer Amplification (SISPA) [44] . Universal primer-PCR is a virus discovery technique that uses universal primers designed on conserved parts of a specific viral family, which can be used to detect novel variants of this viral family. Random priming-based PCR is a technique that randomly amplifies all nucleic acids present in samples, after which the resulting PCR products can be cloned and sequenced. SISPA and VIDISCA are virus discovery techniques that are based on digestion with restriction enzymes, after which adaptors can be ligated. These methods have been successful in the discovery of novel viruses, but there are some limitations. Universal primers are useful for discovering novel viruses of a chosen family, but the primers, based on our present knowledge of the viral family, may not fit on all unknown variants. Random priming PCR, SISPA and VIDISCA are sequence independent amplification techniques. The disadvantage of random priming PCR, SISPA and VIDISCA is that the virus needs to be present at a high concentration, while the host background DNA and/or RNA should be minimal and preferably not complex. In recent years, sequence independent amplification techniques improved considerably by coupling these techniques to next-generation sequencing platforms and as a result several novel viruses have been described in gastroenteritis cases, such as cosavirus [45] , Saffold virus [46] , klassevirus/salivirus [47, 48] , polyomavirus [49] , bufavirus [50] , tusavirus [51] , and recovirus [52] . Although these viruses are found in individuals with diarrhea, for most of them the degree of circulation (prevalence) and the ability to cause morbid conditions or disease (pathogenesis) remains to be determined, as described below (also see Table 1 ). Only found in low prevalence; **: Only limited data is available about this virus; ***: Antibodies against astrovirus HMO-C were observed whereas no antibodies against astrovirus HMO-A were found (HMO = human-mink-ovine-like astrovirus); -No published data available;ˆPicobirnavirus, tusavirus and recovirus were identified in the gastrointestinal tract after next-generation sequencing, but no information regarding antibody response or association with diarrhea is available. In the last decade, two novel clades of astroviruses have been discovered in stool samples from patients with diarrhea that are genetically far distinct from the classical astroviruses. The first clade consists of the VA-1, VA-2, VA-3, VA-4, and VA-5 astroviruses, which are genetically related to feline and porcine astroviruses, while the second clade consists of the MLB1, MLB2 and MLB3 astroviruses and form a separate cluster [55, 57, [74] [75] [76] [77] [78] . For these novel clades the pathogenesis remains to be determined since the viruses have been identified in patients with and without diarrhea, and in some studies the viruses were associated with diarrhea whilst in others no association could be found [55] [56] [57] . In addition an antibody response was observed against some but not all novel astrovirus types [54, 58] . Recently, astrovirus MLB2 has also been detected in blood plasma of a febrile child [79] and astrovirus VA1 in a frontal cortex biopsy specimen from a patient with encephalitis [80] , suggesting that astrovirus infection may not be limited to the gastrointestinal tract. In 2008, Saffold virus was detected in a stool sample from a pediatric patient with fever of unknown origin [46] . Although Saffold virus type 3 was cultured on a human epithelial cervical carcinoma (HeLa) cell line, cytopathic effects were observed and neutralizing antibodies have been found in serum samples [59] , subsequent case-control studies showed that the virus was not significantly associated with diarrhea [53, 60, 61] . Additionally, in 2008 cosavirus was identified in a patient with diarrhea [45] . However, a case-control study showed that this virus was also detected in a substantial amount of individuals without diarrhea and is not associated with diarrhea [32, 62, 63] . Klassevirus/salivirus was identified in 2009 in two fecal samples from infants with gastrointestinal disorders [47, 48] . In two studies the detection of this virus was associated with diarrhea [48, 53] , while in another study no association with disease was found [65] . Serological evidence of human klassevirus infection was obtained, suggesting that the virus infects human cells [64] . With the use of next-generation sequencing techniques, three novel polyomaviruses were also identified in human fecal samples. MW polyomavirus was identified in the stool of a healthy child from Malawi in 2012 [49] , and in the same year MX polyomavirus was found in stool samples of patients with and without diarrhea from Mexico, United States and Chili [68] . One year later, STL polyomavirus was found in the stool of a healthy child from Malawi [71] . An antibody response against MX polyomavirus [66] and MW polyomavirus [69] was observed, although MW polyomavirus [67] and STL polyomavirus [70] were not significantly associated with diarrhea in two independent case-control studies. Bufavirus is a member of the Parvoviridae and was first described in 2012 [50] . Two case-controls in Thailand and in Turkey showed that the virus was only found in patients with diarrhea and not in controls [72, 73] ; however, because of the low prevalence (respectively 0.3% in Thailand and 1.4% in Turkey), no significant association with disease was found. Tusavirus, another recently described member of the Parvoviridae, was identified in the feces of a child from Tunisia with unexplained diarrhea [51] , and thus far this is the only study describing this virus. Recovirus is a novel member of the Caliciviridae and was found in diarrhea samples from Bangladesh [52] . Similar to tusavirus, this is the only study describing this virus thus far. The identification of the above-mentioned novel viruses certainly increased our knowledge about viruses that can be found in the gastrointestinal tract of humans, yet it is unknown how many of these novel viruses are actually enteropathogens. Human stool contains a wide variety of viruses which can be derived from different hosts: Besides genuine human viruses, plant dietary viruses [32, 81] and animal dietary viruses [82] can also be found in human stool, as well as bacteriophages and viruses infecting protozoa [32] . Even viruses derived from other parts of the body can be found in fecal samples, such as the John Cunningham Polyoma virus originating from the kidney ending up in feces via urine [83] , and rhinoviruses [84] , bocaviruses [85] and coronaviruses [86] originating from the respiratory tract and probably swallowed. Furthermore, viruses infecting blood cells such as human immunodeficiency virus (HIV)-1 can also be detected in fecal samples [87] . Therefore, once a novel virus has been identified in human stool samples it is does not indicate that this virus is replicating in human intestinal cells. Koch recognized as early as 1891 that associating the presence of a certain agent with a certain disease is complex, and he therefore postulated guidelines that should be followed before an agent can be classified as a pathogen [88] . His postulates can be summarized in three points: (1) The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease; (2) the microbe occurs in no other disease as a fortuitous and nonpathogenic parasite; and (3), after being fully isolated from the body and repeatedly grown in pure culture, the microbe can induce the disease anew. If a microbe has fulfilled these three postulates it can be stated that "the occurrence of the microbe in the disease can no longer be accidental, but in this case no other relation between it and the disease except that the microbe is the cause of the disease can be considered". For enteric viruses, however, these postulates are not applicable. Firstly, the enteric viruses are not easily cultured [89] [90] [91] , and, secondly, prolonged sheading of viral agents and asymptomatic infection have been described [92] , reviewed in the literature [93] . Although attempts have been made to adjust the Koch's postulates specifically for viruses and the current methodologies deployed [94] [95] [96] , fulfilling these postulates is still not feasible on most occasions due to the lack of an efficient cell culture system, difficulties in antigen synthesis and high levels of viral genetic diversity within viral groups, reviewed in the literature [97] . Several approaches have been made to develop a methodology that adds more significance to the discovery of a novel virus. One approach is based on the enrichment of immunogenic viruses before next-generation sequencing by making use of autologous antibody capture prior to sequencing. This method was tested and validated on several fecal samples containing adenovirus, sapovirus and norovirus, and has shown to enrich immunogenic viruses, while plant viruses and bacteriophages were not enriched after antibody capture [98] . Another method to enrich for relevant viruses prior to next-generation sequencing is the so-called virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) which uses~2 million probes which cover the genomes of all members of the viral taxa known to infect vertebrates [99] . However, both methods have limitations: For the antibody capture method, viruses need to be present in high viral loads, and convalescent blood, serum or plasma needs to be available. A disadvantage of the VirCapSeq-VERT technique is that completely novel viruses, e.g., viruses from a novel virus family, will not be identified. The most straightforward method to demonstrate association with disease is using case-control studies. In order to perform such studies, matched stool samples have to be collected in case and control groups from the same geographical locations in the same period of the year. Additionally, whereas in recent years case-control studies have been performed using conventional real-time PCRs (RT-PCR), in the future, sequence independent next-generation sequencing techniques can be used for such case-control studies. Since it allows detection of virtually all nucleic acids, next-generation sequencing has several advantages compared to specific RT-PCRs. Next-generation sequencing prevents the necessity to perform numerous RT-PCRs to screen for all viruses suspected to be associated with disease, and novel variants of currently known viral families or novel virus species can be detected which can be particularly beneficial if only few reference genomes are available. The major benefit of such a database is that in the immediate future the most important question can be answered if a novel virus is identified in diarrhea cases: Is the virus likely to cause disease? In conclusion, the long list of viruses identified in the gastrointestinal tract is most probably not final yet. It is to be expected that several novel viruses will be described in the near future, since detection of these agents using the current next-generation sequence technologies is no longer a difficulty. Therefore, adding relevance to the discovery of novel viruses should be the main goal for future studies.
What is Koch's first postulate?
false
907
{ "text": [ "The microbe occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease" ], "answer_start": [ 15166 ] }
1,690
Viruses and Evolution – Viruses First? A Personal Perspective https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433886/ SHA: f3b9fc0f8e0a431366196d3e835e1ec368b379d1 Authors: Moelling, Karin; Broecker, Felix Date: 2019-03-19 DOI: 10.3389/fmicb.2019.00523 License: cc-by Abstract: The discovery of exoplanets within putative habitable zones revolutionized astrobiology in recent years. It stimulated interest in the question about the origin of life and its evolution. Here, we discuss what the roles of viruses might have been at the beginning of life and during evolution. Viruses are the most abundant biological entities on Earth. They are present everywhere, in our surrounding, the oceans, the soil and in every living being. Retroviruses contributed to about half of our genomic sequences and to the evolution of the mammalian placenta. Contemporary viruses reflect evolution ranging from the RNA world to the DNA-protein world. How far back can we trace their contribution? Earliest replicating and evolving entities are the ribozymes or viroids fulfilling several criteria of life. RNA can perform many aspects of life and influences our gene expression until today. The simplest structures with non-protein-coding information may represent models of life built on structural, not genetic information. Viruses today are obligatory parasites depending on host cells. Examples of how an independent lifestyle might have been lost include mitochondria, chloroplasts, Rickettsia and others, which used to be autonomous bacteria and became intracellular parasites or endosymbionts, thereby losing most of their genes. Even in vitro the loss of genes can be recapitulated all the way from coding to non-coding RNA. Furthermore, the giant viruses may indicate that there is no sharp border between living and non-living entities but an evolutionary continuum. Here, it is discussed how viruses can lose and gain genes, and that they are essential drivers of evolution. This discussion may stimulate the thinking about viruses as early possible forms of life. Apart from our view “viruses first”, there are others such as “proteins first” and “metabolism first.” Text: Mycoplasma mycoides by systematic deletion of individual genes resulted in a synthetic minimal genome of 473 genes (Hutchison et al., 2016) . Can one consider simpler living entities? There are elements with zero genes that fulfill many criteria for early life: ribozymes, catalytic RNAs closely related to viroids. They were recovered in vitro from 10 15 molecules (aptamers), 220 nucleotides in length, by 10 rounds of selection. Among the many RNA species present in this collection of quasispecies RNAs were catalytically active members, enzymatically active ribozymes. The sequence space for 220-mer RNAs is about 3 × 10 132 (Eigen, 1971; Wilson and Szostak, 1999; Brackett and Dieckmann, 2006) . The selected ribozymes were able to replicate, cleave, join, and form peptide bonds. They can polymerize progeny chemically, allow for mutations to occur and can evolve. One molecule serves as catalyst, the other one as substrate. Replication of ribozymes was demonstrated in the test tube (Lincoln and Joyce, 2009) . Ribozymes can form peptide bonds between amino acids (Zhang and Cech, 1997) . Thus, small peptides were available by ribozyme activity. Consequently, an RNA modification has been proposed as peptide nucleic acid (PNA), with more stable peptide bonds instead of phosphodiester bonds (Zhang and Cech, 1997; Joyce, 2002) . Replication of RNA molecules can be performed chemically from RNA without polymerase enzymes. In addition, deoxyribozymes can form from ribonucleotides (Wilson and Szostak, 1999) . Thus, DNA can arise from RNA chemically, without the key protein enzyme, the reverse transcriptase. An entire living world is possible from non-coding RNA (ncRNA) before evolution of the genetic code and protein enzymes. Ribozymes naturally consist of circular single-stranded RNAs (Orgel, 2004) . They lack the genetic triplet code and do not encode proteins. Instead, they exhibit structural information by hairpin-loops that form hydrogen bonds between incomplete double strands, and loops free to interact with other molecules. They represent a quasispecies in which many species of RNA may form, such as ribozymes, tRNA-like molecules, and other ncRNAs. RNAs within such a pool can bind amino acids. Ninety different amino acids have been identified on the Murchison meteorite found in Australia, while on Earth only about 20 of them are used for protein synthesis (Meierhenrich, 2008) . Where formation of ribozymes occurred on the early Earth is a matter of speculation. The hydrothermal vents such as black smokers in the deep ocean are possibilities where life may have started (Martin et al., 2008) . There, temperature gradients and clay containing minerals such as magnesium or manganese are available. Pores or niches offer possibilities for concentration of building blocks, which is required for chemical reactions to occur. Interestingly, also ice is a candidate for ribozyme formation and chemical reactions. Ice crystals displace the biomolecules into the liquid phase, which leads to concentration, creating a quasicellular compartmentalization where de novo synthesis of nucleotide precursors is promoted. There, RNA and ribozymes can emerge, which are capable of self-replication (Attwater et al., 2010) . tRNA-amino acid complexes can find RNAs as "mRNAs." Such interactions could have contributed to the evolution of the genetic code. This sequence of events can lead to primitive ribosome precursors. Ribozymes are the essential catalytic elements in ribosomes: "The ribosome is a ribozyme" (Cech, 2000) , supplemented with about a hundred scaffold proteins later during evolution. The proteins have structural functions and contribute indirectly to enzymatic activity. Are these ribosomebound ribozymes fossils from the early Earth? Small peptides can be formed by ribozymes before ribosomes evolved, whereby single or dimeric amino acids may originate from the universe (Meierhenrich, 2008) . Small peptides with basic amino acids can increase the catalytic activity of ribozymes as shown in vitro (Müller et al., 1994) . Such proteins are known as RNA-binding proteins from RNA viruses that protect the RNA genome, with motifs such as RAPRKKG of the nucleocapsid NCp7 of HIV (Schmalzbauer et al., 1996) . Peptides can enhance the catalytic activity of ribozymes up to a 100-fold (Müller et al., 1994) . Such peptides of RNA viruses serve as chaperones that remove higher ordered RNA structures, allowing for more efficient interaction of RNA molecules and increasing transcription rates of RNA polymerases (Müller et al., 1994) . Ribonucleoproteins may have also been functionally important during the evolution of ribosomes (Harish and Caetano-Anolles, 2012) . These pre-ribosomal structures are also similar to precursorlike structures of retroviruses. Reverse transcription can be performed by ribozymes chemically. This action does not necessarily require a protein polymerase such as the reverse transcriptase. Similarly, deoxyribonucleotides can arise by removal of an oxygen without the need of a protein enzyme (a reductase) as today, and allow for DNA polymerization (Wilson and Szostak, 1999; Joyce, 2002) . The same elements of the precursors for ribosomes are also building blocks of retroviruses, which may have a similar evolutionary origin (Moelling, 2012 (Moelling, , 2013 . tRNAs serve as primers for the reverse transcriptase, and the sequence of promoters of transposable elements are derived from tRNAs (Lander et al., 2001) . The ribozymes developed into more complex self-cleaving group II introns with insertion of genes encoding a reverse transcriptase and additional proteins (Moelling and Broecker, 2015; Moelling et al., 2017) (Figure 1) . It came as a surprise that the genomes of almost all species are rich in ncDNA, transcribed into ncRNAs but not encoding proteins, as evidenced, for instance, by the "Encyclopedia of DNA Elements" (ENCODE) project. ncDNA amounts to more than 98% of the human DNA genome (Deveson et al., 2017) . Higher organisms tend to have more non-coding information, which allows for more complex modes of gene regulation. The ncRNAs are regulators of the protein-coding sequences. Highly complex organisms such as humans typically have a high number of ncRNA and regulatory mechanisms. ncRNA can range from close to zero in the smallest bacteria such as Pelagibacter ubique to about 98% in the human genome. RNA viruses such as the retrovirus HIV harbor ncRNAs for gene regulation such as the trans-activating response element (TAR), the binding site for the Tat protein for early viral gene expression. Tat has a highly basic domain comprising mostly Lys and Arg residues, resembling other RNA binding proteins. ncRNA also serves on viral RNA genomes as ribosomal entry sites, primer binding sites or packaging signals. DNA synthesis depends on RNA synthesis as initial event, with RNA primers as starters for DNA replication, inside of cells as FIGURE 1 | A compartment is shown with essential components of life as discussed in the text. Non-coding RNA (ncRNA), ribozymes or viroids, can perform many steps for life without protein-coding genes but only by structural information. Individual amino acids are indicated as black dots and may be available on Earth from the universe. DNA may have existed before retroviruses. The compartment can be interpreted as pre-virus or pre-cell. Viroid, green; RNA, red; DNA, black. well as during retroviral replication, proving a requirement of RNA (Flint, 2015) . The number of mammalian protein-coding genes is about 20,000. Surprisingly, this is only a fifth of the number of genes of bread wheat (Appels et al., 2018) . Tulips, maize and other plants also have larger genomes, indicating that the number of genes does not necessarily reflect the complexity of an organism. What makes these plant genomes so large, is still an open question. Could the giant genomes possibly be the result to breeding of plants by farmers or gardeners? According to Szostak there are molecules which appear like relics from the RNA world such as acetyl-CoA or vitamin B12, both of which are bound to a ribonucleotide for no obvious reason -was it "forgotten" to be removed? (Roberts and Szostak, 1997; Szostak et al., 2001; Szostak, 2011) . Perhaps the connected RNA serves as structural stabilizer. Lipid vesicles could have formed the first compartments and enclosed ribozymes, tRNAs with selected amino acids, and RNA which became mRNA. Is this a pre-cell or pre-virus (Figure 1) ? Patel et al. (2015) demonstrated that the building blocks of life, ribonucleotides, lipids and amino acids, can be formed from C, H, O, P, N, S in a "one pot" synthesis. This study can be regarded as a follow-up study of the classical Urey-Miller in vitro synthesis of biomolecules (Miller, 1953; Miller and Urey, 1959) . Transition from the RNA to the DNA world was promoted by the formation of the reverse transcriptase. The enzyme was first described in retroviruses but it is almost ubiquitous and found in numerous cellular species, many of which with unknown functions (Simon and Zimmerly, 2008; Lescot et al., 2016) . It is an important link between the RNA and the DNA worlds. The name reverse transcriptase is historical and irritating because it is the "real" transcriptase during the transition from the RNA to the DNA world. Similarly, the ribonuclease H (RNase H) is an essential enzyme of retroviruses (Mölling et al., 1971) . The RNase H turned out to be one of the five most frequent and ancient proteins (Ma et al., 2008 ) that belongs to a superfamily of more than sixty different unique representatives and 152 families with numerous functions (Majorek et al., 2014) . Some of the many tRNAs can become loaded with amino acids. There are viruses containing tRNA-like structures (TLS), resembling these early RNAs (Dreher, 2009) . The TLS of these viruses typically bind to a single amino acid. TLS-viruses include plant viruses, such as Turnip yellow mosaic virus, in Peanut clump virus, Tobacco mosaic virus (TMV), and Brome mosaic virus. Only half a tRNA is found in Narnaviruses of fungi. The amino acids known to be components of tRNA-like viruses are valine, histidine and tyrosine. The structures were also designated as "mimicry, " enhancing translation (Dreher, 2009 (Dreher, , 2010 . They look like "frozen" precursor-like elements for protein synthesis. This combination of a partial tRNA linked to one amino acid can be interpreted as an evolutionary early step toward protein synthesis, trapped in a viral element. Ribozymes are related to the protein-free viroids. Viroids are virus-like elements that belong to the virosphere, the world of viruses (Chela-Flores, 1994) . Viroids lack protein coats and therefore were initially not designated as viruses but virus-like viroids when they were discovered in 1971 by Theodor Diener. He described viroids as "living fossils" (Diener, 2016) (Figure 2) . From infected potatoes, Diener isolated the Potato spindle tuber viroid (PSTVd) whose genome was about a 100-fold smaller than those of viruses known at that time. The viroids known today are ranging from 246 to 467 nucleotides. They contain circular single-stranded RNA, are protein-free and self-replicating with no genetic information, but only structural FIGURE 2 | Viroids are hairpin-loop structures and are shown schematically and as electron micrograph. Viroids are, like ribozymes, without genetic information and play major biological roles today in plant diseases, in carnation flowers, in liver cancer, as catalyst of protein synthesis in ribosomes and as circular regulatory RNAs, as "sponges" for other regulatory RNAs. information in the form of hairpin-loops (Riesner et al., 1979) . They can generate copies of themselves in the appropriate environment. They were designated as the "frontiers of life" (Flores et al., 2014) . The knowledge of virus composition was based on TMV and its crystallization by Wendell Stanley in 1935 (Pennazio and Roggero, 2000) . The genome of TMV is protein-coding singlestranded RNA of about 6,400 nucleotides that is enclosed by a rod-like protein coat. Viroids, in contrast, do not encode proteins and lack coats but they are closely related to viruses. Viroids can lose their autonomy and rely on host RNA polymerases to replicate, are capable of infecting plants and many are economically important pathogens. There are two families, the nucleus-replicating Pospiviroidae such as PSTVd and the chloroplast-replicating Avsunviroidae like the Avocado sunblotch viroid (ASBVd). Their replication requires host enzymes. Thus, autonomy is replaced by dependence on host enzymes and an intracellular lifestyle. Most viroids are often enzymatically active ribozymes -yet they are examples that this trait can get lost as a result of changing environmental conditions. Loss of ribozyme activity is a functional, not a genetic loss. Only the nuclear variants, the Pospiviroidae, can lose their ribozyme activity and use the cellular RNase III enzyme for their replication. In contrast, the Avsunviroidae are still active hammerhead ribozymes. Thus, inside the nucleus of a host cell, the enzymatic RNA function can become unnecessary. Not genes, but a function, the catalytic activity, gets lost. Viroids did apparently not gain genes but cooperated for a more complex lifestyle. For example, Carnation small viroid-like RNA (CarSV RNA) cooperates with a retrovirus and is accompanied by a homologous DNA generated by a reverse transcriptase. This enzyme presumably originates from a pararetrovirus of plants. Pararetroviruses package virus particles at a different stage during replication than retroviruses, the DNA, not the RNA. This unique combination between two viral elements has so far only been detected with CarSV in carnation flowers (Flores et al., 2005 (Flores et al., , 2014 . Why did such a cooperation evolve -perhaps by breeding gardeners? RNA is sensitive to degradation; therefore, genetic increase and growth of the genome may not be favorable energetically -at least not in plants. Gain of function is, in this case, cooperation. The circular RNA (circRNA) is related to ribozymes/viroids as a chief regulator of other regulatory RNAs, a "sponge" absorbing small RNAs. Micro RNAs (miRNAs) are post-transcriptional regulators that are affected by the presence of circRNAs. circRNAs were detected in human and mouse brains and testes as well as in plants. They can bind 70 conserved miRNAs in a cell and amount up to 25,000 molecules (Hansen et al., 2013) . Their structure is reminiscent of catalytically active ribozymes. There is an exceptional viroid that gained coding information and entered the human liver (Taylor, 2009) . The viroid is known as hepatitis delta virus (HDV). It has the smallest genome of any known animal virus of about 1,680 nucleotides. It has properties typical of viroids, since it contains circRNA, forms similar hairpin-loops and replicates in the nucleus using host enzymes. Two polymerases have to redirect their specificity from DNA to RNA to generate the HDV genome and antigenome. Both of them have ribozyme activity. In contrast to other ribozymes, HDV encodes a protein, the hepatitis delta antigen (HDVAg) that occurs in two forms, the small-HDVAg (24 kDa) supporting replication and the large-HDVAg (27 kDa) that helps virion assembly. The gene was presumably picked up from the host cell by recombination of HDV's mRNA intermediate with a host mRNA. Transmission depends on a helper virus, the Hepatitis B virus (HBV), which delivers the coat (Taylor, 2009 ) Does packaging by a helper virus protect the genome and thereby allow for a larger viroid to exist? In plants, viroids may not be able to become bigger possibly due to their sensitivity to degradation -but they cannot become much smaller either. Only a single viroid is known that is completely composed of protein-coding RNA with triplets (AbouHaidar et al., 2014). Viroids and related replicating RNAs are error-prone replicating units and the error frequency imposes a certain minimal size onto them, as they would otherwise become extinct. This mechanism has been described as "error catastrophe, " which prevents survival (Eigen, 1971 (Eigen, , 2013 . The viroids and related RNAs are the smallest known replicons. Smaller ones would become extinct in the absence of repair systems. In summary, RNA can catalyze many reactions. Protein enzymes which may have evolved later have higher catalytic activities. Ribozymes are carriers of information, but do not require coding genes. Information is stored in their sequence and structure. Thus, replication of an initial RNA is followed by flow of information, from DNA to RNA to protein, as described the Central Dogma (Crick, 1968) . Even an information flow from protein to DNA has been described for some archaeal proteins (Béguin et al., 2015) . The DNA-protein world contains numerous ncRNAs with key functions. ncRNA may serve as a model compound for the origin of life on other planets. Hereby not the chemical composition of this molecule is of prime relevance, but its simplicity and multifunctionality. Furthermore, RNA is software and hardware in a single molecule, which makes it unique in our world. There are other scenarios besides the here discussed "virus-first, " such as "protein-first", "metabolism-fist" or the "lipid world" (Segré et al., 2001; Andras and Andras, 2005; Vasas et al., 2010; Moelling, 2012) . Some of these alternative concepts were built on phylogenomics, the reconstruction of the tree of life by genome sequencing (Delsuc et al., 2005) . Surprisingly, it was Sir Francis Crick, one of the discoverers of the DNA double-helix, who stated that he would not be surprised about a world completely built of RNA. A similar prediction was made by Walter Gilbert (Crick, 1968; Gilbert, 1986) . What a vision! Our world was almost 50 years later defined as "RNAprotein" world (Altman, 2013) . One can speculate our world was built of ribozymes or viroids, which means "viruses first." ncRNAs appear as relics from the past RNA world, before DNA, the genetic code and proteins evolved. However, ncRNA is essential in our biological DNA world today. It is possible to produce such ncRNA today in the test tube by loss of genic information from protein-coding RNA. This reduction to ncRNA was demonstrated in vitro with phage RNA. Phage Qβ genomic RNA, 4,217 nucleotides in length, was incubated in the presence of Qβ replicase, free nucleotides and salts, a rich milieu in the test tube. The RNA was allowed to replicate by means of the Qβ replicase. Serial transfer of aliquots to fresh medium led to ever faster replication rates and reduction of genomic size, down to 218 nucleotides of ncRNA in 74 generations. This study demonstrated that, depending on environmental conditions, an extreme gene reduction can take place. This experiment performed in 1965 was designated as "Spiegelman's Monster." Coding RNA became replicating ncRNA (Spiegelman et al., 1965; Kacian et al., 1972) ! Manfred Eigen extended this experiment and demonstrated further that a mixture containing no RNA to start with but only ribonucleotides and the Qβ replicase can under the right conditions in a test tube spontaneously generate self-replicating ncRNA. This evolved into a form similar to Spiegelman's Monster. The presence of the replicase enzyme was still necessary in these studies. Furthermore, a change in enzyme concentration and addition of short RNAs or an RNA intercalator influenced the arising RNA population (Sumper and Luce, 1975; Eigen, 2013) . Thus, the complexity of genomes depends on the environment: poor conditions lead to increased complexity and rich environments to reduced complexity. The process demonstrated in this experiment with viral components indicates that reversion to simplicity, reduction in size, loss of genetic information and speed in replication can be major forces of life, even though this appears to be like a reversion of evolution. The experiment can perhaps be generalized from the test tube to a principle, that the most successful survivors on our planet are the viruses and microorganisms, which became the most abundant entities. Perhaps life can start from there again. These studies raise the question of how RNA molecules can become longer, if the small polymers become smaller and smaller, replicate faster and outcompete longer ones. This may be overcome by heat flow across an open pore in submerged rocks, which concentrates replicating oligonucleotides from a constant feeding flow and selection for longer strands. This has been described for an increase from 100 to 1,000 nucleotides in vitro. RNA molecules shorter than 75 nucleotides will die out (Kreysing et al., 2015) . Could a poor environment lead to an increase of complexity? This could be tested. Ribozymes were shown to grow in size by uptake of genes, as demonstrated for HDV (Taylor, 2009 ). An interesting recent unexpected example supporting the notion that environmental conditions influence genetic complexity, is the human gut microbiome. Its complexity increases with diverse food, while uniform rich food reduces its diversity and may lead to diseases such as obesity. Colonization of the human intestinal tract starts at birth. A few dozen bacterial and viral/phage species are conserved between individuals (core sequences) as a stable composition (Broecker et al., 2016c . Dysbiosis has been observed in several chronic diseases and in obesity, a loss of bacterial richness and diversity. Nutrition under affluent conditions with sugar-rich food contributes to obesity, which results in a significant reduction of the complexity of the microbiome. This reduction is difficult to revert (Cotillard et al., 2013; Le Chatelier et al., 2013) . The gut microbiome in human patients with obesity is reminiscent of the gene reduction described in the Spiegelman's Monster experiment: reduction of genes in a rich environment. The reduction of the complexity of the microbiome is in part attributed to the action of phages, which under such conditions, defined as stress, lyse the bacteria. Fecal microbiota transplantation can even be replaced by soluble fractions containing phages or metabolites from the donor without bacteria (Ott et al., 2017) . Analogously, the most highly complex microbiomes are found in indigenous human tribes in Africa, which live on a broad variety of different nutrients. It is a slow process, though, to increase gut microbiota complexity by diverse nutrition. The obesity-associated microbiota that survive are fitter and more difficult to counteract. Urbanization and westernization of the diet is associated with a loss of microbial biodiversity, loss of microbial organisms and genes (Segata, 2015) . To understand the mechanism and driving force for genome reduction, deletion rates were tested by insertion of an indicator gene into the Salmonella enterica genome. The loss of the indicator gene was monitored by serial passage in rich medium. After 1,000 generations about 25% of the deletions caused increased bacterial fitness. Deletions resulted in smaller genomes with reduced or absence of DNA repair genes (Koskiniemi et al., 2012) . Gene loss conferred a higher fitness to the bacteria under these experimental conditions. The recently discovered mimiviruses and other giant viruses are worth considering for understanding the evolution of life with respect to the contribution of viruses. Their hosts are, for example, Acanthamoeba, Chlorella, and Coccolithus algae (Emiliania huxleyi), but also corals or sponges as discussed more recently. Mimiviruses were first discovered in cooling water towers in Bradford, United Kingdom in 2003 with about 1,000 genes, most of which unrelated to previously known genes. Mimiviruses have received attention because they contain elements that were considered hallmarks of living cells, not of viruses, such as elements required for protein synthesis, tRNAs and amino acid transferases. The mimiviruses harbor these building blocks as incomplete sets not sufficient for independent protein synthesis as bacteria or archaea can perform, preventing them from leading an autonomous life (La Scola et al., 2003 Scola et al., , 2008 . They are larger than some bacteria. Giant viruses can be looked at as being on an evolutionary path toward a cellular organism. Alternatively, they may have evolved from a cellular organism by loss of genetic information (Nasir and Caetano-Anolles, 2015) . Giant viruses have frequently taken up genes from their hosts by horizontal gene transfer (HGT) (La Scola et al., 2008; Nasir and Caetano-Anolles, 2015; Colson et al., 2018) . A graph on genome sizes shows that mimiviruses and bacteria overlap in size, indicating a continuous transition between viruses and bacteria and between living and non-living worlds (based on Holmes, 2011) (Figure 3) . Other giant viruses, such as megaviruses, were discovered in the ocean of Chile with 1,120 genes. Most recently the Klosneuvirus was identified in the sewage of the monastery Klosterneuburg in Austria in 2017 with 1.57 million (mio) basepairs (Mitch, 2017) . Pithovirus sibericum is the largest among giant viruses discovered to date with a diameter of 1.5 microns, a genome of 470,000 bp with 467 putative genes, 1.6 microns in length, and it is presumably 30,000 years old as it was recovered from permafrost in Siberia (Legendre et al., 2014) . The smaller Pandoraviruses with 1 micron in length have five times larger genomes, 2,500,000 bp (Philippe et al., 2013) (Figure 3) . The giant viruses can even be hosts to smaller viruses, the virophages, reminiscent of bacteriophages, the viruses of bacteria. These virophages such as Sputnik are only 50 nm in size with 18,343 bp of circular dsDNA and 21 predicted proteincoding genes. They replicate in viral factories and consume the resources of the mimivirus, thereby destroying it. Some, virophages can even integrate into the genome of the cellular host and can be reactivated when the host is infected by giant viruses. Thus, giant viruses suggest that viruses are close to living entities or may have been alive (La Scola et al., 2008; Fischer and Hackl, 2016) . In biology it is common to distinguish between living and dead matter by the ability to synthesize proteins and replicate autonomously. The giant viruses may be considered as missing link between the two, because they harbor "almost" the protein synthesis apparatus. The transition from living to the non-living world is continuous, not separated by a sharp borderline (Figure 3) . Viruses are not considered alive by most of the scientific community and as written in textbooks, because they cannot replicate autonomously. Yet some of the giant viruses are equipped with almost all components of the protein synthesis machinery close to bacteria suggesting that they belong to the living matter (Schulz et al., 2017) . The ribozymes may have been the earliest replicating entity. Perhaps also other viruses were initially more independent of the early Earth than they are today. As described in Figure 1 there may have been initially no major difference between an early virus or an early cell. Only later viruses may have given up their autonomous replication and became parasites -as has been described for some bacteria (see below). Efforts have been made to identify the smallest living cell that is still autonomously replicating. Among the presumably smallest naturally occurring bacteria is Pelagibacter ubique of the SAR11 clade of bacteria (Giovannoni, 2017) , which was discovered in 1990. It is an alpha-proteobacterium with 1,389 genes present ubiquitously in all oceans. It can reach up to 10 28 free living cells in total and represents about 25% of microbial plankton cells. Very little of its DNA is non-coding. It harbors podophage-type phages, designated as "pelagiphage" (Zhao et al., 2013) . This small bacterium was designated as the most common organism on the planet. Why is it so successful? This autonomous bacterium is smaller than some parasitic giant viruses. Craig Venter, who first succeeded in sequencing the human genome, tried to minimize the putative smallest genome of a living species, from Mycoplasma mycoides, a parasitic bacterium that lives in ruminants (Gibson et al., 2008 (Gibson et al., , 2010 . His group synthesized a genome of 531,000 bp with 473 genes, 149 of them (32%) with unknown functions (Hutchison et al., 2016) . Among the smallest parasitic living organisms is Nanoarchaeum equitans. It is a thermophile archaeon which lives at 80 • C and at pH 6 with 2% salt (Huber et al., 2003) . Its genome has a size of 490,000 bp and encodes 540 genes. N. equitans is an obligate symbiont of a bigger archaeon, Ignicoccus riding on it as on a horse, hence the name (Huber et al., 2003) . The world of viruses covers a range of three logs in size of their genomes: from zero genes to about 2,500 genes amounting to about 2,500,000 bp of DNA. The zero-gene viroids are about 300 bases in length (Figure 3) . The virosphere is the most successful reservoir of biological entities on our planet in terms of numbers of particles, speed of replication, growth rates, and sequence space. There are about 10 33 viruses on our planet and they are present in every single existing species (Suttle, 2005) . There is no living species without viruses! Viruses also occur freely in the oceans, in the soil, in clouds up to the stratosphere and higher, to at least 300 km in altitude. They populate the human intestine, birth canal, and the outside of the body as protective layer against microbial populations. Microbes contain phages that are activated during stress conditions such as lack of nutrients, change in temperatures, lack of space and other changes of environmental conditions. One of the most earth-shaking papers of this century was the publication of the human genome sequence (Lander et al., 2001) . About half, possibly even two-thirds of the sequence are composed of more or less complete endogenous retroviruses (ERVs) and related retroelements (REs) (de Koning et al., 2011) . REs amplify via copy-and-paste mechanisms involving a reverse transcriptase step from an RNA intermediate into DNA. In addition, DNA transposable elements (TEs) move by a cutand-paste mechanism. The origin of REs is being discussed as remnants of ancient retroviral germline infections that became evolutionarily fixed in the genome. About 450,000 human ERV (HERV) elements constitute about 8% of the human genome consisting of hallmark retroviral elements like the gag, pol, env genes and flanking long terminal repeats (LTR) that act as promoters (Lander et al., 2001) . Howard Temin, one of the discoverers of the reverse transcriptase, in 1985 already described endogenous retrovirus-like elements, which he estimated to about 10% of the human and mouse genome sequence (Temin, 1985) . The actual number is about 45% as estimated today (Lander et al., 2001) . In some genes such as the Protein Kinase Inhibitor B (PKIB) gene we determined about 70% retrovirusrelated sequences (Moelling and Broecker, 2015) . Is there a limit? Could it have been 100%? Retroviruses are estimated to have entered the lineage of the mammalian genome 550 million years ago (MYA) (Hayward, 2017) . Older ERV sequences may exist but are unrecognizable today due to the accumulation of mutations. ERVs undergo mutations, deletions or homologous recombination events with large deletions and can become as short as solo LTR elements, which are a few hundred bp in length -the left-overs from full-length retroviral genomes of about 10,000 bp. The LTR promoters can deregulate neighboring genes. Homologous recombination events may be considered as gene loss or gene reduction events. It is the assumption that the ERVs, which were no longer needed for host cell defense, were no longer selected for by evolution and consequently deleted as unnecessary consumers of energy. Eugene Koonin points out that infection and integration are unique events occurring at a fast pace, while loss and gene reduction may take much longer time frames (Wolf and Koonin, 2013) . A frequent gene reduction of eukaryotic genomes is the loss of the viral envelope protein encoded by the env gene. Without a coat, retroviruses can no longer leave the cell and infect other cells. They lose mobility and become obligatory intracellular elements. Helper viruses can supply envelope proteins in trans and mobilize the viruses. TEs or REs can be regarded as examples of coat-free intracellular virus relics -or could it have been the other way round, perhaps precursors of full-length retroviruses? These elements can be amplified intracellularly and modify the host genomes by integration with the potential danger of gene disruption and genetic changes. REs can lead to gene duplications and pseudogene development, with one copy for stable conservation of acquired functions and the other one for innovations (Cotton and Page, 2005) . Such duplications constitute large amounts of mammalian genomes (Zhang, 2003) . Retroviruses have an RNase H moiety duplication, one of which serves as a catalytically inactive linker between the RT polymerase and the enzymatically active RNase H (Xiong and Eickbush, 1990; Malik and Eickbush, 2001; Moelling and Broecker, 2015; Moelling et al., 2017) . This gene duplication dates back to 500 mio years (Cotton and Page, 2005) . Gene duplications are a common cause of cancer, which often occurs only in the genome of the cancer cell itself, less affecting offsprings. Myc, Myb, ErbB2, Ras, and Raf are oncogenes amplified in diverse types of human cancers (Vogelstein and Kinzler, 2002) . The ability of retroviruses to integrate makes them distinct from endosymbionts which stay separate. Yet the net result is very similar, acquisition of new genetic information, which is transmitted to the next generation, if the germline is infected and endogenization of the virus occurred. Viral integration is not limited to eukaryotic cells but also a mechanism in prokaryotes for maintenance of the lysogenic state of phages inside bacteria. Also, for other eukaryotic viruses such as HBV, the envelope surface antigen BHsAg can be deleted, which leads to an obligatory intracellular life style for the virus, which especially in the presence of HCV promotes cancer (Yang et al., 2016) . HIV has been shown to rapidly lose one of its auxiliary genes, nef, originally for negative factor. The gene was lost within a rather low number of passages of the virus grown under tissue culture conditions by selection for high virus titer producing cells. Deletion of nef resulted in a significant increase of the virus titer in culture -hence the name. The nef gene product was of no need inside tissue culture cells, rather it was inhibitory for replication. However, it is essential for pathogenicity in animals, and subsequently nef was reinterpreted as "necessary factor" (Flint, 2015) . Also, the human hosts of HIV can lose a significant terminal portion of a seven transmembrane receptor in lymphocytes, the primary target cell for HIV entry and for virus uptake. This molecule, the CCR5 cytokine receptor is truncated by 32 carboxy-terminal amino acids (CCR5-32), disabling the receptor functionally. The allele frequency of the mutant CCR5-32 mutant is about 10% in the European population, making these people resistant to HIV infections (Solloch et al., 2017) . This gene loss in Europeans has been shown to make the individuals resistant not only against HIV infection but also against malaria. This may have been the selective pressure in the past before HIV/AIDS arose. No side effect for humans lacking this gene has been described (Galvani and Slatkin, 2003) . Viruses have been proven to be drivers of evolution (Villarreal and Witzany, 2010) , including the human genome, which by at least 45% is composed of sequences related to retroviruses. In addition, endogenized retroviruses supplied the syncytin genes that are essential for the development of the mammalian placenta, and allowed the growth of embryos without its rejection by the maternal immune system (Dupressoir et al., 2012) . Thus, the same property which causes immunodeficiency in HIV-infected patients and leads to AIDS causes syncytia formation, cell fusion after infection by a retrovirus. Viruses have also been proposed to be at the origin of the evolution of adaptive immunity (Villarreal, 2009 ). Thus, viruses shaped genomes by supplying essential genes and mechanisms. Endogenization of retroviruses has occurred in the mammalian genomes for at least 550 mio years (Hayward, 2017) . If the integrated ERVs did not provide any selective advantage, they deteriorated and accumulated mutations with loss of function. This was directly proven by reconstruction of an infectious retrovirus from the consensus sequence of 9 defective endogenous virus sequences, designated as Phoenix. The virus was expressed from a constructed synthetic DNA clone in cell culture and formed virus particles identified by high resolution microscopic analysis (Dewannieux and Heidmann, 2013) . The koalas in Australia are currently undergoing endogenization of a retrovirus (koala retrovirus, KoRV) in "real time" and demonstrate possible consequences for immunity. In the early 1900s, some individuals were transferred to islands, including Kangaroo Island, close to the Australian mainland for repopulation purposes, as koalas were threatened to become extinct. Today, the majority of the koala population is infected by KoRV, which is closely related to the Gibbon ape leukemia virus (GALV). Yet, koalas isolated on Kangaroo Island are KoRV negative, which allows dating the introduction of KoRV into the koala population to about one hundred years ago. Many of the infected koalas fell ill and died, yet some populations became resistant within about 100 years, corresponding to about 10 generations. The koalas likely developed resistance due to the integrated DNA proviruses. The retrovirus is transmitted as exogenous as well as endogenous virus, similar to the Jaagsiekte sheep retrovirus (JSRV), whereby the endogenized viruses protect with a viral gene product, such as Env, against de novo infections by "superinfection exclusion" (Tarlinton, 2012) . The contribution of retroviruses to the antiviral defense is striking, since all retroviral genes have analogous genes in the siRNA/RNAi defense mechanism of eukaryotic cells (Moelling et al., 2006) . Retroviruses can protect against infection by other related viruses, for example, by expressing Env proteins that block cellsurface receptors (Villarreal, 2011) . A comparable mechanism protects bacterial cells against DNA phages, by integrated phage DNA fragments that are transcribed into mRNA and hybridize to incoming new DNA phages and thereby lead to their destruction by hybrid-specific nucleases, CRISPR/Cas immunity (Charpentier and Doudna, 2013) . It is often not realized that immunity acquisition in bacteria and mammalian cells follow analogous mechanisms (Figure 4) . Integration of retroviruses normally occurs in somatic cells after infection as an obligatory step during the viral life cycle. Infection of germline cells can lead to transmission to the next generation and ultimately result in inherited resistance. Endogenized retroviruses likely caused resistance FIGURE 4 | Viruses protect against viruses: retroviruses protect a cell against a new infection by a similar virus designated as "superinfection exclusion" or viral interference. This is mediated by viral gene products such as proteins or nucleic acids. Similarly, phages protect against phages: superinfection of bacteria is prevented by CRISPR/Cas RNA originating from previous infections. The mechanisms of defense against viruses and phages are analogous. Protection by viruses or phages against superinfections represents cellular defense and acquired immunity. The four examples are discussed in the text. to the exogenous counterparts. Similarly, resistance to Simian Immune Deficiency virus (SIV) in some monkey species may be explained by endogenization (Li et al., 2017 (Li et al., , 2018 . In the case of phages and their prokaryotic hosts the mechanism is described as CRISPR/Cas, which follow analogous principles of "endogenization" of incoming genetic material for subsequent exclusion. One may speculate that HIV may also eventually become endogenized into the human genome. There is some evidence that HIV can infect human germline cells and can be transmitted to the embryonic genome (Wang et al., 2011) . How long this may take is not known -10 generations? The loss of function of ERVs can occur by mutations, deletions of the env or other genes and ultimately all coding genes by homologous recombination, leaving behind only one LTR. The number of retrovirus-like elements add up to about 450,000, corresponding to 8% of the human genome (Lander et al., 2001; Cordaux and Batzer, 2009 ). The promoter regions were analyzed for their contribution to cancer by activating neighboring genes -as a consequence of a former retrovirus infection. Indeed, activated cellular genes by "downstream promotion" were identified in animal studies with activation of the myc gene as one of many examples, leading to chronic, not acute development of cancer (Ott et al., 2013) . As a general mechanism for human cancer today the LTRs are, however, not identified as a major culprit. Most of the ERVs we find today have been integrated during evolution in introns or other regions where their presence is relatively harmless. Did the other ones result in death of the carriers which disappeared? The effects of LTRs on the expression levels of neighboring host genes was studied with the endogenous human virus, HERV-K, as a possible cause of cancer, but this appears not to be a general phenomenon (Broecker et al., 2016b) . As shown for the koalas, ERVs can confer immunity to viral infections (Feschotte and Gilbert, 2012) . A related ERV, HERV-H, was shown to produce an RNA that keeps early embryonic cells pluripotent and even revert adult cells to regain pluripotency (Grow et al., 2015) . Thus, the role of ERVs may be more complex than we presently know. Transposable elements and REs that lost the ability of cellular transmission by deletion of the coat protein majorly contribute to genetic complexity of host cells. They are "locked" inside the cells and are major drivers of the increase of genetic complexity (Cordaux and Batzer, 2009 ). One could speculate that these intracellular elements are replicationincompetent retroviruses lacking coats (Lander et al., 2001) . Bats transmit viruses such as Ebola and SARS coronavirus without suffering from disease (Beltz, 2018) . Even RNA viruses such as Bornaviruses have been shown to integrate by illegitimate reverse transcription, possibly also supplying immunity against superinfection (Katzourakis and Gifford, 2010) . There are two prominent events that significantly contributed to the success of life and the formation of cells. Both of them are associated with gene reduction. This phenomenon may play a role for the evolution of viruses from autonomous to parasitic lifestyles. In the 1960s Lynn Margulis proposed an extracellular origin for mitochondria (Margulis, 1970 (Margulis, , 1993 ). An ancestral cell, perhaps an archaeon, was infected by an anaerobic bacterium, which gave rise to mitochondria. Similarly, cyanobacteria formed the chloroplasts in modern plant cells. Mitochondria arose around 1.45 billion years ago (BYA) (Embley and Martin, 2006) . Mitochondria and chloroplasts are the most striking examples for a change in lifestyle from autonomous bacteria to endosymbionts. This transition is often considered as extremely rare and a hallmark of evolution of life on our planet. However, there are many other obligate intracellular parasites such as Rickettsia, Chlamydia trachomatis, Coxiella burnetii (the causative agent of Q fever), Mycobacterium leprae, M. tuberculosis, and M. mycoides (Beare et al., 2006) . The change of lifestyle of the endosymbionts in the two cases of mitochondria and chloroplasts is striking. Both of them drastically reduced their genetic make-up. Mitochondria contain less than 37 genes, left from the original about 3,000 genes. Is endogenization of retroviruses, the ERVs, which are integrated into germline cells, related to endosymbiosis? Are these endosymbionts models for the transition from autonomous lifestyle to a parasitic life-which may have taken place with viruses? A more recent typical example for a reductive evolution are Rickettsia. These bacteria were assumed for some time to be viruses because of their obligatory intracellular parasitic existence. Rickettsia have evolved from autonomously replicating bacteria. Reductive evolution of endosymbionts can yield bacteria with tiny genomes on the expense of autonomous extracellular life. Their genomes are 1.11 mio bp in length with about 834 protein-coding genes, and loss of 24% by reductive evolution (Ogata et al., 2001) . Rickettsia may have some relationship with cyanobacteria, which are considered as the major symbionts. Can one speculate that viruses may have been autonomous entities initially? Viroids may have undergone transition from autonomy to parasites, just as shown for mitochondria, chloroplasts or Rickettsia? To which extent have viruses been autonomous and independent of cellular metabolisms originally -and contributed to the origin of cells? Could they only later have lost their autonomy and become parasitic? Viruses are minimalistic in their composition and must have undergone stringent gene reductions (Flint, 2015) . How small can their genomes become? Most coding RNA viruses still contain regulatory elements, ncRNA at the 3 and 5 terminal regions for ribosomal entry, protein synthesis, transcriptional regulation, and others. A subgroup of retroviruses is an interesting example in respect to simultaneous loss and gain of genetic information. The oncogenic retroviruses or tumorviruses can recombine with cellular genes which under the promoters of retroviruses can become oncogenes and drivers of cancer. About a hundred oncogenes have been selected for in the laboratories and studied over decades for understanding the molecular mechanisms of cancer. Selection for growth advantages of the host cells led to the discovery of the fastest growth-promoting oncogenes we know today, such as Ras, Raf, ErbB or Myc, which are in part successful targets for anticancer drugs (Moelling et al., 1984) . These oncogenes were in most cases taken up by the retroviruses at the expense of structural (gag), replicating (pol) or envelope (env) genes, and are often expressed as fusion proteins with Gag. Thus, oncogenic retroviruses are obligatory intracellular defective viruses and were selected for in the laboratory by researchers for the oncogenes with the most potent growth promoting ability. They need the supply of replicatory genes in trans from co-infecting helper viruses to infect other cells (Flint, 2015) . Retroviruses are able to pick up cellular genes, transfer and integrate them into neighboring cells. Some strains of Rous sarcoma virus maintain replication competent when carrying the cell-derived src (for sarcoma) oncogene encoding a protein of 536 amino acids that apparently can fit into the retroviral particle along with the full-size viral genome (Broecker et al., 2016a) . Spatial reasons may have influenced the formation of oncogenic retroviruses and limited their size and thereby led to their defective phenotypes. There are indications that the uncontrolled activity of (retro)transposons in germline cells can result in diseases such as male infertility -presumably by "error catastrophe, " caused by too many transposition events. In mammals, piRNAs tame transposon activity by means of the RNase H activity of PIWI proteins during spermatogenesis (Girard et al., 2006) . Only a minority of viruses are pathogens; most of them do not cause diseases. On the contrary, they are most important as drivers of evolution, as transmitters of genetic material, as innovative agents. In particular, the RNA viruses are the most innovative ones. Some of them are pathogenic and dangerous, such as HIV or influenza virus, or viroids in plants. RNA viruses are able to change so rapidly that the host immune system is unable to counteract the infection. Pathogenicity arises when environmental conditions change, for instance, when a virus enters a new organism or species. Increase of cellular complexity by viruses is an important feature of evolution. Such major evolutionary changes are recently taken as arguments against the evolutionary theory by Charles Darwin who considered gradual changes, small increments by mutations as the main basis for selection and evolution. New criticism is addressing this thinking, considering larger changes as evolutionary drivers. Such changes arise by many complex phenomena such as endosymbiosis, infection by prokaryotes, viruses and fungi, recombination of genes, HGT, infections, sex. Dramatic changes such as endosymbiosis or pathogen infections extend Darwin's concept of evolution. There are numerous examples for the contribution of viruses to the evolution of life since at least as long as 550 MYA (Hayward, 2017) . But genetic noise through random mutations does not allow us to go back to the origin of life. It may not be impossible that the earliest compartment was indistinguishable, either a pre-cell or a pre-virus. By analogy one may speculate that at some point autonomous viruses gave up independence for an obligatory intracellular life -as has been described for mitochondria and chloroplasts but also intracellular bacteria such as Rickettsia. This speculation is based on the concept that early life must have started simple and with high genetic variability and then became more complex. But complexity can be given up for a less energy consuming lifestyle with small genomes and high speed of replication (Moelling, 2012 (Moelling, , 2013 . Therefore, the question may be repeated: "Are viruses our oldest ancestors?" Some fossil life can be partially reproduced in vitro by Spiegelman's Monster and Eigen's follow-up experiments, explaining the great surviving potential of simple ncRNA. Viruses can be pathogens, but their recognition as primarily causing diseases is wrong. This notion is based on the history of viruses in medicine, as explained in a book entitled "Viruses: More Friends Than Foes" (Moelling, 2017) . The scenario described here focuses on viruses as drivers of evolution. The early RNA world gained interest 20-30 years ago as evidenced by the references provided above. Surprisingly, there are scientists who still believe in the "pansperm hypothesis" and think that retroviruses are of extraterrestric origin (Steele et al., 2018) . The recent interest in the origin of life arose from the newly discovered exoplanets whose number increases daily -and which may be as numerous as 10 25 . Thus, pure statistics make some people believe that there is extraterrestrial life. The extraterrestric life is mimicked in laboratories on Earth with many assumptions -perhaps this overview stimulates some thinking. The discussion presented here should be taken as concept about simple replicating and evolving entities possibly arising from different building blocks in other environments, with structure being more relevant than sequence.
What contributed to a large part of mammalian genomic sequence?
false
1,176
{ "text": [ "Retroviruses" ], "answer_start": [ 731 ] }
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 the MERS LRT disease involve?
false
4,183
{ "text": [ "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." ], "answer_start": [ 1123 ] }
2,459
No credible evidence supporting claims of the laboratory engineering of SARS-CoV-2 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054935/ SHA: 5a9154aee79901dd8fecd58b7bcd9b7351102d24 Authors: Liu, Shan-Lu; Saif, Linda J.; Weiss, Susan R.; Su, Lishan Date: 2020-02-26 DOI: 10.1080/22221751.2020.1733440 License: cc-by Abstract: nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 (COVID-19) (https://globalbiodefense. com/novel-coronavirus-covid-19-portal/). According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity [4, 5] . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV (RaTG13) was recently reported, which shared ∼96% homology with the SARS-CoV-2 [4] . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV [4] , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (https:// www.nature.com/articles/d41586-020-00364-2). Another claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 (>5,000 nucleotides). The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice (hence M15), due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells [10, 11] . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry [8] . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice [7] . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy (https://www.nih.gov/about-nih/who-weare/nih-director/statements/nih-lifts-funding-pausegain-function-research). The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups [5, 14] , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv (a manuscript sharing site prior to any peer review), claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author(s). Susan R. Weiss http://orcid.org/0000-0002-8155-4528
what is the clinical manifestation similar to?
false
4,574
{ "text": [ "to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV." ], "answer_start": [ 859 ] }
2,620
Estimating the Unreported Number of Novel Coronavirus (2019-nCoV) Cases in China in the First Half of January 2020: A Data-Driven Modelling Analysis of the Early Outbreak https://doi.org/10.3390/jcm9020388 SHA: bf20dda99538a594eafc258553634fd9195104cb Authors: Zhao, Shi; Musa, Salihu S.; Lin, Qianying; Ran, Jinjun; Yang, Guangpu; Wang, Weiming; Lou, Yijun; Yang, Lin; Gao, Daozhou; He, Daihai; Wang, Maggie H. Date: 2020 DOI: 10.3390/jcm9020388 License: cc-by Abstract: Background: In December 2019, an outbreak of respiratory illness caused by a novel coronavirus (2019-nCoV) emerged in Wuhan, China and has swiftly spread to other parts of China and a number of foreign countries. The 2019-nCoV cases might have been under-reported roughly from 1 to 15 January 2020, and thus we estimated the number of unreported cases and the basic reproduction number, R0, of 2019-nCoV. Methods: We modelled the epidemic curve of 2019-nCoV cases, in mainland China from 1 December 2019 to 24 January 2020 through the exponential growth. The number of unreported cases was determined by the maximum likelihood estimation. We used the serial intervals (SI) of infection caused by two other well-known coronaviruses (CoV), Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) CoVs, as approximations of the unknown SI for 2019-nCoV to estimate R0. Results: We confirmed that the initial growth phase followed an exponential growth pattern. The under-reporting was likely to have resulted in 469 (95% CI: 403&minus;540) unreported cases from 1 to 15 January 2020. The reporting rate after 17 January 2020 was likely to have increased 21-fold (95% CI: 18&minus;25) in comparison to the situation from 1 to 17 January 2020 on average. We estimated the R0 of 2019-nCoV at 2.56 (95% CI: 2.49&minus;2.63). Conclusion: The under-reporting was likely to have occurred during the first half of January 2020 and should be considered in future investigation. Text: A novel coronavirus (2019-nCoV) infected pneumonia infection, which is deadly [1] , was first identified in Wuhan, China in December 2019 [2] . The virus causes a range of symptoms including fever, cough, and shortness of breath [3] . The cumulative number of reported cases slowly increased to cumulative 41 cases by 1 January 2020, and rapidly increased after 16 January 2020. As of 26 January 2020, the still ongoing outbreak had resulted in 2066 (618 of them are in Wuhan) confirmed cases and 56 (45 of them were in Wuhan) deaths in mainland China [4] , and sporadic cases exported from Wuhan were reported in Thailand, Japan, Republic of Korea, Hong Kong, Taiwan, Australia, and the United States, please see the World Health Organization (WHO) news release via https://www.who.int/csr/don/en/ from 14 to 21 January 2020. Using the number of cases exported from Wuhan to other countries, a research group at Imperial College London estimated that there had been 4000 (95%CI: 1000-9700) cases in Wuhan with symptoms onset by 18 January 2020, and the basic reproduction number (R 0 ) was estimated at 2.6 (95%CI: 1.5-3.5) [5] . Leung et al. drew a similar conclusion and estimated the number of cases exported from Wuhan to other major cities in China [6] , and the potentials of travel related risks of disease spreading was also indicated by [7] . Due to an unknown reason, the cumulative number of cases remained at 41 from 1 to 15 January 2020 according to the official report, i.e., no new case was reported during these 15 days, which appears inconsistent with the following rapid growth of the epidemic curve since 16 January 2020. We suspect that the 2019-nCoV cases were under-reported roughly from 1 to 15 January 2020. In this study, we estimated the number of unreported cases and the basic reproduction number, R 0 , of 2019-nCoV in Wuhan from 1 to 15 January 2020 based on the limited data in the early outbreak. The time series data of 2019-nCoV cases in mainland China were initially released by the Wuhan Municipal Health Commission from 10 to 20 January 2020 [8] , and later by the National Health Commission of China after 21 January 2020 [9] . The case time series data in December 2019 were obtained from a published study [3] . All cases were laboratory confirmed following the case definition by the national health commission of China [10] . We chose the data up to 24 January 2020 instead of to the present study completion date. Given the lag between timings of case confirmation and news release of new cases, the data of the most recent few days were most likely to be tentative, and thus they were excluded from the analysis to be consistent. We suspected that there was a number of cases, denoted by ξ, under-reported from 1 to 15 January 2020. The cumulative total number of cases, denoted by C i , of the i-th day since 1 December 2019 is the summation of the cumulative reported, c i , and cumulative unreported cases, Ξ i . We have C i = c i + Ξ i , where c i is observed from the data, and Ξ i is 0 for i before 1 January and ξ for i after 15 January 2020. Following previous studies [11, 12] , we modelled the epidemic curve, i.e., the C i series, as an exponential growing Poisson process. Since the data from 1 to 15 January 2020 appeared constant due to unclear reason(s), we removed these data from the fitting of exponential growth. The ξ and the intrinsic growth rate (γ) of the exponential growth were to be estimated based on the log-likelihood, denoted by , from the Poisson priors. The 95% confidence interval (95% CI) of ξ was estimated by the profile likelihood estimation framework with cutoff threshold determined by a Chi-square quantile [13] , χ 2 pr = 0.95, df = 1 . With γ estimated, the basic reproduction number could be obtained by R 0 = 1/M(−γ) with 100% susceptibility for 2019-nCoV presumed at this early stage. Here, the function M(·) was the Laplace transform, i.e., the moment generating function, of the probability distribution for the serial interval (SI) of the disease [11, 14] , denoted by h(k) and k is the mean SI. Since the transmission chain of 2019-nCoV remained unclear, we adopted the SI information from Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), which share the similar pathogen as 2019-nCoV [15] [16] [17] . We modelled h(k) as Gamma distributions with mean of 8.0 days and standard deviation (SD) of 3.6 days by averaging the SI mean and SD of SARS, mean of 7.6 days and SD of 3.4 days [18] , and MERS, mean of 8.4 days and SD of 3.8 days [19] . We were also interested in inferring the patterns of the daily number of cases, denoted by ε i for the i-th day, and thus it is obviously that C i = C i−1 + ε i . A simulation framework was developed for the iterative Poisson process such that E[ denoted the expectation. The simulation was implemented starting from 1 January 2020 with a cumulative number of cases seed of 40, the same as reported on 31 December 2019. We conducted 1000 samples and calculated the median and 95% CI. The number of 2019-nCoV unreported cases was estimated at 469 (95% CI: 403-540), see Figure 1a , which was significantly larger than 0. This finding implied the occurrence of under-reporting between 1 and 15 January 2020. After accounting for the effect of under-reporting, the R 0 was estimated at 2.56 (95% CI: 2.49-2.63), see Figure 1b , which is consistent with many existing online preprints with range from 2 to 4 [5, [20] [21] [22] . With the R 0 of 2.56 and ξ of 469, the exponential growing framework fitted the cumulative total number of cases (C i ) remarkably well, see Figure 1c iterative Poisson process such that denoted the expectation. The simulation was implemented starting from 1 January 2020 with a cumulative number of cases seed of 40, the same as reported on 31 December 2019. We conducted 1000 samples and calculated the median and 95% CI. The number of 2019-nCoV unreported cases was estimated at 469 (95% CI: 403−540), see Figure 1a , which was significantly larger than 0. This finding implied the occurrence of under-reporting between 1 and 15 January 2020. After accounting for the effect of under-reporting, the R0 was estimated at 2.56 (95% CI: 2.49−2.63), see Figure 1b , which is consistent with many existing online preprints with range from 2 to 4 [5, [20] [21] [22] . With the R0 of 2.56 and ξ of 469, the exponential growing framework fitted the cumulative total number of cases (Ci) remarkably well, see Figure 1c , referring to McFadden's pseudo-R-squared of 0.99. show the exponential growth fitting results of the cumulative number of cases (Ci) and the daily number of cases (εi) respectively. In panels (c) and (d), the gold squares are the reported cases, the blue bold curve represents the median of the fitting results, the dashed blue curves are the 95% CI of the fitting results, and the purple shading area represents the time window from 1 to 15 January 2020. In panel (c), the blue dots are the cumulative total, i.e., reported and unreported, number of cases. In panel (d), the grey curves are the 1000 simulation samples. Our estimation of R0 rely on the SI of 2019-nCoV, which remains unknown as of 26 January 2020. In this work, we employed the SIs of SARS and MERS as approximations to that of 2019-nCoV. The determination of SI requires the knowledge of the chain of disease transmission that needs a sufficient number of patient samples and periods of time for follow-up [23] , and thus this is unlikely to be achieved shortly. However, using SIs of SARS and MERS as approximation could provide an panels (a,b) , the green shading area represents the 95% CI (on the horizontal axis), and the vertical green line represents the maximum likelihood estimate (MLE) of the number of unreported cases. With the MLE of R 0 at 2.56, panels (c,d) show the exponential growth fitting results of the cumulative number of cases (C i ) and the daily number of cases (ε i ) respectively. In panels (c,d), the gold squares are the reported cases, the blue bold curve represents the median of the fitting results, the dashed blue curves are the 95% CI of the fitting results, and the purple shading area represents the time window from 1 to 15 January 2020. In panel (c), the blue dots are the cumulative total, i.e., reported and unreported, number of cases. In panel (d), the grey curves are the 1000 simulation samples. Our estimation of R 0 rely on the SI of 2019-nCoV, which remains unknown as of 26 January 2020. In this work, we employed the SIs of SARS and MERS as approximations to that of 2019-nCoV. The determination of SI requires the knowledge of the chain of disease transmission that needs a sufficient number of patient samples and periods of time for follow-up [23] , and thus this is unlikely to be achieved shortly. However, using SIs of SARS and MERS as approximation could provide an insight into the transmission potential of 2019-nCoV at the early outbreak. We note that slightly varying the mean and SD of SI would not affect our main conclusions. The R 0 of 2019-nCoV was estimated at 2.56 (95% CI: 2.49-2.63), and it is generally in line with those of SARS, i.e., 2-5 [19, 24, 25] , and MERS, i.e., 2.7-3.9 [26] . For the simulated daily number of cases (ε i ), see Figure 1d , we found that ε i matched the observed daily number after 17 January 2020, but was significantly larger than the observations from 1 to 17 January 2020. This finding implied that under-reporting was likely to have occurred in the first half of January 2020. We estimated that the reporting rate after 17 January 2020 increased 21-fold (95% CI: [18] [19] [20] [21] [22] [23] [24] [25] compared to the situation from 1 to 17 January 2020 on average. One of the possible reasons was that the official diagnostic protocol was released by WHO on 17 January 2020 [27] , and the diagnosis and reporting efforts of 2019-nCoV infections probably increased. Thereafter, the daily number of newly reported cases started increasing rapidly after 17 January 2020, see Figure 1d . We conducted additional sensitivity analysis by varying the starting date of the under-reporting time window, e.g., 1 January 2020 in the main results, from 2 December 2019 to 3 January 2020, and we report our estimates largely hold. The exact value of the reporting rate was difficult to determine due to lack of serological surveillance data. The reporting rate can be determined if serological surveillance data are available for a population; we would know who was infected (seropositive) and who was not (seronegative), with high confidence. The reporting rate is the ratio of reported cases over the number of seropositive individuals. It was statistically evident that increasing in reporting was likely, and thus it should be considered in the future investigation of this outbreak. Previous preprint suggested cumulative cases of 1723 (95% CI: 427-4471) as of 12 January 2020, and 4000 (95% CI: 1000-9700) as of 18 January 2020 based on the aggregated international export cases [5] . Our analysis yielded cumulative cases of 280 (95% CI: 128-613) as of 12 January 2020, and 609 (95% CI: 278-1333) as of 18 January 2020 based on the exponential growing mechanistic in the early outbreak. Although our estimate case number appeared to have a lower mean than those estimated by Imai et al. [5] , they are not statistically different. This study applied a different screening effort to detect the 2019-nCoV cases from that in Imai et al. [5] . Imai et al. assumed the average screening effort at overseas airports that covered travelers arriving from Wuhan. Whereas we assumed a constant screening effort applied in Wuhan at the same point of time, and then a number of cases (i.e., ξ) should have been reported yet failed to be reported in the first half of January 2020 due to all sorts of reasons. It is not surprising that different assumptions yielded different results, and this difference in screening effort also partly explained why the detected cases out of China mainly presented mild symptoms. Thus, it was reasonable that our estimates appeared lower than those estimated by Imai et al. [5] . It must be emphasized that such a gap in the knowledge would be resolved by serological survey study (for a large population to approximate the actual positive rate) or an explicit estimation of the actual reporting rate. Under-reporting was likely to have occurred and resulted in 469 (95% CI: 403-540) unreported cases from 1 to 15 January 2020. The reporting rate after 17 January 2020 was likely to have increased 21-fold (95% CI: 18-25) compared with the situation from 1 to 17 January 2020 on average, and it should be considered in future investigation. We estimated the R 0 at 2019-nCoV to be 2.56 (95% CI: 2.49-2.63). Author Contributions: All authors conceived the study, carried out the analysis, discussed the results, drafted the first manuscript. All authors have read and agreed to the published version of the manuscript.
What is R0?
false
1,877
{ "text": [ "basic reproduction number," ], "answer_start": [ 836 ] }
1,686
Nucleolar Protein Trafficking in Response to HIV-1 Tat: Rewiring the Nucleolus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499507/ SHA: efa871aeaf22cbd0ce30e8bd1cb3d1afff2a98f9 Authors: Jarboui, Mohamed Ali; Bidoia, Carlo; Woods, Elena; Roe, Barbara; Wynne, Kieran; Elia, Giuliano; Hall, William W.; Gautier, Virginie W. Date: 2012-11-15 DOI: 10.1371/journal.pone.0048702 License: cc-by Abstract: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production. Text: The nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (NORs) formed by clusters of hundreds of rDNA gene repeats organised in tandem head-to-tail repeat [1, 2] . A membrane-less organelle originally described as the ''Ribosome Factory'', the nucleolus is dedicated to RNA-polymerase-I-directed rDNA transcription, rRNA processing mediated by small nucleolar ribonucleoproteins (soRNPs) and ribosome assembly. Ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60S) and small (40S) ribosomal subunits, which are subsequently exported to the cytoplasm. This fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] . The nucleolus is the site of additional RNA processing, including mRNA export and degradation, the maturation of uridine-rich small nuclear RNPs (U snRNPs), which form the core of the spliceosome, biogenesis of t-RNA and microRNAs (miRNAs) [4] . The nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] . The concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . Collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions. Of importance, the nucleolus is also the target of viruses including HIV-1, hCMV, HSV and KSHV, as part of their replication strategy [2, 17] . Proteomics studies analysing the nucleoli of cells infected with Human respiratory syncytial virus (HRSV), influenza A virus, avian coronavirus infectious bronchitis virus (IBV) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . Interestingly, both HIV-1 regulatory proteins Tat and Rev localise to the nucleoplasm and nucleolus. Both their sequences encompass a nucleolar localisation signal (NoLS) overlapping with their nuclear localisation signal (NLS), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . Furthermore, Tat and Rev interact with the nucleolar antigen B23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . Nevertheless, a recent study described that in contrast to Jurkat T-cells and other transformed cell lines where Tat is associated with the nucleus and nucleolus, in primary T-cells Tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . While the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing Tat and Rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . Importantly, two major studies by Machienzi et al. have revealed important functional links between HIV-1 replication and the nucleolus [49, 50] . First, they could inhibit HIV-1 replication and Tat transactivation function employing a TAR decoy specifically directed to the nucleolus. Furthermore, using a similar approach, with an anti-HIV-1 hammerhead ribozyme fused to the U16 small nucleolar RNA and therefore targeted to the nucleolus, they could dramatically suppress HIV-1 replication. Collectively, these findings strongly suggest that HIV-1 transcripts and Tat nucleolar trafficking are critical for HIV-1 replication. However the nature of these contributions remains to be elucidated. In this report, we systematically analysed the nucleolar proteome perturbations occurring in Jurkat T-cells constitutively expressing HIV-1 Tat, using a quantitative mass spectrometry approach. Following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon Tat expression, we focussed on the Tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, DNA replication and repair and metabolism and discuss their potential involvement in HIV-1 pathogenesis. In this study, we investigated the quantitative changes in the nucleolar proteome of Jurkat T cells constitutively expressing HIV-1 Tat (86aa) versus their Tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (SILAC) technology, followed by ESI tandem mass spectrometry and implemented the experimental approach described in Figure 1A . First, using retroviral gene delivery, we transduced HIV-1 Tat fused to a tandem affinity purification (TAP) tag (consisting of two protein G and a streptavidin binding peptide) or TAP tag alone (control vector) in Jurkat leukemia T cell clone E6-1 and sorted the transduced cells (GFP positive) by FACS. This resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( Figure 1B) . The functionality of TAP-Tat was confirmed by transfecting Jurkat TAP-Tat and TAP cells with a luciferase reporter gene vector under the control of the HIV-1 LTR (pGL3-LTR) [36] . TAP-Tat up regulated gene expression from the HIV-1 LTR by up to 28 fold compared to control ( Figure 1C ). To further address the functionality of Tat fused to TAP, we compared Jurkat TAP-Tat with Jurkat-tat, a cell line stably expressing untagged Tat [51] . Both cell line exhibited comparable HIV-1 LTR activity following transfection with pGL3-LTR ( Figure S1 ). Next, Tat expression and subcellular localization was verified by subcellular fractionation followed by WB analysis ( Figure 1E ). TAP-Tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. These observations were further validated by immunofluorescence microscopy ( Figure 1E ). Of note, Jurkat-tat presented similar patterns for Tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by WB analysis (Figure S2 and S3). We next compared the growth rate and proliferation of the Jurkat TAP and TAP-Tat cell lines (Materials and Methods S1), which were equivalent ( Figure S4A ). Similarly, FACS analysis confirmed that the relative populations in G1, S, and G2/M were similar for Jurkat TAP-Tat and TAP cells ( Figure S4B ). We labeled Jurkat TAP-Tat and Jurkat TAP cells with light (R0K0) and heavy (R6K6) isotope containing arginine and lysine, respectively. Following five passages in their respective SILAC medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( Figure 1A ) [52] . Each step of the procedure was closely monitored by microscopic examination. To assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by Western Blot analysis ( Figure 1D ). Nucleolin (110 kDa) and Fibrillarin (FBL) (34 kDa), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. Of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. This distribution pattern for nucleolin appears to be specific for Jurkat T-cells as show previously [52, 53] . The nuclear protein PARP-1 (Poly ADPribose polymerase 1) (113 kDa) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. Alpha-tubulin (50 kDa) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. Collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination. Subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. Comparative quantitative proteomic analysis was performed using MaxQuant to analyse the ratios in isotopes for each peptide identified. A total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. The fully annotated list of the quantified nucleolar proteins is available in Table S1 and the raw data from the mass spectrometry analysis was deposited in the Tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. We annotated the quantified proteins using the ToppGene Suite tools [54] and extracted Gene Ontology (GO) and InterPro annotations [55] . The analysis of GO biological processes ( Figure 1F ) revealed that the best-represented biological processes included transcription (24%), RNA processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of Jurkat T-cell nucleolar proteome [52] . Subcellular distribution analysis ( Figure 1F ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. The subcellular distribution was similar to our previous analysis of the Jurkat T-cell nucleolar proteome [52] . Table S1 . The distribution of protein ratios are represented in Figure 1G as log 2 (abundance change). The SILAC ratios indicate changes in protein abundance in the nucleolar fraction of Jurkat TAP-Tat cells in comparison with Jurkat TAP cells. The distribution of the quantified proteins followed a Gaussian distribution ( Figure 1G ). A total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon Tat expression (Table 1) . Of these, 30 proteins were enriched, whereas 19 proteins were depleted. Cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (NPM1/ B23), C23, FBL, nucleolar protein P120 (NOL1), and nucleolar protein 5A (NOL5A). The distinct ratios of protein changes upon Tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus. We performed WB analysis to validate the SILAC-based results obtained by our quantitative proteomic approach ( Figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon Tat expression, including 9 enriched (HSP90b, STAT3, pRb, CK2a, CK2a', HSP90a, Transportin, ZAP70, DDX3), and 3 depleted (ILF3, BOP1, and SSRP1) proteins. In addition, we also tested by WB analysis, protein abundance not affected by Tat expression (Importin beta, FBL, B23, C23). These results highlight the concordance in the trend of the corresponding SILAC ratios, despite some differences in the quantitative ranges. Of note, using WB, we could observe a change of intensity for protein with a SILAC fold change as low as 1.25-fold. Of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. On the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in Table 1 . Alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. Therefore, we next focused on both enriched or depleted individual proteins with activities associated with HIV-1 or Tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes. We initially focused on signaling proteins interacting with Tat and/or associated HIV-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by Tat expression. Phospho-protein phosphatases. Phospho-protein phosphatase PP1 and PP2A are essential serine/threonine phosphatases [56, 57] . Importantly, PP1 accounts for 80% of the Ser/Thr phosphatase activity within the nucleolus. In our study, PP1 was found to be potentially enriched by 1.52-fold in the nucleolus of Jurkat cells expressing Tat, which supports previous studies describing the nuclear and nucleolar targeting of PP1a by HIV-1 Tat and how PP1 upregulates HIV-1 transcription [58, 59, 60, 61, 62] . PP1 c was also identified as part of the in vitro nuclear interactome [63] . Similarly, PPP2CA, the PP2A catalytic subunit (1.29-fold) and its regulatory subunit PP2R1A (1.27-fold) were similarly enriched upon Tat expression. Interestingly, Tat association with the PP2A subunit promoters results in the overexpression and up regulation of PP2A activity in lymphocytes [64, 65] . Furthermore, PP2A contributes to the regulation of HIV-1 transcription and replication [61, 66] . Retinoblastoma Protein. The tumour suppressor gene pRb protein displayed a 1.4-fold change in the nucleolus upon Tat expression [67] . Furthermore, WB analysis confirmed the distinct translocation of pRb from the nucleoplasm to the nucleolus by Tat ( Figure 2 ). Depending on the cell type, pRb can be hyperphosphorylated or hypophosphorylated upon Tat expression and can negatively or positively regulate Tat-mediated transcription respectively [68, 69, 70] . Interestingly, the hyperphosphorylation of pRB triggers in its translocation into the nucleolus [71] . Phosphorylation of pRB is also associated with an increase in ribosomal biogenesis and cell growth [72] . STAT3. The transcription factor signal transducer and activator of transcription 3 (STAT3) was significantly enriched (1.86-fold) in the nucleolar fraction by Tat constitutive expression. Furthermore, WB analysis indicated that Tat expression could promote the relocalisation of STAT3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2) . Interestingly, previous studies have demonstrated Tat-mediated activation of STAT3 signaling, as shown by its phosphorylation status [73] . Interestingly, STAT3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] . YBX1. YBX1, the DNA/RNA binding multifunctional protein was enriched by 1.38-fold in the nucleolus of Jurkat cells upon Tat expression. Interestingly, YBX1 interacts with Tat and TAR and modulates HIV-1 gene expression [63, 75] . ZAP70. The protein tyrosine kinase ZAP70 (Zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of Jurkat cells expressing Tat [76] . Furthermore, WB analysis revealed that Tat expression could promote the relocalisation of ZAP70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( Figure 2 ). Of note, ZAP70 is part of the in vitro nuclear Tat interactome [63] . Matrin 3. The inner nuclear matrix protein, Matrin 3 (MATR3), presented a 1.39-fold change in the nucleolus of Jurkat cells expressing Tat. It localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . Matrin 3 has been identified as part of the in vitro HIV-1 Tat nuclear interactome [63] . Two recent studies have described Matrin 3 as part of ribonucleoprotein complexes also including HIV-1 Rev and (Rev Response Element) RRE-containing HIV-1 RNA, and promoting HIV-1 post-transcriptional regulation [78, 79, 80] . CASP10. The pro-apototic signaling molecule, Caspase 10 (CASP10), was significantly depleted from the nucleolus of Jurkat-Tat cells (0.82-fold) [81] . Importantly, Tat expression downregulates CASP10 expression and activity in Jurkat cells [82] . ADAR1. Adenosine deaminase acting on RNA (ADAR1), which converts adenosines to inosines in double-stranded RNA, was significantly depleted from the nucleolus of Jurkat-Tat cells (0.78-fold). Interestingly, ADAR1 over-expression up-regulates HIV-1 replication via an RNA editing mechanism [83, 84, 85, 86, 87, 88] . Furthermore, ADAR1 belongs to the in vitro HIV-1 Tat nuclear interactome [63] . To underline the structural and functional relationships of the nucleolar proteins affected by HIV-1 Tat, we constructed a network representation of our dataset. We employed Cytoscape version 2.6.3 [89] and using the MiMI plugin [90] to map previously characterised interactions, extracted from protein interaction databases (BIND, DIP, HPRD, CCSB, Reactome, IntAct and MINT). This resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( Figure 3A ). Centrality analysis revealed a threshold of 23.7 interactions per protein. Topology analysis using the CentiScaPe plugin [91] showed that the node degree distribution follows a power law ( Figure S5 ), characteristic of a scale-free network. Importantly, when we analysed the clustering coefficient distribution ( Figure S6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around HIV-1 Tat. Furthermore, node degree connection analysis of our network identified HIV-1 Tat as the most connected protein ( Figure S6 ). Specifically, the topology analysis indicated that the values for Tat centralities were the highest (Node degree, stress, radiality, closeness, betweeness and centroid), characterising Tat as the main hub protein of the nucleolar network. Indeed, a total of 146 proteins have been previously described to interact with Tat ( Figure 3B , Table S2 ). These proteins are involved in a wide range of cellular processes including chromosomal organization, DNA and RNA processing and cell cycle control. Importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold). In parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( Figure 4) . Ribosomal biogenesis. We initially focused on ribosome biogenesis, the primary function of the nucleolus. We could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by Tat expression (Figure 4 ). While some ribosomal proteins remained unaffected, Tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except RPL35A, for which Tat expression caused a marked decrease at the nucleolar level (0.29-fold). Similarly, several proteins involved in rRNA processing exhibited an overall increase in nucleolar accumulation upon Tat expression. These include human canonical members of the L7ae family together with members participating in Box C/D, H/ACA and U3 snoRNPs ( Figure 4) . Conversely, BOP1, a component of the PeBoW (Pescadillo Bop1 WDR12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of Jurkat TAP-Tat cells (0.81-fold) and this was confirmed by WB analysis (Figure 2 ) [93] . Nevertheless, the other PeBoW complex components, Pes1 (0.94-fold) and WDR12 (1.1fold), were not affected by Tat expression. Of note, we did not detect change in the abundance of protein participating in rDNA transcription such as RNAPOLI, UBF. Spliceosome. We identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . These proteins include the small nuclear ribonucleoproteins U1, U2 and U5, Sm D1, D2, D3, F and B, and the heterogeneous nuclear ribonucleoproteins. Our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of HIV-1 Tat in Jurkat T-cells (Figure 3 and 4) . The only three proteins that were significantly depleted from the nucleolus upon expression of HIV-1 Tat were RBMX (0.89-fold), HNRNPA2B1 (0.84-fold) and SNRPA (0.81-fold). Several investigations showed expression alteration in cellular splicing factors in HIV-1 infected cells [95, 96] . Molecular chaperones. We have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of T-cells upon Tat expression (Figure 3 and 4) , many of which were previously characterised as part of the Tat nuclear interactome [63] . Several heat-shock proteins including DNAJs, specific HSP90, HSP70 and HSP40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of Jurkat cells expressing Tat ( Figure 4 ). As shown by WB, while HSP90a and b are mostly cytoplasmic, Tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (Figure 2) . Similarly, heat-shock can cause the HSP90 and HSP70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . In a recent study, Fassati's group has shown that HSP90 is present at the HIV-1 promoter and may directly regulate viral gene expression [102] . We also observed the coordinated increased abundance of class I (GroEL and GroES) and class II (chaperonin containing TCP-1 (CTT)) chaperonin molecules (Figure 3 and 4) upon Tat expression. Ubiquitin-proteasome pathway. The ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . Importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . The 26S proteasome is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). Alternatively, CP can associate with the 11S RP to form the immunoproteasome. All the quantified proteins in our study are part of the 19S regulatory complex and include PSMD2 (1.5-fold), PSMD3 (1.32-fold), PSMD11 (1.25-fold) and PSMD13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of Tat (Figure 4) . Interestingly, Tat interacts with distinct subunits of the proteasome system, including the 19S, 20S and 11S subunits. The consequences of these interactions include the competition of Tat with 11S RP or 19S RP for binding to the 20S CP, which resulted in the inhibition of the 20S peptidase activity [106, 107, 108, 109, 110, 111] . Furthermore, Tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic T-lymphocytes [112] . Importantly, a recent study demonstrated that in the absence of Tat, proteasome components are associated to the HIV-1 promoter and proteasome activity limits transcription [113] . Addition of Tat promoted the dissociation of the 19S subunit from the 20S proteasome, followed by the distinct enrichment of the 19S-like complex in nuclear extracts together with the Tat-mediated recruitment of the 19S subunits to the HIV-1 promoter, which facilitated its transcriptional elongation [113] . We also quantified UBA1 (1.36-fold), the E3 ubiquitin-protein ligase UHRF1 (1.13-fold), UBC (1-fold) and two Ubiquitinspecific-peptidases, USP30 (1.28-fold) and USP20 (0.06-fold) (Figure 4) . DNA replication and repair. Upon HIV-1 Tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with DNA replication and repairs pathways (Figure 4) . Tat induced the coordinated enrichment of the miniature chromosome maintenance MCM2-7 complex (from 1.23-to 3.30fold, respectively) [114] . MCM7, 6 and 3 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . The structural maintenance of chromosomes 2, SMC2, was enriched (1.35-fold) in the nucleolar fraction by Tat expression. SMC2 was identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . While replication factor C1 (RFC1) and RFC2 (1.31-and 1.28-fold respectively) displayed an increased fold change and RFC5/3 were not affected, RFC4 was severely depleted (0.69-fold) from the nucleolar fraction upon Tat expression [115] . RFC1 and RFC2 were identified as part of the in vitro nuclear interactome of HIV-1 Tat [63] . Tat induced the enrichment of XRCC6 (1.27-fold) and XRCC5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous DNA end joining (NHEJ) [116] . XRCC6 associates with viral preintegration complexes containing HIV-1 Integrase and also interact with Tat and TAR [117, 118, 119] . Furthermore, in a ribozyme-based screen, XRCC5 (Ku80) knockdown decreased both retroviral integration and Tatmediated transcription [120] . As part of the base excision repair (BER), we have identified a major apurinic/apyrimidinic endonuclease 1 (APEX1) (1.29-fold) . Importantly, in a siRNA screen targeting DNA repair factors, APEX1 knockdown was found to inhibit HIV-1 infection by more 60% [121] . The high mobility group (HMG) protein, HMGA1 (1.30-fold), was enriched in the nucleolus following Tat expression [122] . HMGA1 interact with HIV-1 Integrase and is part of the HIV-1 pre-integration complex [123, 124] . Importantly, HMGA1 has been identified in a proteomic screen, as a cellular cofactor interacting with the HIV-1 59leader [125] . Metabolism. Our proteomic data suggest that Tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (Figure 4 and Figure S7 ). Notably, in T cells expressing Tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with Tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase B (LDHB) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.17-fold) and phosphoglyceric acid mutase (PGAM1) (0.89-fold) ( Figure 4 and Figure S7 ). Briefly, GPI catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. Subsequently, PFKP catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. At the end of the glycolytic pathway, PKM2, in its tetrameric form, is known to generate ATP and pyruvate, while LDHB diverts the majority of the pyruvate to lactate production and regeneration of NAD+ in support to continued glycolysis, a phenomenon described for proliferative Tcells [126] . Of note, in highly proliferating cells, PKM2 can be found in its dimeric form and its activity is altered. This upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. As part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (G6PD) (2.11-fold), which branches of the glycolysis pathway to generate NADPH, ribose-5phosphate an important precursor for the synthesis of nucleotides. Consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. More specifically, IMPDH2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (PRPS2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (CTPS) (1.74-fold) which catalyses the conversion of UTP to CTP and the ribonucleotide reductase large subunit (RRM1) (1.56-fold). In parralel, we noted the increased abundance of the phosphoserine aminotransferase PSAT1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro. The host-virus interface is a fundamental aspect in defining the molecular pathogenesis of HIV-1 [127, 128, 129, 130, 131, 132, 133] . Indeed, with its limited repertoire of viral proteins, HIV-1 relies extensively on the host cell machinery for its replication. Several recent studies have capitalized on the recent advances in the ''OMICS'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . HIV-1 Tat is essential for viral replication and orchestrates HIV-1 gene expression. The viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . We and others have employed system-level approaches to investigate Tat interplay with the host cell machinery, which have characterised HIV-1 Tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . Here, we have investigated the nucleolar proteins trafficking in response to HIV-1 Tat expression in T-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by Tat in the fine-tuning of protein availability and function. We have developed for this study, a cellular model using Jurkat T-cells stably expressing Tat fused in its N-ternminal to TAP-tag. Jurkat T-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. Importantly, they have been widely employed to evaluate Tat-mediated pathogenesis using system-wide approaches and to analyse T-cell key cellular signaling pathways and functions [144, 150, 151, 152] . Indeed, we have found them particularly suited for prolongued in vitro culture in SILAC medium and subsequent isolation of their nucleolus followed by MS analysis, which requires up to 85 millions of cells. We fused Tat to the TAP tag to enable future downstream applications such as Tandem affinity purification or Chromatin IP analysis. Importantly, we have confirm that N-terminal TAP-tag did not interfere with Tat function nor its localisation in Jurkat cells, when compared to untagged-Tat. Of note, Tat subcellular distribution can vary according to the cell type employed. While Tat is known to accumulate in the nucleus and nucleolus in Jurkat cells and other transformed cell lines, in primary T-cells, Tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . These differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by Kuusisto et al. [39] . Furthermore, Stauber and Pavlakis have suggested that Tat nucleolar localisation could be the results of Tat overexpression [31] . Here, we have selected and employed a polyclonal population of Jurkat T-cells expressing Tat at different levels. We propose that this heterogeneity in Tat expression levels might reflect Tat stochastic expression described during viral replication [153] . Using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. The extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. Nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with HIV-1 pathogenesis. HIV-1 Tat is expressed early following HIV-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. In this context and based on our results, we propose that Tat could participate in shaping the intracellular environment and metabolic profile of T cells to favor host biosynthetic activities supporting robust virions production. Indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. With the notable exeption of RPL35A nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (NHP2L1, RLP14, RPL17, RPL27, RPS2, RPL13). Furthermore, this effect appears to be specific to HIV-1 Tat since transcription inhibition by Actinomycin D resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . Moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . Whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. Nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. In parralel, we observed the concordant modulation of pathways regulating protein homeostasis. We noted the significant nucleolar accumulation of multiple molecular chaperones including the HSPs, the TCP-1 complex, and CANX/CALR molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . These observations further support previous studies describibing the modulation of the proteasomal activity by Tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . We also observed the concomitant depletion of CASP10 in the nucleolus of Jurkat TAP-Tat. It has been suggested that CASP10 could be targeted to the nucleolus to inhibit protein synthesis [154] . Interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by Banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . As viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by Tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability. Coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. Similarly, these pathways are elevated in proliferative T-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the Warburg effect [156, 157, 158, 159] . There, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the TCA cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl CoA and NADPH for redox homeostasis. Consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including IMPH2, a rate limiting enzyme known to control the pool of GTP. Similarly, we noted the nucleolar enrichment of PSAT1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . Collectively, we propose that by controlling protein homeostasis and metabolic pathways, Tat could meet both the energetic and biosynthetic demand of HIV-1 productive infection. Of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. Nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . Furthermore glycolytic enzymes, such as PKM2, LDH, phosphoglycerate kinase, GAPDH, and aldolase, also have been reported to display nuclear localization and bind to DNA [162] . More specifically, PKM2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] . HIV-1 Tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote TCR signaling [164] . We have observed the nucleolar enrichment by Tat of key proximal or downstream components of T-cell signaling pathways, including ZAP70, ILF3 and STAT3, which play crucial roles in T-cell development and activation. We had previously identified them as T-cell specific components of the nucleolus, and IF studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . Our results further support that Tat could contribute to the dysregulation of TCR-derived signals and that the nucleolus could represent an important spatial link for TCR signaling molecules. We observed the coordinated nucleolar enrichment of key components of the DNA replication, recombination and repair pathways by Tat. These include XRCC5 and XRCC6, HMGA1, APEX1, MCM2-7, SMC2, RFC1 and RFC2, while RFC4 was found to be significantly depleted. Interestingly, these cofactors have been associated with the efficiency of retroviral DNA integration into the host DNA or the integrity of integrated provirus [166] . Whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined. The mechanisms of Tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with Tat, since subcellular fractionation combined with WB analysis showed that the pattern and extent of subcellular redistribution between proteins varied. We could observe cases where Tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (DDX3, TNPO1). Alternatively, Tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (pRb). Additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon Tat expression (STAT3, ZAP70 and HSP90). Finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (SSRP1). It remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. Conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. Remarkably, we identified several known HIV-1 Tat partners involved in HIV-1 pathogenesis, which suggests that Tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. Tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. This is exemplified by the following enriched proteins, pRb, PP1 and STAT3, for which phosphorylation is induced by Tat. Importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by Tat. Moreover, our data indicates that serine/threonine kinases (CK2 a') and phosphatases (PP1) were significantly enriched in the nucleolar fractions of Jurkat TAP-Tat. These enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. In addition, Tat significantly decreased the levels of SUMO-2 in the nucleolus. Similarly, SUMO-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . Given the potential importance of post-translational modifications, including phosphorylation in the Tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach. The control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. Ribosomal proteins are degraded by the Ubiquitin-Proteasome pathway to ensure their abundance matches up with rRNA transcription levels. Conversely, heat shock proteins HSP90s protect them from degradation. Interestingly, our data showing that Tat modulation the abundance proteins associated with the Ubiquitin-proteasome and heat-shock pathway. This could contribute to the observed enrichment of ribosomal proteins by Tat. Nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of Tat-mediated prevention of their export to the cytoplasm. Interestingly, using a different cellular system, a drosophila melanogaster Tat transgenic strain, Ponti et al, analysed the effects of Tat on ribosome biogenesis, following 3 days heat shock treatment to induce Tat expression under the control of the hsp70 promoter [167] . Following Tat expression, they observed a defect in pre-rRNA processing associated with a decrease in the level of 80S ribosomes [167] . Nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours. While previous system-level studies have monitored the effects of HIV-1 Tat expression on T cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to HIV-1 Tat expression. Using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to Tat constitutive expression. Our findings underscore that Tat expressing T-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. Importantly, we noted the functional relationship of nucleolar proteins of our dataset with HIV-1 pathogenesis and HIV-1 Tat in particular. This further increases our confidence in our experimental strategy and suggests a role for Tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. Ultimatly, our study provides new insights on the importance of Tat in the cross talk between nucleolar functions and viral pathogenesis. Importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with HIV-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention. Jurkat T-cells, clone E6.1 (ATCC), Jurkat NTAP-Tat and Jurkat NTAP were maintained in RPMI-1640 medium supplemented with 10% (v/v) foetal bovine serum (Gibco, EU approved), and antibiotics. Phoenix-GP cells (G.P. Nolan; www.stanford.edu/ group/nolan/), were maintained in DMEM medium supplemented with 10% (v/v) foetal bovine serum (GIBCO, EU approved). Cells were counted using Scepter TM 2.0 Cell Counter (Millipore). The sequence of HIV-1 Tat (HIV-1 HXB2, 86 amino acids) was sub-cloned into pENTR 2B vector (Invitrogen, A10463). Using the Gateway technology (Invitrogen), we introduced the HIV-1 Tat sequence into the plasmid pCeMM-NTAP(GS)-Gw [168] . Phoenix cells (G.P. Nolan; www.stanford.edu/group/ nolan/), were transfected using Fugene 6 (Roche) with 5 mg of the plasmid NTAP-Tat or NTAP and 3 mg of the pMDG-VSVG. Viral supernatants were collected after 48 h, filtered and used to transduce the Jurkat cell lines. The construct is termed NTAP-Tat, the empty vector was termed NTAP. Using retroviral gene delivery, we stably transduced Jurkat cells (clone E6.1 (ATCC)). The positive clones named Jurkat NTAP-Tat and Jurkat NTAP were sorted to enrich the population of cells expressing GFP using the BC MoFlo XDP cell sorter (Beckman Coulter). Sub-cellular fractions (10 mg) were resolved by SDS-PAGE and transferred onto BioTrace PVDF membranes (Pall corporation). The following primary antibodies were used: a-Tubulin (Sc 5286), C23 (Sc 6013), and Fibrillarin (Sc 25397) were from Santa Cruz Biotechnology, and PARP (AM30) from Calbiochem, mouse anti-ZAP 70 (05-253, Millipore), rabbit anti-STAT3 (06-596, Millipore), rabbit anti-ILF3 (ab92355, Abcam), rabbit anti-HSP90 beta (ab32568, Abcam), mouse anti-ADAR1 (ab88574, Abcam), rabbit anti-HDAC1 (ab19845, Abcam), rabbit anti-SSRP1 (ab21584, Abcam) rabbit anti-BOP1 (ab86982, Abcam), mouse anti-KpNB1 (ab10303, Abcam), rabbit anti-HIV-1 Tat (ab43014, Abcam), rabbit anti-CK2A (ab10466, Abcam), rabbit anti-DDX3X (ab37160, Abcam), mouse anti-TNPO1 (ab2811, Abcam), mouse anti-HSP90A (CA1023, MERCK), and rabbit-anti RB1 (sc-102, Santa Cruz).The following secondary antibodies were used ECL: Anti-mouse IgG and ECL Anti-rabbit IgG (GE Healthcare), and Donkey anti-goat IgG (Sc 2020) (Santa Cruz Biotechnology). For SILAC analysis SILAC-RPMI R0K0 and SILAC-RPMI R6K6 (Dundee cells) media supplemented with 10% dialyzed FBS (GIBCO, 26400-036) were used. The Jurkat cells expressing NTAP-Tat and NTAP were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. Cells viability was checked with Trypan Blue (0.4% solution, SIGMA) and further confirmed using PI staining and FACS analysis. Cells were mixed to the ratio 1:1 to obtain 140610 6 cells. Nucleoli were isolated from the mixed cell population as previously described in Jarboui et al., [165] . Nucleolar extracts (100 mg) were resuspended in 50 mM ammonium bicarbonate and in solution trypsin digested as previously described in Jarboui et al. [165] . Sample was run on a Thermo Scientific LTQ ORBITRAP XL mass spectrometer connected to an Eksigent NANO LC.1DPLUS chromatography system incorporating an auto-sampler. Sample was loaded onto a Biobasic C18 PicofritTM column (100 mm length, 75 mm ID) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nL min-1. The mass spectrometer was operated in positive ion mode with a capillary temperature of 200uC, a capillary voltage of 46V, a tube lens voltage of 140V and with a potential of 1800 V applied to the frit. All data was acquired with the mass spectrometer operating in automatic data dependent switching mode. A high resolution MS scan was performed using the Orbitrap to select the 5 most intense ions prior to MS/MS analysis using the Ion trap. The incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''Heavy'' medium as described in [169] . Our analysis showed that we had an incorporation efficiency .95% (data not shown). The MS/MS spectra were searched for peptides identification and quantification using the MaxQuant software [170] (version 1.1.1.36), the Human IPI Database (version 3.83) and the Andromeda search engine associated to MaxQuant [171] . Standard settings were used for MaxQuant with the Acetyl (Protein N-term) as variable modification and Carbamidomethyl (Cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. Initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 Da, respectively. Each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. Proteins were identified with the minimum of one peptide with a false discovery rate less than 1%. Gene ontology, KEGG pathway and Pfam terms were extracted from UNIPROT entries using Perseus, a software from the MaxQuant Data analysis package (http://www.maxquant.org ), and the ToppGene suite tools [54] . The Jurkat NTAP-Tat and Jurkat NTAP were transfected using the Amaxa electroporation system (Amaxa biosystem) with the pGL3 (pGL3-LTR) (Promega) as recommended by Amaxa Biosystem. Dual-luciferase assays (Promega) were performed according to the manufacturer's instructions. Luciferase activity was measured and normalized against the total amount of proteins as quantified by the BCA protein quantification kit (Pierce, Thermo Scientific). To preserve their original shape, we performed immunostaining of Jurkat cells in suspension. Cells were fixed in 2% PFA for 10 min at RT, permeabilised in 0.5% Triton X-100 for 15 min at RT and blocked with 5% FCS. Cells were incubated with the rabbit HIV-1 Tat antibody (ab43014, Abcam) followed by the secondary antibody anti-Rabbit alexa fluor 647 (A-21246, Invitrogen). Cells were allowed to attach to Cell-Tak (BD) coated Silanised Slides (DaoCytomation), and stained with DAPI. Images were captured with a Carl Zeiss Confocal Microscope equipped with a Plan-Apochromat 63X/1.4 oil DIC objective. The proteomics RAW Data file from the mass spectrometry analysis was deposited to the Tranche repository(https:// proteomecommons.org/tranche/) [172] . The file can be accessed and downloaded using the following hash key: (R3O5SV5Z6HvWqrBNDhp21tXFetluDWYxvwMIfU-h6e1kMgarauCSq4dlNcxeUvFOHDEzLeDcg4X5Y8reSb6-MUA6wM1kIAAAAAAAAB/w = = ). Materials and Methods S1 Description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis. (DOCX)
What is shown in Table S1?
false
5,145
{ "text": [ "The fully annotated list of the quantified nucleolar proteins" ], "answer_start": [ 11360 ] }
2,642
First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, 24 January to 21 February 2020 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068164/ SHA: ce358c18aac69fc83c7b2e9a7dca4a43b0f60e2e Authors: Spiteri, Gianfranco; Fielding, James; Diercke, Michaela; Campese, Christine; Enouf, Vincent; Gaymard, Alexandre; Bella, Antonino; Sognamiglio, Paola; Sierra Moros, Maria José; Riutort, Antonio Nicolau; Demina, Yulia V.; Mahieu, Romain; Broas, Markku; Bengnér, Malin; Buda, Silke; Schilling, Julia; Filleul, Laurent; Lepoutre, Agnès; Saura, Christine; Mailles, Alexandra; Levy-Bruhl, Daniel; Coignard, Bruno; Bernard-Stoecklin, Sibylle; Behillil, Sylvie; van der Werf, Sylvie; Valette, Martine; Lina, Bruno; Riccardo, Flavia; Nicastri, Emanuele; Casas, Inmaculada; Larrauri, Amparo; Salom Castell, Magdalena; Pozo, Francisco; Maksyutov, Rinat A.; Martin, Charlotte; Van Ranst, Marc; Bossuyt, Nathalie; Siira, Lotta; Sane, Jussi; Tegmark-Wisell, Karin; Palmérus, Maria; Broberg, Eeva K.; Beauté, Julien; Jorgensen, Pernille; Bundle, Nick; Pereyaslov, Dmitriy; Adlhoch, Cornelia; Pukkila, Jukka; Pebody, Richard; Olsen, Sonja; Ciancio, Bruno Christian Date: 2020-03-05 DOI: 10.2807/1560-7917.es.2020.25.9.2000178 License: cc-by Abstract: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters’ index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. Text: In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters' index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases. A cluster of pneumonia of unknown origin was identified in Wuhan, China, in December 2019 [1] . On 12 January 2020, Chinese authorities shared the sequence of a novel coronavirus termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from some clustered cases [2] . Since then, the disease caused by SARS-CoV-2 has been named coronavirus disease 2019 (COVID -19) . As at 21 February 2020, the virus had spread rapidly mostly within China but also to 28 other countries, including in the World Health Organization (WHO) European Region [3] [4] [5] . Here we describe the epidemiology of the first cases of COVID-19 in this region, excluding cases reported in the United Kingdom (UK), as at 21 February 2020. The study includes a comparison between cases detected among travellers from China and cases whose infection was acquired due to subsequent local transmission. On 27 January 2020, the European Centre for Disease Prevention and Control (ECDC) and the WHO Regional Office for Europe asked countries to complete a WHO standard COVID-19 case report form for all confirmed and probable cases according to WHO criteria [6] [7] [8] . The overall aim of surveillance at this time was to support the global strategy of containment of COVID-19 with rapid identification and follow-up of cases linked to affected countries in order to minimise onward transmission. The surveillance objectives were to: describe the key epidemiological and clinical characteristics of COVID-19 cases detected in Europe; inform country preparedness; and improve further case detection and management. Data collected included demographics, history of recent travel to affected areas, close contact with a probable or confirmed COVID-19 case, underlying conditions, signs and symptoms of disease at onset, type of specimens from which the virus was detected, and clinical outcome. The WHO case definition was adopted for surveillance: a confirmed case was a person with laboratory confirmation of SARS-CoV-2 infection (ECDC recommended two separate SARS-CoV-2 RT-PCR tests), irrespective of clinical signs and symptoms, whereas a probable case was a suspect case for whom testing for SARS-CoV-2 was inconclusive or positive using a pan-coronavirus assay [8] . By 31 January 2020, 47 laboratories in 31 countries, including 38 laboratories in 24 European Union and European Economic Area (EU/EEA) countries, had diagnostic capability for SARS-CoV-2 available (close to 60% of countries in the WHO European Region), with cross-border shipment arrangements in place for many of those lacking domestic testing capacity. The remaining six EU/EEA countries were expected to have diagnostic testing available by mid-February [9] . As at 09:00 on 21 February 2020, 47 confirmed cases of COVID-19 were reported in the WHO European Region and one of these cases had died [4] . Data on 38 of these cases (i.e. all except the nine reported in the UK) are included in this analysis. The first three cases detected were reported in France on 24 January 2020 and had onset of symptoms on 17, 19 and 23 January respectively [10] . The first death was reported on 15 February in France. As at 21 February, nine countries had reported cases ( Figure) : Belgium (1), Finland (1), France (12), Germany (16), Italy (3), Russia (2), Spain (2), Sweden (1) and the UK (9 -not included further). The place of infection (assessed at national level based on an incubation period presumed to be up to 14 days [11] , travel history and contact with probable or confirmed cases as per the case definition) was reported for 35 cases (missing for three cases), of whom 14 were infected in China (Hubei province: 10 cases; Shandong province: one case; province not reported for three cases). The remaining 21 cases were infected in Europe. Of these, 14 were linked to a cluster in Bavaria, Germany, and seven to a cluster in Haute-Savoie, France [12, 13] . Cases from the Bavarian cluster were reported from Germany and Spain, whereas cases from the Haute-Savoie cluster were reported from France All but two cases were hospitalised (35 of 37 where information on hospitalisation was reported), although it is likely that most were hospitalised to isolate the person rather than because of severe disease. The time from onset of symptoms to hospitalisation (and isolation) ranged between 0 and 10 days with a mean of 3.7 days (reported for 29 cases). The mean number of days to hospitalisation was 2.5 days for cases imported from China, but 4.6 days for those infected in Europe. This was mostly a result of delays in identifying the index cases of the two clusters in France and Germany. In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six took only a mean of 2 days to be hospitalised. Symptoms at the point of diagnosis were reported for 31 cases. Two cases were asymptomatic and remained so until tested negative. The asymptomatic cases were tested as part of screening following repatriation and during contact tracing respectively. Of the remaining 29, 20 reported fever, 14 reported cough and eight reported weakness. Additional symptoms reported included headaches (6 cases), sore throat (2), rhinorrhoea (2), shortness of breath (2), myalgia (1), diarrhoea (1) and nausea (1). Fever was reported as the sole symptom for nine cases. In 16 of 29 symptomatic cases, the symptoms at diagnosis were consistent with the case definition for acute respiratory infection [16] , although it is possible that cases presented additional symptoms after diagnosis and these were not reported. Data on pre-existing conditions were reported for seven cases; five had no pre-existing conditions while one was reported to be obese and one had pre-existing cardiac disease. No data on clinical signs e.g. dyspnea etc. were reported for any of the 38 cases. All hospitalised cases had a benign clinical evolution except four, two reported in Italy and two reported in France, all of whom developed viral pneumonia. All three cases who were aged 65 years or over were admitted to intensive care and required respiratory support and one French case died. The case who died was hospitalised for 21 days and required intensive care and mechanical ventilation for 19 days. The duration of hospitalisation was reported for 16 cases with a median of 13 days (range: 8-23 days). As at 21 February 2020, four cases were still hospitalised. All cases were confirmed according to specific assays targeting at least two separate genes (envelope (E) gene as a screening test and RNA-dependent RNA polymerase (RdRp) gene or nucleoprotein (N) gene for confirmation) [8, 17] . The specimen types tested were reported for 27 cases: 15 had positive nasopharyngeal swabs, nine had positive throat swabs, three cases had positive sputum, two had a positive nasal swab, one case had a positive nasopharyngeal aspirate and one a positive endotracheal aspirate. As at 09:00 on 21 February, few COVID-19 cases had been detected in Europe compared with Asia. However the situation is rapidly developing, with a large outbreak recently identified in northern Italy, with transmission in several municipalities and at least two deaths [18] . As at 5 March 2020, there are 4,250 cases including 113 deaths reported among 38 countries in the WHO European region [19] . In our analysis of early cases, we observed transmission in two broad contexts: sporadic cases among travellers from China (14 cases) and cases who acquired infection due to subsequent local transmission in Europe (21 cases). Our analysis shows that the time from symptom onset to hospitalisation/case isolation was about 3 days longer for locally acquired cases than for imported cases. People returning from affected areas are likely to have a low threshold to seek care and be tested when symptomatic, however delays in identifying the index cases of the two clusters in France and Germany meant that locally acquired cases took longer to be detected and isolated. Once the exposure is determined and contacts identified and quarantined (171 contacts in France and 200 in Germany for the clusters in Haute-Savoie and Bavaria, respectively), further cases are likely to be rapidly detected and isolated when they develop symptoms [15, 20] . In the German cluster, for example, the first three cases detected locally were hospitalised in a mean of 5.7 days, whereas the following six were hospitalised after a mean of 2 days. Locally acquired cases require significant resources for contact tracing and quarantine, and countries should be prepared to allocate considerable public health resources during the containment phase, should local clusters emerge in their population. In addition, prompt sharing of information on cases and contacts through international notification systems such as the International Health Regulations (IHR) mechanism and the European Commission's European Early Warning and Response System is essential to contain international spread of infection. All of the imported cases had a history of travel to China. This was consistent with the epidemiological situation in Asia, and supported the recommendation for testing of suspected cases with travel history to China and potentially other areas of presumed ongoing community transmission. The situation has evolved rapidly since then, however, and the number of countries reporting COVID-19 transmission increased rapidly, notably with a large outbreak in northern Italy with 3,089 cases reported as at 5 March [18, 19] . Testing of suspected cases based on geographical risk of importation needs to be complemented with additional approaches to ensure early detection of local circulation of COVID-19, including through testing of severe acute respiratory infections in hospitals irrespectively of travel history as recommended in the WHO case definition updated on 27 February 2020 [21] . The clinical presentation observed in the cases in Europe is that of an acute respiratory infection. However, of the 31 cases with information on symptoms, 20 cases presented with fever and nine cases presented only with fever and no other symptoms. These findings, which are consistent with other published case series, have prompted ECDC to include fever among several clinical signs or symptoms indicative for the suspected case definition. Three cases were aged 65 years or over. All required admission to intensive care and were tourists (imported cases). These findings could reflect the average older age of the tourist population compared with the local contacts exposed to infection in Europe and do not allow us to draw any conclusion on the proportion of severe cases that we could expect in the general population of Europe. Despite this, the finding of older individuals being at higher risk of a severe clinical course is consistent with the evidence from Chinese case series published so far although the majority of infections in China have been mild [22, 23] . This preliminary analysis is based on the first reported cases of COVID-19 cases in the WHO European Region. Given the small sample size, and limited completeness for some variables, all the results presented should be interpreted with caution. With increasing numbers of cases in Europe, data from surveillance and investigations in the region can build on the evidence from countries in Asia experiencing more widespread transmission particularly on disease spectrum and the proportion of infections with severe outcome [22] . Understanding the infection-severity is critical to help plan for the impact on the healthcare system and the wider population. Serological studies are vital to understand the proportion of cases who are asymptomatic. Hospital-based surveillance could help estimate the incidence of severe cases and identify risk factors for severity and death. Established hospital surveillance systems that are in place for influenza and other diseases in Europe may be expanded for this purpose. In addition, a number of countries in Europe are adapting and, in some cases, already using existing sentinel primary care based surveillance systems for influenza to detect community transmission of SARS-CoV-2. This approach will be used globally to help identify evidence of widespread community transmission and, should the virus spread and containment no longer be deemed feasible, to monitor intensity of disease transmission, trends and its geographical spread. Additional research is needed to complement surveillance data to build knowledge on the infectious period, modes of transmission, basic and effective reproduction numbers, and effectiveness of prevention and case management options also in settings outside of China. Such special studies are being conducted globally, including a cohort study on citizens repatriated from China to Europe, with the aim to extrapolate disease incidence and risk factors for infection in areas with community transmission. Countries together with ECDC and WHO, should use all opportunities to address these questions in a coordinated fashion at the European and global level. provided input to the outline, multiple versions of the manuscript and gave approval to the final draft.
How can present systems of surveillance be used?
false
3,844
{ "text": [ "Established hospital surveillance systems that are in place for influenza and other diseases in Europe may be expanded for this purpose. In addition, a number of countries in Europe are adapting and, in some cases, already using existing sentinel primary care based surveillance systems for influenza to detect community transmission of SARS-CoV-2" ], "answer_start": [ 14099 ] }
2,634
Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067204/ SHA: c097a8a9a543d69c34f10e5c3fd78019e560026a Authors: Chan, Jasper Fuk-Woo; Kok, Kin-Hang; Zhu, Zheng; Chu, Hin; To, Kelvin Kai-Wang; Yuan, Shuofeng; Yuen, Kwok-Yung Date: 2020-01-28 DOI: 10.1080/22221751.2020.1719902 License: cc-by Abstract: A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus (2019-nCoV) was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet(s) containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection. Text: Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses that belong to the subfamily Coronavirinae, family Coronavirdiae, order Nidovirales. There are four genera of CoVs, namely, Alphacoronavirus (αCoV), Betacoronavirus (βCoV), Deltacoronavirus (δCoV), and Gammacoronavirus (γCoV) [1] . Evolutionary analyses have shown that bats and rodents are the gene sources of most αCoVs and βCoVs, while avian species are the gene sources of most δCoVs and γCoVs. CoVs have repeatedly crossed species barriers and some have emerged as important human pathogens. The best-known examples include severe acute respiratory syndrome CoV (SARS-CoV) which emerged in China in 2002-2003 to cause a large-scale epidemic with about 8000 infections and 800 deaths, and Middle East respiratory syndrome CoV (MERS-CoV) which has caused a persistent epidemic in the Arabian Peninsula since 2012 [2, 3] . In both of these epidemics, these viruses have likely originated from bats and then jumped into another amplification mammalian host [the Himalayan palm civet (Paguma larvata) for SARS-CoV and the dromedary camel (Camelus dromedarius) for MERS-CoV] before crossing species barriers to infect humans. Prior to December 2019, 6 CoVs were known to infect human, including 2 αCoV (HCoV-229E and HKU-NL63) and 4 βCoV (HCoV-OC43 [ HCoV-OC43 and HCoV-HKU1 usually cause self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and elderly [4] . In contrast, SARS-CoV (lineage B βCoV) and MERS-CoV (lineage C βCoV) may cause severe lower respiratory tract infection with acute respiratory distress syndrome and extrapulmonary manifestations, such as diarrhea, lymphopenia, deranged liver and renal function tests, and multiorgan dysfunction syndrome, among both immunocompetent and immunocompromised hosts with mortality rates of ∼10% and ∼35%, respectively [5, 6] . On 31 December 2019, the World Health Organization (WHO) was informed of cases of pneumonia of unknown cause in Wuhan City, Hubei Province, China [7] . Subsequent virological testing showed that a novel CoV was detected in these patients. As of 16 January 2020, 43 patients have been diagnosed to have infection with this novel CoV, including two exported cases of mild pneumonia in Thailand and Japan [8, 9] . The earliest date of symptom onset was 1 December 2019 [10] . The symptomatology of these patients included fever, malaise, dry cough, and dyspnea. Among 41 patients admitted to a designated hospital in Wuhan, 13 (32%) required intensive care and 6 (15%) died. All 41 patients had pneumonia with abnormal findings on chest computerized tomography scans [10] . We recently reported a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan [11] . In the present study, we analyzed a 2019-nCoV complete genome from a patient in this familial cluster and compared it with the genomes of related βCoVs to provide insights into the potential source and control strategies. The complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank (accession no. MN975262) ( Table 1 ). The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup. Phylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees [12] . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches [13] . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site [14] . All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA X [15] . Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOX-SHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction (PSIPRED) [16] . For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOX-SHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/). Secondary structure prediction in the 5 ′ -untranslated region (UTR) and 3 ′ -UTR was performed using the RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/ RNAWebSuite/RNAfold.cgi) with minimum free energy (MFE) and partition function in Fold algorithms and Table 2 . Putative functions and proteolytic cleavage sites of 16 nonstructural proteins in orf1a/b as predicted by bioinformatics. Putative function/domain Amino acid position Putative cleave site complex with nsp3 and 6: DMV formation complex with nsp3 and 4: DMV formation short peptide at the end of orf1a basic options. The human SARS-CoV 5 ′ -and 3 ′ -UTR were used as references to adjust the prediction results. The single-stranded RNA genome of the 2019-nCoV was 29891 nucleotides in size, encoding 9860 amino acids. The G + C content was 38%. Similar to other (Table 2 ). There are no remarkable differences between the orfs and nsps of 2019-nCoV with those of SARS-CoV (Table 3) . The major distinction between SARSr-CoV and SARS-CoV is in orf3b, Spike and orf8 but especially variable in Spike S1 and orf8 which were previously shown to be recombination hot spots. Spike glycoprotein comprised of S1 and S2 subunits. The S1 subunit contains a signal peptide, followed by an N-terminal domain (NTD) and receptor-binding domain (RBD), while the S2 subunit contains conserved fusion peptide (FP), heptad repeat (HR) 1 and 2, transmembrane domain (TM), and cytoplasmic domain (CP). We found that the S2 subunit of 2019-nCoV is highly conserved and shares 99% identity with those of the two bat SARS-like CoVs (SL-CoV ZXC21 and ZC45) and human SARS-CoV (Figure 2 ). Thus the broad spectrum antiviral peptides against S2 would be an important preventive and treatment modality for testing in animal models before clinical trials [18] . Though the S1 subunit of 2019-nCoV shares around 70% identity to that of the two bat SARS-like CoVs and human SARS-CoV (Figure 3(A) ), the core domain of RBD (excluding the external subdomain) are highly conserved (Figure 3(B) ). Most of the amino acid differences of RBD are located in the external subdomain, which is responsible for the direct interaction with the host receptor. Further investigation of this soluble variable external subdomain region will reveal its receptor usage, interspecies transmission and pathogenesis. Unlike 2019-nCoV and human SARS-CoV, most known bat SARSr-CoVs have two stretches of deletions in the spike receptor binding domain (RBD) when compared with that of human SARS-CoV. But some Yunnan strains such as the WIV1 had no such deletions and can use human ACE2 as a cellular entry receptor. It is interesting to note that the two bat SARS-related coronavirus ZXC21 and ZC45, being closest to 2019-nCoV, can infect suckling rats and cause inflammation in the brain tissue, and pathological changes in lung & intestine. However, these two viruses could not be isolated in Vero E6 cells and were not investigated further. The two retained deletion sites in the Spike genes of ZXC21 and ZC45 may lessen their likelihood of jumping species barriers imposed by receptor specificity. A novel short putative protein with 4 helices and no homology to existing SARS-CoV or SARS-r-CoV protein was found within Orf3b ( Figure 4 ). It is notable that SARS-CoV deletion mutants lacking orf3b replicate to levels similar to those of wildtype virus in several cell types [19] , suggesting that orf3b is dispensable for viral replication in vitro. But orf3b may have a role in viral pathogenicity as Vero E6 but not 293T cells transfected with a construct expressing Orf3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time points [20] . Orf3b was also shown to inhibit expression of IFN-β at synthesis and signalling [21] . Subsequently, orf3b homologues identified from three bat SARSrelated-CoV strains were C-terminally truncated and lacked the C-terminal nucleus localization signal of SARS-CoV [22] . IFN antagonist activity analysis demonstrated that one SARS-related-CoV orf3b still possessed IFN antagonist and IRF3-modulating activities. These results indicated that different orf3b proteins display different IFN antagonist activities and this function is independent of the protein's nuclear localization, suggesting a potential link between bat SARS-related-CoV orf3b function and pathogenesis. The importance of this new protein in 2019-nCoV will require further validation and study. Orf8 orf8 is an accessory protein found in the Betacoronavirus lineage B coronaviruses. Human SARS-CoVs isolated from early-phase patients, all civet SARS-CoVs, and other bat SARS-related CoVs contain fulllength orf8 [23] . However, a 29-nucleotide deletion, Bat SL-CoV ZXC21 2018 Bat which causes the split of full length of orf8 into putative orf8a and orf8b, has been found in all SARS-CoV isolated from mid-and late-phase human patients [24] . In addition, we have previously identified two bat SARS-related-CoV (Bat-CoV YNLF_31C and YNLF_34C) and proposed that the original SARS-CoV full-length orf8 is acquired from these two bat SARS-related-CoV [25] . Since the SARS-CoV is the closest human pathogenic virus to the 2019-nCoV, we performed phylogenetic analysis and multiple alignments to investigate the orf8 amino acid sequences. The orf8 protein sequences used in the analysis derived from early phase SARS-CoV that includes full-length orf8 (human SARS-CoV GZ02), the mid-and late-phase SARS-CoV that includes the split orf8b (human SARS-CoV Tor2), civet SARS-CoV (paguma SARS-CoV), two bat SARS-related-CoV containing full-length orf8 (bat-CoV YNLF_31C and YNLF_34C), 2019-nCoV, the other two closest bat SARS-related-CoV to 2019-nCoV SL-CoV ZXC21 and ZC45), and bat SARS-related-CoV HKU3-1 ( Figure 5(A) ). As expected, orf8 derived from 2019-nCoV belongs to the group that includes the closest genome sequences of bat SARS-related-CoV ZXC21 and ZC45. Interestingly, the new 2019-nCoV orf8 is distant from the conserved orf8 or Figure 5(B) ) which was shown to trigger intracellular stress pathways and activates NLRP3 inflammasomes [26] , but this is absent in this novel orf8 of 2019-nCoV. Based on a secondary structure prediction, this novel orf8 has a high possibility to form a protein with an alpha-helix, following with a betasheet(s) containing six strands ( Figure 5(C) ). The genome of 2019-nCoV has overall 89% nucleotide identity with bat SARS-related-CoV SL-CoVZXC21 (MG772934.1), and 82% with human SARS-CoV BJ01 2003 (AY278488) and human SARS-CoV Tor2 (AY274119). The phylogenetic trees constructed using the amino acid sequences of orf1a/b and the 4 structural genes (S, E, M, and N) were shown (Figure 6(A-E) ). For all these 5 genes, the 2019-nCoV was clustered with lineage B βCoVs. It was most closely related to the bat SARS-related CoVs ZXC21 and ZC45 found in Chinese horseshoe As shown in Figure 7 (A-C), the SARS-CoV 5 ′ -UTR contains SL1, SL2, SL3, SL4, S5, SL5A, SL5B, SL5C, SL6, SL7, and SL8. The SL3 contains trans-cis motif [27] . The SL1, SL2, SL3, SL4, S5, SL5A, SL5B, and SL5C structures were similar among the 2019-nCoV, human SARS-CoV and the bat SARS-related ZC45. In the 2019-nCoV, part of the S5 found was inside Figure 7 Continued the orf1a/b (marked in red), which was similar to SARS-CoV. In bat SARS-related CoV ZC45, the S5 was not found inside orf1a/b. The 2019-nCoV had the same SL6, SL7, and SL8 as SARS-CoV, and an additional stem loop. Bat SARS-related CoV ZC45 did not have the SARS-COV SL6-like stem loop. Instead, it possessed two other stem loops in this region. All three strains had similar SL7 and SL8. The bat SARS-like CoV ZC45 also had an additional stem loop between SL7 and SL8. Overall, the 5 ′ -UTR of 2019-nCoV was more similar to that of SARS-CoV than the bat SARS-related CoV ZC 45. The biological relevance and effects of virulence of the 5 ′ -UTR structures should be investigated further. The 2019-nCoV had various 3 ′ -UTR structures, including BSL, S1, S2, S3, S4, L1, L2, L3, and HVR (Figure 7(D-F) ). The 3 ′ -UTR was conserved among 2019-nCoV, human SARS-CoV and SARS-related CoVs [27] . In summary, 2019-nCoV is a novel lineage B Betacoronavirus closely related to bat SARS-related coronaviruses. It also has unique genomic features which deserves further investigation to ascertain their roles in viral replication cycle and pathogenesis. More animal sampling to determine its natural animal reservoir and intermediate animal host in the market is important. This will shed light on the evolutionary history of this emerging coronavirus which has jumped into human after the other two zoonotic Betacoroanviruses, SARS-CoV and MERS-CoV.
What was the authors' recent report on?
false
3,705
{ "text": [ "a familial cluster of 2019-nCoV infection in a Shenzhen family with travel history to Wuhan" ], "answer_start": [ 4684 ] }
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?
false
1,239
{ "text": [ "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" ], "answer_start": [ 2740 ] }
2,459
No credible evidence supporting claims of the laboratory engineering of SARS-CoV-2 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054935/ SHA: 5a9154aee79901dd8fecd58b7bcd9b7351102d24 Authors: Liu, Shan-Lu; Saif, Linda J.; Weiss, Susan R.; Su, Lishan Date: 2020-02-26 DOI: 10.1080/22221751.2020.1733440 License: cc-by Abstract: nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 (COVID-19) (https://globalbiodefense. com/novel-coronavirus-covid-19-portal/). According to what has been reported [1] [2] [3] , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome (SARS) caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity [4, 5] . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV (RaTG13) was recently reported, which shared ∼96% homology with the SARS-CoV-2 [4] . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide (nt) variations (SNVs) identified across the genome [6] . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV [4] , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species (bats) are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published (https:// www.nature.com/articles/d41586-020-00364-2). Another claim in Chinese social media points to a Nature Medicine paper published in 2015 [7] , which reports the construction of a chimeric CoV with a bat CoV S gene (SHC014) in the backbone of a SARS CoV that has adapted to infect mice (MA15) and is capable of infecting human cells [8] . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 (>5,000 nucleotides). The mouse-adapted SARS virus (MA15) [9] was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice (hence M15), due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells [10, 11] . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans [6, 12] . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry [8] . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV [13] , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice [7] . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function (GOF) studies under the US government-mandated pause policy (https://www.nih.gov/about-nih/who-weare/nih-director/statements/nih-lifts-funding-pausegain-function-research). The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups [5, 14] , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv (a manuscript sharing site prior to any peer review), claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random [15] . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author(s). Susan R. Weiss http://orcid.org/0000-0002-8155-4528
How much similarity the SARS-COV-2 genome sequence has with SARS-COV?
false
3,592
{ "text": [ "∼80% identity with SARS-CoV" ], "answer_start": [ 975 ] }
2,653
Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048229/ SHA: da81f0d3a12ab7faa09148acb6564271474e9e02 Authors: Zhang, Wei; Du, Rong-Hui; Li, Bei; Zheng, Xiao-Shuang; Yang, Xing-Lou; Hu, Ben; Wang, Yan-Yi; Xiao, Geng-Fu; Yan, Bing; Shi, Zheng-Li; Zhou, Peng Date: 2020-02-17 DOI: 10.1080/22221751.2020.1729071 License: cc-by Abstract: In December 2019, a novel coronavirus (2019-nCoV) caused an outbreak in Wuhan, China, and soon spread to other parts of the world. It was believed that 2019-nCoV was transmitted through respiratory tract and then induced pneumonia, thus molecular diagnosis based on oral swabs was used for confirmation of this disease. Likewise, patient will be released upon two times of negative detection from oral swabs. However, many coronaviruses can also be transmitted through oral–fecal route by infecting intestines. Whether 2019-nCoV infected patients also carry virus in other organs like intestine need to be tested. We conducted investigation on patients in a local hospital who were infected with this virus. We found the presence of 2019-nCoV in anal swabs and blood as well, and more anal swab positives than oral swab positives in a later stage of infection, suggesting shedding and thereby transmitted through oral–fecal route. We also showed serology test can improve detection positive rate thus should be used in future epidemiology. Our report provides a cautionary warning that 2019-nCoV may be shed through multiple routes. Text: Coronaviruses (CoVs) belong to the subfamily Orthocoronavirinae in the family Coronaviridae and the order Nidovirales. A human coronavirus (SARS-CoV) caused the severe acute respiratory syndrome coronavirus (SARS) outbreak in 2003. Most recently, an SARS-related CoV was implicated as the etiological agent responsible for the outbreak in Wuhan, central China. This outbreak is estimated to have started on 12th December 2019 and 17,332 laboratory confirmed cases with 361 deaths as of 3rd February 2020 in China [1] . The virus has spread to 23 other countries by travellers from Wuhan [1] . Typical symptoms are fever, malaise, shortness of breath and in severe cases, pneumonia [2] [3] [4] . The disease was first called unidentified viral pneumonia. We quickly identified the etiological agent, termed 2019-nCoV (virus name designated by the World Health Organization). The newly identified virus is an SARS-related virus (SARSr-CoV) but shares only 74.5% genome identity to SARS-CoV [2] . We developed molecular detection tools based on viral spike genes. Our previous studies indicate that qPCR method can be used for the detection of 2019-nCoV in oral swabs or in bronchoalveolar lavage fluid (BALF) [5] . Additionally, we developed IgM and IgG detection methods using a cross-reactive nucleocapsid protein (NP) from another SARSr-CoV Rp3 [6] , which is 92% identical to 2019-nCoV NP. Using these serological tools, we demonstrate viral antibody titres increase in patients infected with 2019-nCoV [5] . Like SARS-CoV, 2019-nCoV induced pneumonia through respiratory tract by clinical observation. Therefore, the presence of viral antigen in oral swabs was used as detection standard for 2019-nCoV. Similarly, two times of oral swabs negative in a 24-h interval was considered as viral clearance by patients officially. Here we launched an investigation of 2019-nCoV in a Wuhan hospital, aiming to investigate the other possible transmission route of this virus. Human samples, including oral swabs, anal swabs and blood samples were collected by Wuhan pulmonary hospital with the consent from all patients and approved by the ethics committee of the designated hospital for emerging infectious diseases. Two investigations were performed. In the first investigation, we collected samples from 39 patients, 7 of which were in severe conditions. In the second investigation, we collected samples from 139 patients, yet their clinical records were not available. We only showed patients who were viral nucleotide detection positive. Patients were sampled without gender or age preference unless where indicated. For swabs, 1.5 ml DMEM+2% FBS medium was added in each tube. Supernatant was collected after 2500 rpm, 60 s vortex and 15-30 min standing. Supernatant from swabs were added to lysis buffer for RNA extraction. Serum was separated by centrifugation at 3000 g for 15 min within 24 h of collection, followed by 56°C 30 min inactivation, and then stored at 4°C until use. Whenever commercial kits were used, manufacturer's instructions were followed without modification. RNA was extracted from 200 μl of samples with the High Pure Viral RNA Kit (Roche). RNA was eluted in 50 μl of elution buffer and used as the template for RT-PCR. QPCR detection method based on 2019-nCoV S gene can be found in the previous study [5] . In brief, RNA extracted from above used in qPCR by HiScript® II One Step qRT-PCR SYBR® Green Kit (Vazyme Biotech Co., Ltd). The 20 μl qPCR reaction mix contained 10 μl 2× One Step SYBR Green Mix, 1 μl One Step SYBR Green Enzyme Mix, 0.4 μl 50 × ROX Reference Dye 1, 0.4 μl of each primer (10 μM) and 2 μl template RNA. Amplification was performed as follows: 50°C for 3 min, 95°C for 30 s followed by 40 cycles consisting of 95°C for 10 s, 60°C for 30 s, and a default melting curve step in an ABI 7500 machine. In-house anti-SARSr-CoV IgG and IgM ELISA kits were developed using SARSr-CoV Rp3 NP as antigen, which shared above 90% amino acid identity to all SARSr-CoVs, as reported previously [5] . For IgG test, MaxiSorp Nunc-immuno 96 well ELISA plates were coated (100 ng/well) overnight with recombinant NP. Human sera were used at 1:20 dilution for 1 h at 37°C. An anti-Human IgG-HRP conjugated monoclonal antibody (Kyab Biotech Co., Ltd, Wuhan, China) was used at a dilution of 1:40,000. The OD value (450-630) was calculated. For IgM test, Maxi-Sorp Nunc-immuno 96 wellELISA plates were coated (500 ng/well) overnight with anti-human IgM (µ chain). Human sera were used at 1:100 dilution for 40 min at 37°C, followed by anti-Rp3 NP-HRP conjugated (Kyab Biotech Co., Ltd, Wuhan, China) at a dilution of 1:4000. The OD value (450-630) was calculated. In the first investigation, we aimed to test whether viral positive can be found in anal swab and blood as well as oral swabs. We conducted a molecular investigation to patients in Wuhan pulmonary hospital, who were detected as oral swabs positive for 2019-nCoV upon admission. We collected blood, oral swabs and anal swabs for 2019-nCoV qPCR test using previously established method [5] . We found 15 patients who still carry virus following days of medical treatments. Of these patients, 8 were oral swabs positive (53.3%), 4 were anal swabs positive (26.7%), 6 blood positives (40%) and 3 serum positives (20%). Two patients were positive by both oral swab and anal swab, yet none of the blood positive was also swabs positive. Not surprisingly, all serum positives were also whole serum positive (Table 1 ). In summary, viral nucleotide can be found in anal swab or blood even if it cannot be detected in oral swabs. It should be noted that although swabs may be negative, the patient might still be viremic. We then did another investigation to find out the dynamic changes of viral presence in two consecutive studies in both oral and anal swabs in another group of patients. The target patients were those who received around 10 days of medical treatments upon admission. We tested for both viral antibody and viral nucleotide levels by previously established method [5] . We showed that both IgM and IgG titres were relatively low or undetectable in day 0 (the day of first sampling). On day 5, an increase of viral antibodies can be seen in nearly all patients, which was normally considered as a transition from earlier to later period of infection ( Figure 1 and supplementary table 1 ). IgM positive rate increased from 50% (8/16) to 81% (13/16), whereas IgG positive rate increased from 81% (13/16) to 100% (16/16). This is in contrast to a relatively low detection positive rate from molecular test (below). For molecular detection, we found 8 oral swabs positive (50%) and 4 anal swabs (25%) in these 16 people on day 0. On day 5, we were only able to find 4 oral swabs positive (25%). In contrast, we found 6 anal swabs positive (37.5%). When counting all swab positives together, we found most of the positives came from oral swab (8/10, 80%) on day 0. However, this trend appears to change on day 5. We found more (6/8, 75%) anal swab positive than oral swab positive (4/8, 50%). Another observation is the reoccurrence of virus in 6 patients who were detected negative on day 0. Of note, 4 of these 6 viral positives were from anal swabs ( Table 2) . These data suggested a shift from more oral positive during early period (as indicated by antibody titres) to more anal positive during later period might happen. Within 1 month of the 2019-nCoV disease outbreak, we rapidly developed molecular and serological detection tools. This is the first molecular and serological study on this virus after the initial identification of 2019-NCoV from 7 patients diagnosed with unidentified viral pneumonia [5] . We detected the virus in oral swabs, anal swabs and blood, thus infected patients can potentially shed this pathogen through respiratory, fecal-oral or body fluid routes. In addition, we successfully applied serology test a large population and showed which could greatly improved detection positive rate. We show that the current strategy for the detection of viral RNA in oral swabs used for 2019-nCoV diagnosis is not perfect. The virus may be present in anal swabs or blood of patients when oral swabs detection negative. In SARS-CoV and MERS-CoV infected patients, intestinal infection was observed at later stages of infection [7] [8] [9] . However, patients infected with 2019-nCoV may harbour the virus in the intestine at the early or late stage of disease. It is also worth to note none of the patients with viremia blood had positive swabs. These patients would likely be considered as 2019-nCoV negative through routine surveillance, and thus pose a threat to other people. In contrast, we found viral antibodies in near all patients, indicating serology should be considered for 2019-nCoV epidemiology. A possible shift from oral positive during early infection to anal swab positive during late infection can be observed. This observation implied that we cannot discharge a patient purely based on oral swabs negative, who may still shed the virus by oral-fecal route. Above all, we strongly suggest using viral IgM and IgG serological test to confirm an infection, considering the unreliable results from oral swabs detection. In summary, we provide a cautionary warning that 2019-nCoV may be transmitted through multiple routes. Both molecular and serological tests are needed to definitively confirm a virus carrier.
Is oral swab for detecting 2019-nCOV infection, sufficient?
false
884
{ "text": [ "patients infected with 2019-nCoV may harbour the virus in the intestine at the early or late stage of disease. It is also worth to note none of the patients with viremia blood had positive swabs. These patients would likely be considered as 2019-nCoV negative through routine surveillance, and thus pose a threat to other people." ], "answer_start": [ 9970 ] }
1,719
Virus-Vectored Influenza Virus Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/ SHA: f6d2afb2ec44d8656972ea79f8a833143bbeb42b Authors: Tripp, Ralph A.; Tompkins, S. Mark Date: 2014-08-07 DOI: 10.3390/v6083055 License: cc-by Abstract: Despite the availability of an inactivated vaccine that has been licensed for >50 years, the influenza virus continues to cause morbidity and mortality worldwide. Constant evolution of circulating influenza virus strains and the emergence of new strains diminishes the effectiveness of annual vaccines that rely on a match with circulating influenza strains. Thus, there is a continued need for new, efficacious vaccines conferring cross-clade protection to avoid the need for biannual reformulation of seasonal influenza vaccines. Recombinant virus-vectored vaccines are an appealing alternative to classical inactivated vaccines because virus vectors enable native expression of influenza antigens, even from virulent influenza viruses, while expressed in the context of the vector that can improve immunogenicity. In addition, a vectored vaccine often enables delivery of the vaccine to sites of inductive immunity such as the respiratory tract enabling protection from influenza virus infection. Moreover, the ability to readily manipulate virus vectors to produce novel influenza vaccines may provide the quickest path toward a universal vaccine protecting against all influenza viruses. This review will discuss experimental virus-vectored vaccines for use in humans, comparing them to licensed vaccines and the hurdles faced for licensure of these next-generation influenza virus vaccines. Text: Seasonal influenza is a worldwide health problem causing high mobility and substantial mortality [1] [2] [3] [4] . Moreover, influenza infection often worsens preexisting medical conditions [5] [6] [7] . Vaccines against circulating influenza strains are available and updated annually, but many issues are still present, including low efficacy in the populations at greatest risk of complications from influenza virus infection, i.e., the young and elderly [8, 9] . Despite increasing vaccination rates, influenza-related hospitalizations are increasing [8, 10] , and substantial drug resistance has developed to two of the four currently approved anti-viral drugs [11, 12] . While adjuvants have the potential to improve efficacy and availability of current inactivated vaccines, live-attenuated and virus-vectored vaccines are still considered one of the best options for the induction of broad and efficacious immunity to the influenza virus [13] . The general types of influenza vaccines available in the United States are trivalent inactivated influenza vaccine (TIV), quadrivalent influenza vaccine (QIV), and live attenuated influenza vaccine (LAIV; in trivalent and quadrivalent forms). There are three types of inactivated vaccines that include whole virus inactivated, split virus inactivated, and subunit vaccines. In split virus vaccines, the virus is disrupted by a detergent. In subunit vaccines, HA and NA have been further purified by removal of other viral components. TIV is administered intramuscularly and contains three or four inactivated viruses, i.e., two type A strains (H1 and H3) and one or two type B strains. TIV efficacy is measured by induction of humoral responses to the hemagglutinin (HA) protein, the major surface and attachment glycoprotein on influenza. Serum antibody responses to HA are measured by the hemagglutination-inhibition (HI) assay, and the strain-specific HI titer is considered the gold-standard correlate of immunity to influenza where a four-fold increase in titer post-vaccination, or a HI titer of ≥1:40 is considered protective [4, 14] . Protection against clinical disease is mainly conferred by serum antibodies; however, mucosal IgA antibodies also may contribute to resistance against infection. Split virus inactivated vaccines can induce neuraminidase (NA)-specific antibody responses [15] [16] [17] , and anti-NA antibodies have been associated with protection from infection in humans [18] [19] [20] [21] [22] . Currently, NA-specific antibody responses are not considered a correlate of protection [14] . LAIV is administered as a nasal spray and contains the same three or four influenza virus strains as inactivated vaccines but on an attenuated vaccine backbone [4] . LAIV are temperature-sensitive and cold-adapted so they do not replicate effectively at core body temperature, but replicate in the mucosa of the nasopharynx [23] . LAIV immunization induces serum antibody responses, mucosal antibody responses (IgA), and T cell responses. While robust serum antibody and nasal wash (mucosal) antibody responses are associated with protection from infection, other immune responses, such as CD8 + cytotoxic lymphocyte (CTL) responses may contribute to protection and there is not a clear correlate of immunity for LAIV [4, 14, 24] . Currently licensed influenza virus vaccines suffer from a number of issues. The inactivated vaccines rely on specific antibody responses to the HA, and to a lesser extent NA proteins for protection. The immunodominant portions of the HA and NA molecules undergo a constant process of antigenic drift, a natural accumulation of mutations, enabling virus evasion from immunity [9, 25] . Thus, the circulating influenza A and B strains are reviewed annually for antigenic match with current vaccines, Replacement of vaccine strains may occur regularly, and annual vaccination is recommended to assure protection [4, 26, 27] . For the northern hemisphere, vaccine strain selection occurs in February and then manufacturers begin production, taking at least six months to produce the millions of vaccine doses required for the fall [27] . If the prediction is imperfect, or if manufacturers have issues with vaccine production, vaccine efficacy or availability can be compromised [28] . LAIV is not recommended for all populations; however, it is generally considered to be as effective as inactivated vaccines and may be more efficacious in children [4, 9, 24] . While LAIV relies on antigenic match and the HA and NA antigens are replaced on the same schedule as the TIV [4, 9] , there is some suggestion that LAIV may induce broader protection than TIV due to the diversity of the immune response consistent with inducing virus-neutralizing serum and mucosal antibodies, as well as broadly reactive T cell responses [9, 23, 29] . While overall both TIV and LAIV are considered safe and effective, there is a recognized need for improved seasonal influenza vaccines [26] . Moreover, improved understanding of immunity to conserved influenza virus antigens has raised the possibility of a universal vaccine, and these universal antigens will likely require novel vaccines for effective delivery [30] [31] [32] . Virus-vectored vaccines share many of the advantages of LAIV, as well as those unique to the vectors. Recombinant DNA systems exist that allow ready manipulation and modification of the vector genome. This in turn enables modification of the vectors to attenuate the virus or enhance immunogenicity, in addition to adding and manipulating the influenza virus antigens. Many of these vectors have been extensively studied or used as vaccines against wild type forms of the virus. Finally, each of these vaccine vectors is either replication-defective or causes a self-limiting infection, although like LAIV, safety in immunocompromised individuals still remains a concern [4, 13, [33] [34] [35] . Table 1 summarizes the benefits and concerns of each of the virus-vectored vaccines discussed here. There are 53 serotypes of adenovirus, many of which have been explored as vaccine vectors. A live adenovirus vaccine containing serotypes 4 and 7 has been in use by the military for decades, suggesting adenoviruses may be safe for widespread vaccine use [36] . However, safety concerns have led to the majority of adenovirus-based vaccine development to focus on replication-defective vectors. Adenovirus 5 (Ad5) is the most-studied serotype, having been tested for gene delivery and anti-cancer agents, as well as for infectious disease vaccines. Adenovirus vectors are attractive as vaccine vectors because their genome is very stable and there are a variety of recombinant systems available which can accommodate up to 10 kb of recombinant genetic material [37] . Adenovirus is a non-enveloped virus which is relatively stable and can be formulated for long-term storage at 4 °C, or even storage up to six months at room temperature [33] . Adenovirus vaccines can be grown to high titers, exceeding 10 1° plaque forming units (PFU) per mL when cultured on 293 or PER.C6 cells [38] , and the virus can be purified by simple methods [39] . Adenovirus vaccines can also be delivered via multiple routes, including intramuscular injection, subcutaneous injection, intradermal injection, oral delivery using a protective capsule, and by intranasal delivery. Importantly, the latter two delivery methods induce robust mucosal immune responses and may bypass preexisting vector immunity [33] . Even replication-defective adenovirus vectors are naturally immunostimulatory and effective adjuvants to the recombinant antigen being delivered. Adenovirus has been extensively studied as a vaccine vector for human disease. The first report using adenovirus as a vaccine vector for influenza demonstrated immunogenicity of recombinant adenovirus 5 (rAd5) expressing the HA of a swine influenza virus, A/Swine/Iowa/1999 (H3N2). Intramuscular immunization of mice with this construct induced robust neutralizing antibody responses and protected mice from challenge with a heterologous virus, A/Hong Kong/1/1968 (H3N2) [40] . Replication defective rAd5 vaccines expressing influenza HA have also been tested in humans. A rAd5-HA expressing the HA from A/Puerto Rico/8/1934 (H1N1; PR8) was delivered to humans epicutaneously or intranasally and assayed for safety and immunogenicity. The vaccine was well tolerated and induced seroconversion with the intranasal administration had a higher conversion rate and higher geometric meant HI titers [41] . While clinical trials with rAd vectors have overall been successful, demonstrating safety and some level of efficacy, rAd5 as a vector has been negatively overshadowed by two clinical trial failures. The first trial was a gene therapy examination where high-dose intravenous delivery of an Ad vector resulted in the death of an 18-year-old male [42, 43] . The second clinical failure was using an Ad5-vectored HIV vaccine being tested as a part of a Step Study, a phase 2B clinical trial. In this study, individuals were vaccinated with the Ad5 vaccine vector expressing HIV-1 gag, pol, and nef genes. The vaccine induced HIV-specific T cell responses; however, the study was stopped after interim analysis suggested the vaccine did not achieve efficacy and individuals with high preexisting Ad5 antibody titers might have an increased risk of acquiring HIV-1 [44] [45] [46] . Subsequently, the rAd5 vaccine-associated risk was confirmed [47] . While these two instances do not suggest Ad-vector vaccines are unsafe or inefficacious, the umbra cast by the clinical trials notes has affected interest for all adenovirus vaccines, but interest still remains. Immunization with adenovirus vectors induces potent cellular and humoral immune responses that are initiated through toll-like receptor-dependent and independent pathways which induce robust pro-inflammatory cytokine responses. Recombinant Ad vaccines expressing HA antigens from pandemic H1N1 (pH1N1), H5 and H7 highly pathogenic avian influenza (HPAI) virus (HPAIV), and H9 avian influenza viruses have been tested for efficacy in a number of animal models, including chickens, mice, and ferrets, and been shown to be efficacious and provide protection from challenge [48, 49] . Several rAd5 vectors have been explored for delivery of non-HA antigens, influenza nucleoprotein (NP) and matrix 2 (M2) protein [29, [50] [51] [52] . The efficacy of non-HA antigens has led to their inclusion with HA-based vaccines to improve immunogenicity and broaden breadth of both humoral and cellular immunity [53, 54] . However, as both CD8 + T cell and neutralizing antibody responses are generated by the vector and vaccine antigens, immunological memory to these components can reduce efficacy and limit repeated use [48] . One drawback of an Ad5 vector is the potential for preexisting immunity, so alternative adenovirus serotypes have been explored as vectors, particularly non-human and uncommon human serotypes. Non-human adenovirus vectors include those from non-human primates (NHP), dogs, sheep, pigs, cows, birds and others [48, 55] . These vectors can infect a variety of cell types, but are generally attenuated in humans avoiding concerns of preexisting immunity. Swine, NHP and bovine adenoviruses expressing H5 HA antigens have been shown to induce immunity comparable to human rAd5-H5 vaccines [33, 56] . Recombinant, replication-defective adenoviruses from low-prevalence serotypes have also been shown to be efficacious. Low prevalence serotypes such as adenovirus types 3, 7, 11, and 35 can evade anti-Ad5 immune responses while maintaining effective antigen delivery and immunogenicity [48, 57] . Prime-boost strategies, using DNA or protein immunization in conjunction with an adenovirus vaccine booster immunization have also been explored as a means to avoided preexisting immunity [52] . Adeno-associated viruses (AAV) were first explored as gene therapy vectors. Like rAd vectors, rAAV have broad tropism infecting a variety of hosts, tissues, and proliferating and non-proliferating cell types [58] . AAVs had been generally not considered as vaccine vectors because they were widely considered to be poorly immunogenic. A seminal study using AAV-2 to express a HSV-2 glycoprotein showed this virus vaccine vector effectively induced potent CD8 + T cell and serum antibody responses, thereby opening the door to other rAAV vaccine-associated studies [59, 60] . AAV vector systems have a number of engaging properties. The wild type viruses are non-pathogenic and replication incompetent in humans and the recombinant AAV vector systems are even further attenuated [61] . As members of the parvovirus family, AAVs are small non-enveloped viruses that are stable and amenable to long-term storage without a cold chain. While there is limited preexisting immunity, availability of non-human strains as vaccine candidates eliminates these concerns. Modifications to the vector have increased immunogenicity, as well [60] . There are limited studies using AAVs as vaccine vectors for influenza. An AAV expressing an HA antigen was first shown to induce protective in 2001 [62] . Later, a hybrid AAV derived from two non-human primate isolates (AAVrh32.33) was used to express influenza NP and protect against PR8 challenge in mice [63] . Most recently, following the 2009 H1N1 influenza virus pandemic, rAAV vectors were generated expressing the HA, NP and matrix 1 (M1) proteins of A/Mexico/4603/2009 (pH1N1), and in murine immunization and challenge studies, the rAAV-HA and rAAV-NP were shown to be protective; however, mice vaccinated with rAAV-HA + NP + M1 had the most robust protection. Also, mice vaccinated with rAAV-HA + rAAV-NP + rAAV-M1 were also partially protected against heterologous (PR8, H1N1) challenge [63] . Most recently, an AAV vector was used to deliver passive immunity to influenza [64, 65] . In these studies, AAV (AAV8 and AAV9) was used to deliver an antibody transgene encoding a broadly cross-protective anti-influenza monoclonal antibody for in vivo expression. Both intramuscular and intranasal delivery of the AAVs was shown to protect against a number of influenza virus challenges in mice and ferrets, including H1N1 and H5N1 viruses [64, 65] . These studies suggest that rAAV vectors are promising vaccine and immunoprophylaxis vectors. To this point, while approximately 80 phase I, I/II, II, or III rAAV clinical trials are open, completed, or being reviewed, these have focused upon gene transfer studies and so there is as yet limited safety data for use of rAAV as vaccines [66] . Alphaviruses are positive-sense, single-stranded RNA viruses of the Togaviridae family. A variety of alphaviruses have been developed as vaccine vectors, including Semliki Forest virus (SFV), Sindbis (SIN) virus, Venezuelan equine encephalitis (VEE) virus, as well as chimeric viruses incorporating portions of SIN and VEE viruses. The replication defective vaccines or replicons do not encode viral structural proteins, having these portions of the genome replaces with transgenic material. The structural proteins are provided in cell culture production systems. One important feature of the replicon systems is the self-replicating nature of the RNA. Despite the partial viral genome, the RNAs are self-replicating and can express transgenes at very high levels [67] . SIN, SFV, and VEE have all been tested for efficacy as vaccine vectors for influenza virus [68] [69] [70] [71] . A VEE-based replicon system encoding the HA from PR8 was demonstrated to induce potent HA-specific immune response and protected from challenge in a murine model, despite repeated immunization with the vector expressing a control antigen, suggesting preexisting immunity may not be an issue for the replicon vaccine [68] . A separate study developed a VEE replicon system expressing the HA from A/Hong Kong/156/1997 (H5N1) and demonstrated varying efficacy after in ovo vaccination or vaccination of 1-day-old chicks [70] . A recombinant SIN virus was use as a vaccine vector to deliver a CD8 + T cell epitope only. The well-characterized NP epitope was transgenically expressed in the SIN system and shown to be immunogenic in mice, priming a robust CD8 + T cell response and reducing influenza virus titer after challenge [69] . More recently, a VEE replicon system expressing the HA protein of PR8 was shown to protect young adult (8-week-old) and aged (12-month-old) mice from lethal homologous challenge [72] . The VEE replicon systems are particularly appealing as the VEE targets antigen-presenting cells in the lymphatic tissues, priming rapid and robust immune responses [73] . VEE replicon systems can induce robust mucosal immune responses through intranasal or subcutaneous immunization [72] [73] [74] , and subcutaneous immunization with virus-like replicon particles (VRP) expressing HA-induced antigen-specific systemic IgG and fecal IgA antibodies [74] . VRPs derived from VEE virus have been developed as candidate vaccines for cytomegalovirus (CMV). A phase I clinical trial with the CMV VRP showed the vaccine was immunogenic, inducing CMV-neutralizing antibody responses and potent T cell responses. Moreover, the vaccine was well tolerated and considered safe [75] . A separate clinical trial assessed efficacy of repeated immunization with a VRP expressing a tumor antigen. The vaccine was safe and despite high vector-specific immunity after initial immunization, continued to boost transgene-specific immune responses upon boost [76] . While additional clinical data is needed, these reports suggest alphavirus replicon systems or VRPs may be safe and efficacious, even in the face of preexisting immunity. Baculovirus has been extensively used to produce recombinant proteins. Recently, a baculovirus-derived recombinant HA vaccine was approved for human use and was first available for use in the United States for the 2013-2014 influenza season [4] . Baculoviruses have also been explored as vaccine vectors. Baculoviruses have a number of advantages as vaccine vectors. The viruses have been extensively studied for protein expression and for pesticide use and so are readily manipulated. The vectors can accommodate large gene insertions, show limited cytopathic effect in mammalian cells, and have been shown to infect and express genes of interest in a spectrum of mammalian cells [77] . While the insect promoters are not effective for mammalian gene expression, appropriate promoters can be cloned into the baculovirus vaccine vectors. Baculovirus vectors have been tested as influenza vaccines, with the first reported vaccine using Autographa californica nuclear polyhedrosis virus (AcNPV) expressing the HA of PR8 under control of the CAG promoter (AcCAG-HA) [77] . Intramuscular, intranasal, intradermal, and intraperitoneal immunization or mice with AcCAG-HA elicited HA-specific antibody responses, however only intranasal immunization provided protection from lethal challenge. Interestingly, intranasal immunization with the wild type AcNPV also resulted in protection from PR8 challenge. The robust innate immune response to the baculovirus provided non-specific protection from subsequent influenza virus infection [78] . While these studies did not demonstrate specific protection, there were antigen-specific immune responses and potential adjuvant effects by the innate response. Baculovirus pseudotype viruses have also been explored. The G protein of vesicular stomatitis virus controlled by the insect polyhedron promoter and the HA of A/Chicken/Hubei/327/2004 (H5N1) HPAIV controlled by a CMV promoter were used to generate the BV-G-HA. Intramuscular immunization of mice or chickens with BV-G-HA elicited strong HI and VN serum antibody responses, IFN-γ responses, and protected from H5N1 challenge [79] . A separate study demonstrated efficacy using a bivalent pseudotyped baculovirus vector [80] . Baculovirus has also been used to generate an inactivated particle vaccine. The HA of A/Indonesia/CDC669/2006(H5N1) was incorporated into a commercial baculovirus vector controlled by the e1 promoter from White Spot Syndrome Virus. The resulting recombinant virus was propagated in insect (Sf9) cells and inactivated as a particle vaccine [81, 82] . Intranasal delivery with cholera toxin B as an adjuvant elicited robust HI titers and protected from lethal challenge [81] . Oral delivery of this encapsulated vaccine induced robust serum HI titers and mucosal IgA titers in mice, and protected from H5N1 HPAIV challenge. More recently, co-formulations of inactivated baculovirus vectors have also been shown to be effective in mice [83] . While there is growing data on the potential use of baculovirus or pseudotyped baculovirus as a vaccine vector, efficacy data in mammalian animal models other than mice is lacking. There is also no data on the safety in humans, reducing enthusiasm for baculovirus as a vaccine vector for influenza at this time. Newcastle disease virus (NDV) is a single-stranded, negative-sense RNA virus that causes disease in poultry. NDV has a number of appealing qualities as a vaccine vector. As an avian virus, there is little or no preexisting immunity to NDV in humans and NDV propagates to high titers in both chicken eggs and cell culture. As a paramyxovirus, there is no DNA phase in the virus lifecycle reducing concerns of integration events, and the levels of gene expression are driven by the proximity to the leader sequence at the 3' end of the viral genome. This gradient of gene expression enables attenuation through rearrangement of the genome, or by insertion of transgenes within the genome. Finally, pathogenicity of NDV is largely determined by features of the fusion protein enabling ready attenuation of the vaccine vector [84] . Reverse genetics, a method that allows NDV to be rescued from plasmids expressing the viral RNA polymerase and nucleocapsid proteins, was first reported in 1999 [85, 86] . This process has enabled manipulation of the NDV genome as well as incorporation of transgenes and the development of NDV vectors. Influenza was the first infectious disease targeted with a recombinant NDV (rNDV) vector. The HA protein of A/WSN/1933 (H1N1) was inserted into the Hitchner B1 vaccine strain. The HA protein was expressed on infected cells and was incorporated into infectious virions. While the virus was attenuated compared to the parental vaccine strain, it induced a robust serum antibody response and protected against homologous influenza virus challenge in a murine model of infection [87] . Subsequently, rNDV was tested as a vaccine vector for HPAIV having varying efficacy against H5 and H7 influenza virus infections in poultry [88] [89] [90] [91] [92] [93] [94] . These vaccines have the added benefit of potentially providing protection against both the influenza virus and NDV infection. NDV has also been explored as a vaccine vector for humans. Two NHP studies assessed the immunogenicity and efficacy of an rNDV expressing the HA or NA of A/Vietnam/1203/2004 (H5N1; VN1203) [95, 96] . Intranasal and intratracheal delivery of the rNDV-HA or rNDV-NA vaccines induced both serum and mucosal antibody responses and protected from HPAIV challenge [95, 96] . NDV has limited clinical data; however, phase I and phase I/II clinical trials have shown that the NDV vector is well-tolerated, even at high doses delivered intravenously [44, 97] . While these results are promising, additional studies are needed to advance NDV as a human vaccine vector for influenza. Parainfluenza virus type 5 (PIV5) is a paramyxovirus vaccine vector being explored for delivery of influenza and other infectious disease vaccine antigens. PIV5 has only recently been described as a vaccine vector [98] . Similar to other RNA viruses, PIV5 has a number of features that make it an attractive vaccine vector. For example, PIV5 has a stable RNA genome and no DNA phase in virus replication cycle reducing concerns of host genome integration or modification. PIV5 can be grown to very high titers in mammalian vaccine cell culture substrates and is not cytopathic allowing for extended culture and harvest of vaccine virus [98, 99] . Like NDV, PIV5 has a 3'-to 5' gradient of gene expression and insertion of transgenes at different locations in the genome can variably attenuate the virus and alter transgene expression [100] . PIV5 has broad tropism, infecting many cell types, tissues, and species without causing clinical disease, although PIV5 has been associated with -kennel cough‖ in dogs [99] . A reverse genetics system for PIV5 was first used to insert the HA gene from A/Udorn/307/72 (H3N2) into the PIV5 genome between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Similar to NDV, the HA was expressed at high levels in infected cells and replicated similarly to the wild type virus, and importantly, was not pathogenic in immunodeficient mice [98] . Additionally, a single intranasal immunization in a murine model of influenza infection was shown to induce neutralizing antibody responses and protect against a virus expressing homologous HA protein [98] . PIV5 has also been explored as a vaccine against HPAIV. Recombinant PIV5 vaccines expressing the HA or NP from VN1203 were tested for efficacy in a murine challenge model. Mice intranasally vaccinated with a single dose of PIV5-H5 vaccine had robust serum and mucosal antibody responses, and were protected from lethal challenge. Notably, although cellular immune responses appeared to contribute to protection, serum antibody was sufficient for protection from challenge [100, 101] . Intramuscular immunization with PIV5-H5 was also shown to be effective at inducing neutralizing antibody responses and protecting against lethal influenza virus challenge [101] . PIV5 expressing the NP protein of HPAIV was also efficacious in the murine immunization and challenge model, where a single intranasal immunization induced robust CD8 + T cell responses and protected against homologous (H5N1) and heterosubtypic (H1N1) virus challenge [102] . Currently there is no clinical safety data for use of PIV5 in humans. However, live PIV5 has been a component of veterinary vaccines for -kennel cough‖ for >30 years, and veterinarians and dog owners are exposed to live PIV5 without reported disease [99] . This combined with preclinical data from a variety of animal models suggests that PIV5 as a vector is likely to be safe in humans. As preexisting immunity is a concern for all virus-vectored vaccines, it should be noted that there is no data on the levels of preexisting immunity to PIV5 in humans. However, a study evaluating the efficacy of a PIV5-H3 vaccine in canines previously vaccinated against PIV5 (kennel cough) showed induction of robust anti-H3 serum antibody responses as well as high serum antibody levels to the PIV5 vaccine, suggesting preexisting immunity to the PIV5 vector may not affect immunogenicity of vaccines even with repeated use [99] . Poxvirus vaccines have a long history and the notable hallmark of being responsible for eradication of smallpox. The termination of the smallpox virus vaccination program has resulted in a large population of poxvirus-naï ve individuals that provides the opportunity for the use of poxviruses as vectors without preexisting immunity concerns [103] . Poxvirus-vectored vaccines were first proposed for use in 1982 with two reports of recombinant vaccinia viruses encoding and expressing functional thymidine kinase gene from herpes virus [104, 105] . Within a year, a vaccinia virus encoding the HA of an H2N2 virus was shown to express a functional HA protein (cleaved in the HA1 and HA2 subunits) and be immunogenic in rabbits and hamsters [106] . Subsequently, all ten of the primary influenza proteins have been expressed in vaccine virus [107] . Early work with intact vaccinia virus vectors raised safety concerns, as there was substantial reactogenicity that hindered recombinant vaccine development [108] . Two vaccinia vectors were developed to address these safety concerns. The modified vaccinia virus Ankara (MVA) strain was attenuated by passage 530 times in chick embryo fibroblasts cultures. The second, New York vaccinia virus (NYVAC) was a plaque-purified clone of the Copenhagen vaccine strain rationally attenuated by deletion of 18 open reading frames [109] [110] [111] . Modified vaccinia virus Ankara (MVA) was developed prior to smallpox eradication to reduce or prevent adverse effects of other smallpox vaccines [109] . Serial tissue culture passage of MVA resulted in loss of 15% of the genome, and established a growth restriction for avian cells. The defects affected late stages in virus assembly in non-avian cells, a feature enabling use of the vector as single-round expression vector in non-permissive hosts. Interestingly, over two decades ago, recombinant MVA expressing the HA and NP of influenza virus was shown to be effective against lethal influenza virus challenge in a murine model [112] . Subsequently, MVA expressing various antigens from seasonal, pandemic (A/California/04/2009, pH1N1), equine (A/Equine/Kentucky/1/81 H3N8), and HPAI (VN1203) viruses have been shown to be efficacious in murine, ferret, NHP, and equine challenge models [113] . MVA vaccines are very effective stimulators of both cellular and humoral immunity. For example, abortive infection provides native expression of the influenza antigens enabling robust antibody responses to native surface viral antigens. Concurrently, the intracellular influenza peptides expressed by the pox vector enter the class I MHC antigen processing and presentation pathway enabling induction of CD8 + T cell antiviral responses. MVA also induces CD4 + T cell responses further contributing to the magnitude of the antigen-specific effector functions [107, [112] [113] [114] [115] . MVA is also a potent activator of early innate immune responses further enhancing adaptive immune responses [116] . Between early smallpox vaccine development and more recent vaccine vector development, MVA has undergone extensive safety testing and shown to be attenuated in severely immunocompromised animals and safe for use in children, adults, elderly, and immunocompromised persons. With extensive pre-clinical data, recombinant MVA vaccines expressing influenza antigens have been tested in clinical trials and been shown to be safe and immunogenic in humans [117] [118] [119] . These results combined with data from other (non-influenza) clinical and pre-clinical studies support MVA as a leading viral-vectored candidate vaccine. The NYVAC vector is a highly attenuated vaccinia virus strain. NYVAC is replication-restricted; however, it grows in chick embryo fibroblasts and Vero cells enabling vaccine-scale production. In non-permissive cells, critical late structural proteins are not produced stopping replication at the immature virion stage [120] . NYVAC is very attenuated and considered safe for use in humans of all ages; however, it predominantly induces a CD4 + T cell response which is different compared to MVA [114] . Both MVA and NYVAC provoke robust humoral responses, and can be delivered mucosally to induce mucosal antibody responses [121] . There has been only limited exploration of NYVAC as a vaccine vector for influenza virus; however, a vaccine expressing the HA from A/chicken/Indonesia/7/2003 (H5N1) was shown to induce potent neutralizing antibody responses and protect against challenge in swine [122] . While there is strong safety and efficacy data for use of NYVAC or MVA-vectored influenza vaccines, preexisting immunity remains a concern. Although the smallpox vaccination campaign has resulted in a population of poxvirus-naï ve people, the initiation of an MVA or NYVAC vaccination program for HIV, influenza or other pathogens will rapidly reduce this susceptible population. While there is significant interest in development of pox-vectored influenza virus vaccines, current influenza vaccination strategies rely upon regular immunization with vaccines matched to circulating strains. This would likely limit the use and/or efficacy of poxvirus-vectored influenza virus vaccines for regular and seasonal use [13] . Intriguingly, NYVAC may have an advantage for use as an influenza vaccine vector, because immunization with this vector induces weaker vaccine-specific immune responses compared to other poxvirus vaccines, a feature that may address the concerns surrounding preexisting immunity [123] . While poxvirus-vectored vaccines have not yet been approved for use in humans, there is a growing list of licensed poxvirus for veterinary use that include fowlpox-and canarypox-vectored vaccines for avian and equine influenza viruses, respectively [124, 125] . The fowlpox-vectored vaccine expressing the avian influenza virus HA antigen has the added benefit of providing protection against fowlpox infection. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [126] . These poxvirus vectors have the potential for use as vaccine vectors in humans, similar to the first use of cowpox for vaccination against smallpox [127] . The availability of these non-human poxvirus vectors with extensive animal safety and efficacy data may address the issues with preexisting immunity to the human vaccine strains, although the cross-reactivity originally described with cowpox could also limit use. Influenza vaccines utilizing vesicular stomatitis virus (VSV), a rhabdovirus, as a vaccine vector have a number of advantages shared with other RNA virus vaccine vectors. Both live and replication-defective VSV vaccine vectors have been shown to be immunogenic [128, 129] , and like Paramyxoviridae, the Rhabdoviridae genome has a 3'-to-5' gradient of gene expression enabling attention by selective vaccine gene insertion or genome rearrangement [130] . VSV has a number of other advantages including broad tissue tropism, and the potential for intramuscular or intranasal immunization. The latter delivery method enables induction of mucosal immunity and elimination of needles required for vaccination. Also, there is little evidence of VSV seropositivity in humans eliminating concerns of preexisting immunity, although repeated use may be a concern. Also, VSV vaccine can be produced using existing mammalian vaccine manufacturing cell lines. Influenza antigens were first expressed in a VSV vector in 1997. Both the HA and NA were shown to be expressed as functional proteins and incorporated into the recombinant VSV particles [131] . Subsequently, VSV-HA, expressing the HA protein from A/WSN/1933 (H1N1) was shown to be immunogenic and protect mice from lethal influenza virus challenge [129] . To reduce safety concerns, attenuated VSV vectors were developed. One candidate vaccine had a truncated VSV G protein, while a second candidate was deficient in G protein expression and relied on G protein expressed by a helper vaccine cell line to the provide the virus receptor. Both vectors were found to be attenuated in mice, but maintained immunogenicity [128] . More recently, single-cycle replicating VSV vaccines have been tested for efficacy against H5N1 HPAIV. VSV vectors expressing the HA from A/Hong Kong/156/97 (H5N1) were shown to be immunogenic and induce cross-reactive antibody responses and protect against challenge with heterologous H5N1 challenge in murine and NHP models [132] [133] [134] . VSV vectors are not without potential concerns. VSV can cause disease in a number of species, including humans [135] . The virus is also potentially neuroinvasive in some species [136] , although NHP studies suggest this is not a concern in humans [137] . Also, while the incorporation of the influenza antigen in to the virion may provide some benefit in immunogenicity, changes in tropism or attenuation could arise from incorporation of different influenza glycoproteins. There is no evidence for this, however [134] . Currently, there is no human safety data for VSV-vectored vaccines. While experimental data is promising, additional work is needed before consideration for human influenza vaccination. Current influenza vaccines rely on matching the HA antigen of the vaccine with circulating strains to provide strain-specific neutralizing antibody responses [4, 14, 24] . There is significant interest in developing universal influenza vaccines that would not require annual reformulation to provide protective robust and durable immunity. These vaccines rely on generating focused immune responses to highly conserved portions of the virus that are refractory to mutation [30] [31] [32] . Traditional vaccines may not be suitable for these vaccination strategies; however, vectored vaccines that have the ability to be readily modified and to express transgenes are compatible for these applications. The NP and M2 proteins have been explored as universal vaccine antigens for decades. Early work with recombinant viral vectors demonstrated that immunization with vaccines expressing influenza antigens induced potent CD8 + T cell responses [107, [138] [139] [140] [141] . These responses, even to the HA antigen, could be cross-protective [138] . A number of studies have shown that immunization with NP expressed by AAV, rAd5, alphavirus vectors, MVA, or other vector systems induces potent CD8 + T cell responses and protects against influenza virus challenge [52, 63, 69, 102, 139, 142] . As the NP protein is highly conserved across influenza A viruses, NP-specific T cells can protect against heterologous and even heterosubtypic virus challenges [30] . The M2 protein is also highly conserved and expressed on the surface of infected cells, although to a lesser extent on the surface of virus particles [30] . Much of the vaccine work in this area has focused on virus-like or subunit particles expressing the M2 ectodomain; however, studies utilizing a DNA-prime, rAd-boost strategies to vaccinate against the entire M2 protein have shown the antigen to be immunogenic and protective [50] . In these studies, antibodies to the M2 protein protected against homologous and heterosubtypic challenge, including a H5N1 HPAIV challenge. More recently, NP and M2 have been combined to induce broadly cross-reactive CD8 + T cell and antibody responses, and rAd5 vaccines expressing these antigens have been shown to protect against pH1N1 and H5N1 challenges [29, 51] . Historically, the HA has not been widely considered as a universal vaccine antigen. However, the recent identification of virus neutralizing monoclonal antibodies that cross-react with many subtypes of influenza virus [143] has presented the opportunity to design vaccine antigens to prime focused antibody responses to the highly conserved regions recognized by these monoclonal antibodies. The majority of these broadly cross-reactive antibodies recognize regions on the stalk of the HA protein [143] . The HA stalk is generally less immunogenic compared to the globular head of the HA protein so most approaches have utilized -headless‖ HA proteins as immunogens. HA stalk vaccines have been designed using DNA and virus-like particles [144] and MVA [142] ; however, these approaches are amenable to expression in any of the viruses vectors described here. The goal of any vaccine is to protect against infection and disease, while inducing population-based immunity to reduce or eliminate virus transmission within the population. It is clear that currently licensed influenza vaccines have not fully met these goals, nor those specific to inducing long-term, robust immunity. There are a number of vaccine-related issues that must be addressed before population-based influenza vaccination strategies are optimized. The concept of a -one size fits all‖ vaccine needs to be updated, given the recent ability to probe the virus-host interface through RNA interference approaches that facilitate the identification of host genes affecting virus replication, immunity, and disease. There is also a need for revision of the current influenza virus vaccine strategies for at-risk populations, particularly those at either end of the age spectrum. An example of an improved vaccine regime might include the use of a vectored influenza virus vaccine that expresses the HA, NA and M and/or NP proteins for the two currently circulating influenza A subtypes and both influenza B strains so that vaccine take and vaccine antigen levels are not an issue in inducing protective immunity. Recombinant live-attenuated or replication-deficient influenza viruses may offer an advantage for this and other approaches. Vectored vaccines can be constructed to express full-length influenza virus proteins, as well as generate conformationally restricted epitopes, features critical in generating appropriate humoral protection. Inclusion of internal influenza antigens in a vectored vaccine can also induce high levels of protective cellular immunity. To generate sustained immunity, it is an advantage to induce immunity at sites of inductive immunity to natural infection, in this case the respiratory tract. Several vectored vaccines target the respiratory tract. Typically, vectored vaccines generate antigen for weeks after immunization, in contrast to subunit vaccination. This increased presence and level of vaccine antigen contributes to and helps sustain a durable memory immune response, even augmenting the selection of higher affinity antibody secreting cells. The enhanced memory response is in part linked to the intrinsic augmentation of immunity induced by the vector. Thus, for weaker antigens typical of HA, vectored vaccines have the capacity to overcome real limitations in achieving robust and durable protection. Meeting the mandates of seasonal influenza vaccine development is difficult, and to respond to a pandemic strain is even more challenging. Issues with influenza vaccine strain selection based on recently circulating viruses often reflect recommendations by the World Health Organization (WHO)-a process that is cumbersome. The strains of influenza A viruses to be used in vaccine manufacture are not wild-type viruses but rather reassortants that are hybrid viruses containing at least the HA and NA gene segments from the target strains and other gene segments from the master strain, PR8, which has properties of high growth in fertilized hen's eggs. This additional process requires more time and quality control, and specifically for HPAI viruses, it is a process that may fail because of the nature of those viruses. In contrast, viral-vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. There are several viral-based vectors currently employed as antigen delivery systems, including poxviruses, adenoviruses baculovirus, paramyxovirus, rhabdovirus, and others; however, the majority of human clinical trials assessing viral-vectored influenza vaccines use poxvirus and adenovirus vectors. While each of these vector approaches has unique features and is in different stages of development, the combined successes of these approaches supports the virus-vectored vaccine approach as a whole. Issues such as preexisting immunity and cold chain requirements, and lingering safety concerns will have to be overcome; however, each approach is making progress in addressing these issues, and all of the approaches are still viable. Virus-vectored vaccines hold particular promise for vaccination with universal or focused antigens where traditional vaccination methods are not suited to efficacious delivery of these antigens. The most promising approaches currently in development are arguably those targeting conserved HA stalk region epitopes. Given the findings to date, virus-vectored vaccines hold great promise and may overcome the current limitations of influenza vaccines.
What are alphaviruses?
false
1,564
{ "text": [ "positive-sense, single-stranded RNA viruses of the Togaviridae family" ], "answer_start": [ 16320 ] }
1,578
Inhibitory Effect and Possible Mechanism of Action of Patchouli Alcohol against Influenza A (H2N2) Virus https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264369/ SHA: f2d842780b9928cc70f38a4458553f2431877603 Authors: Wu, Huaxing; Li, Beili; Wang, Xue; Jin, Mingyuan; Wang, Guonian Date: 2011-08-03 DOI: 10.3390/molecules16086489 License: cc-by Abstract: In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, "Asian Influenza (1957/H2N2)" and "Hong Kong Influenza (1968/H3N2)" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] . The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] . Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity. Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. Cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell). As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol. To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light. MDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 . The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C. The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data. Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves. Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates. Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee. Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days. The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules [40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories. All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant. In conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view.
What method was used to measure the inhibition of viral replication?
false
4,075
{ "text": [ "MTT method" ], "answer_start": [ 13705 ] }
2,504
Respiratory Viral Infections in Exacerbation of Chronic Airway Inflammatory Diseases: Novel Mechanisms and Insights From the Upper Airway Epithelium https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052386/ SHA: 45a566c71056ba4faab425b4f7e9edee6320e4a4 Authors: Tan, Kai Sen; Lim, Rachel Liyu; Liu, Jing; Ong, Hsiao Hui; Tan, Vivian Jiayi; Lim, Hui Fang; Chung, Kian Fan; Adcock, Ian M.; Chow, Vincent T.; Wang, De Yun Date: 2020-02-25 DOI: 10.3389/fcell.2020.00099 License: cc-by Abstract: Respiratory virus infection is one of the major sources of exacerbation of chronic airway inflammatory diseases. These exacerbations are associated with high morbidity and even mortality worldwide. The current understanding on viral-induced exacerbations is that viral infection increases airway inflammation which aggravates disease symptoms. Recent advances in in vitro air-liquid interface 3D cultures, organoid cultures and the use of novel human and animal challenge models have evoked new understandings as to the mechanisms of viral exacerbations. In this review, we will focus on recent novel findings that elucidate how respiratory viral infections alter the epithelial barrier in the airways, the upper airway microbial environment, epigenetic modifications including miRNA modulation, and other changes in immune responses throughout the upper and lower airways. First, we reviewed the prevalence of different respiratory viral infections in causing exacerbations in chronic airway inflammatory diseases. Subsequently we also summarized how recent models have expanded our appreciation of the mechanisms of viral-induced exacerbations. Further we highlighted the importance of the virome within the airway microbiome environment and its impact on subsequent bacterial infection. This review consolidates the understanding of viral induced exacerbation in chronic airway inflammatory diseases and indicates pathways that may be targeted for more effective management of chronic inflammatory diseases. Text: The prevalence of chronic airway inflammatory disease is increasing worldwide especially in developed nations (GBD 2015 Chronic Respiratory Disease Collaborators, 2017 Guan et al., 2018) . This disease is characterized by airway inflammation leading to complications such as coughing, wheezing and shortness of breath. The disease can manifest in both the upper airway (such as chronic rhinosinusitis, CRS) and lower airway (such as asthma and chronic obstructive pulmonary disease, COPD) which greatly affect the patients' quality of life (Calus et al., 2012; Bao et al., 2015) . Treatment and management vary greatly in efficacy due to the complexity and heterogeneity of the disease. This is further complicated by the effect of episodic exacerbations of the disease, defined as worsening of disease symptoms including wheeze, cough, breathlessness and chest tightness (Xepapadaki and Papadopoulos, 2010) . Such exacerbations are due to the effect of enhanced acute airway inflammation impacting upon and worsening the symptoms of the existing disease (Hashimoto et al., 2008; Viniol and Vogelmeier, 2018) . These acute exacerbations are the main cause of morbidity and sometimes mortality in patients, as well as resulting in major economic burdens worldwide. However, due to the complex interactions between the host and the exacerbation agents, the mechanisms of exacerbation may vary considerably in different individuals under various triggers. Acute exacerbations are usually due to the presence of environmental factors such as allergens, pollutants, smoke, cold or dry air and pathogenic microbes in the airway (Gautier and Charpin, 2017; Viniol and Vogelmeier, 2018) . These agents elicit an immune response leading to infiltration of activated immune cells that further release inflammatory mediators that cause acute symptoms such as increased mucus production, cough, wheeze and shortness of breath. Among these agents, viral infection is one of the major drivers of asthma exacerbations accounting for up to 80-90% and 45-80% of exacerbations in children and adults respectively (Grissell et al., 2005; Xepapadaki and Papadopoulos, 2010; Jartti and Gern, 2017; Adeli et al., 2019) . Viral involvement in COPD exacerbation is also equally high, having been detected in 30-80% of acute COPD exacerbations (Kherad et al., 2010; Jafarinejad et al., 2017; Stolz et al., 2019) . Whilst the prevalence of viral exacerbations in CRS is still unclear, its prevalence is likely to be high due to the similar inflammatory nature of these diseases (Rowan et al., 2015; Tan et al., 2017) . One of the reasons for the involvement of respiratory viruses' in exacerbations is their ease of transmission and infection (Kutter et al., 2018) . In addition, the high diversity of the respiratory viruses may also contribute to exacerbations of different nature and severity (Busse et al., 2010; Costa et al., 2014; Jartti and Gern, 2017) . Hence, it is important to identify the exact mechanisms underpinning viral exacerbations in susceptible subjects in order to properly manage exacerbations via supplementary treatments that may alleviate the exacerbation symptoms or prevent severe exacerbations. While the lower airway is the site of dysregulated inflammation in most chronic airway inflammatory diseases, the upper airway remains the first point of contact with sources of exacerbation. Therefore, their interaction with the exacerbation agents may directly contribute to the subsequent responses in the lower airway, in line with the "United Airway" hypothesis. To elucidate the host airway interaction with viruses leading to exacerbations, we thus focus our review on recent findings of viral interaction with the upper airway. We compiled how viral induced changes to the upper airway may contribute to chronic airway inflammatory disease exacerbations, to provide a unified elucidation of the potential exacerbation mechanisms initiated from predominantly upper airway infections. Despite being a major cause of exacerbation, reports linking respiratory viruses to acute exacerbations only start to emerge in the late 1950s (Pattemore et al., 1992) ; with bacterial infections previously considered as the likely culprit for acute exacerbation (Stevens, 1953; Message and Johnston, 2002) . However, with the advent of PCR technology, more viruses were recovered during acute exacerbations events and reports implicating their role emerged in the late 1980s (Message and Johnston, 2002) . Rhinovirus (RV) and respiratory syncytial virus (RSV) are the predominant viruses linked to the development and exacerbation of chronic airway inflammatory diseases (Jartti and Gern, 2017) . Other viruses such as parainfluenza virus (PIV), influenza virus (IFV) and adenovirus (AdV) have also been implicated in acute exacerbations but to a much lesser extent (Johnston et al., 2005; Oliver et al., 2014; Ko et al., 2019) . More recently, other viruses including bocavirus (BoV), human metapneumovirus (HMPV), certain coronavirus (CoV) strains, a specific enterovirus (EV) strain EV-D68, human cytomegalovirus (hCMV) and herpes simplex virus (HSV) have been reported as contributing to acute exacerbations . The common feature these viruses share is that they can infect both the upper and/or lower airway, further increasing the inflammatory conditions in the diseased airway (Mallia and Johnston, 2006; Britto et al., 2017) . Respiratory viruses primarily infect and replicate within airway epithelial cells . During the replication process, the cells release antiviral factors and cytokines that alter local airway inflammation and airway niche (Busse et al., 2010) . In a healthy airway, the inflammation normally leads to type 1 inflammatory responses consisting of activation of an antiviral state and infiltration of antiviral effector cells. This eventually results in the resolution of the inflammatory response and clearance of the viral infection (Vareille et al., 2011; Braciale et al., 2012) . However, in a chronically inflamed airway, the responses against the virus may be impaired or aberrant, causing sustained inflammation and erroneous infiltration, resulting in the exacerbation of their symptoms (Mallia and Johnston, 2006; Dougherty and Fahy, 2009; Busse et al., 2010; Britto et al., 2017; Linden et al., 2019) . This is usually further compounded by the increased susceptibility of chronic airway inflammatory disease patients toward viral respiratory infections, thereby increasing the frequency of exacerbation as a whole (Dougherty and Fahy, 2009; Busse et al., 2010; Linden et al., 2019) . Furthermore, due to the different replication cycles and response against the myriad of respiratory viruses, each respiratory virus may also contribute to exacerbations via different mechanisms that may alter their severity. Hence, this review will focus on compiling and collating the current known mechanisms of viral-induced exacerbation of chronic airway inflammatory diseases; as well as linking the different viral infection pathogenesis to elucidate other potential ways the infection can exacerbate the disease. The review will serve to provide further understanding of viral induced exacerbation to identify potential pathways and pathogenesis mechanisms that may be targeted as supplementary care for management and prevention of exacerbation. Such an approach may be clinically significant due to the current scarcity of antiviral drugs for the management of viral-induced exacerbations. This will improve the quality of life of patients with chronic airway inflammatory diseases. Once the link between viral infection and acute exacerbations of chronic airway inflammatory disease was established, there have been many reports on the mechanisms underlying the exacerbation induced by respiratory viral infection. Upon infecting the host, viruses evoke an inflammatory response as a means of counteracting the infection. Generally, infected airway epithelial cells release type I (IFNα/β) and type III (IFNλ) interferons, cytokines and chemokines such as IL-6, IL-8, IL-12, RANTES, macrophage inflammatory protein 1α (MIP-1α) and monocyte chemotactic protein 1 (MCP-1) (Wark and Gibson, 2006; Matsukura et al., 2013) . These, in turn, enable infiltration of innate immune cells and of professional antigen presenting cells (APCs) that will then in turn release specific mediators to facilitate viral targeting and clearance, including type II interferon (IFNγ), IL-2, IL-4, IL-5, IL-9, and IL-12 (Wark and Gibson, 2006; Singh et al., 2010; Braciale et al., 2012) . These factors heighten local inflammation and the infiltration of granulocytes, T-cells and B-cells (Wark and Gibson, 2006; Braciale et al., 2012) . The increased inflammation, in turn, worsens the symptoms of airway diseases. Additionally, in patients with asthma and patients with CRS with nasal polyp (CRSwNP), viral infections such as RV and RSV promote a Type 2-biased immune response (Becker, 2006; Jackson et al., 2014; Jurak et al., 2018) . This amplifies the basal type 2 inflammation resulting in a greater release of IL-4, IL-5, IL-13, RANTES and eotaxin and a further increase in eosinophilia, a key pathological driver of asthma and CRSwNP (Wark and Gibson, 2006; Singh et al., 2010; Chung et al., 2015; Dunican and Fahy, 2015) . Increased eosinophilia, in turn, worsens the classical symptoms of disease and may further lead to life-threatening conditions due to breathing difficulties. On the other hand, patients with COPD and patients with CRS without nasal polyp (CRSsNP) are more neutrophilic in nature due to the expression of neutrophil chemoattractants such as CXCL9, CXCL10, and CXCL11 (Cukic et al., 2012; Brightling and Greening, 2019) . The pathology of these airway diseases is characterized by airway remodeling due to the presence of remodeling factors such as matrix metalloproteinases (MMPs) released from infiltrating neutrophils (Linden et al., 2019) . Viral infections in such conditions will then cause increase neutrophilic activation; worsening the symptoms and airway remodeling in the airway thereby exacerbating COPD, CRSsNP and even CRSwNP in certain cases (Wang et al., 2009; Tacon et al., 2010; Linden et al., 2019) . An epithelial-centric alarmin pathway around IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), and their interaction with group 2 innate lymphoid cells (ILC2) has also recently been identified (Nagarkar et al., 2012; Hong et al., 2018; Allinne et al., 2019) . IL-25, IL-33 and TSLP are type 2 inflammatory cytokines expressed by the epithelial cells upon injury to the epithelial barrier (Gabryelska et al., 2019; Roan et al., 2019) . ILC2s are a group of lymphoid cells lacking both B and T cell receptors but play a crucial role in secreting type 2 cytokines to perpetuate type 2 inflammation when activated (Scanlon and McKenzie, 2012; Li and Hendriks, 2013) . In the event of viral infection, cell death and injury to the epithelial barrier will also induce the expression of IL-25, IL-33 and TSLP, with heighten expression in an inflamed airway (Allakhverdi et al., 2007; Goldsmith et al., 2012; Byers et al., 2013; Shaw et al., 2013; Beale et al., 2014; Jackson et al., 2014; Uller and Persson, 2018; Ravanetti et al., 2019) . These 3 cytokines then work in concert to activate ILC2s to further secrete type 2 cytokines IL-4, IL-5, and IL-13 which further aggravate the type 2 inflammation in the airway causing acute exacerbation (Camelo et al., 2017) . In the case of COPD, increased ILC2 activation, which retain the capability of differentiating to ILC1, may also further augment the neutrophilic response and further aggravate the exacerbation (Silver et al., 2016) . Interestingly, these factors are not released to any great extent and do not activate an ILC2 response during viral infection in healthy individuals (Yan et al., 2016; Tan et al., 2018a) ; despite augmenting a type 2 exacerbation in chronically inflamed airways (Jurak et al., 2018) . These classical mechanisms of viral induced acute exacerbations are summarized in Figure 1 . As integration of the virology, microbiology and immunology of viral infection becomes more interlinked, additional factors and FIGURE 1 | Current understanding of viral induced exacerbation of chronic airway inflammatory diseases. Upon virus infection in the airway, antiviral state will be activated to clear the invading pathogen from the airway. Immune response and injury factors released from the infected epithelium normally would induce a rapid type 1 immunity that facilitates viral clearance. However, in the inflamed airway, the cytokines and chemokines released instead augmented the inflammation present in the chronically inflamed airway, strengthening the neutrophilic infiltration in COPD airway, and eosinophilic infiltration in the asthmatic airway. The effect is also further compounded by the participation of Th1 and ILC1 cells in the COPD airway; and Th2 and ILC2 cells in the asthmatic airway. Frontiers in Cell and Developmental Biology | www.frontiersin.org mechanisms have been implicated in acute exacerbations during and after viral infection (Murray et al., 2006) . Murray et al. (2006) has underlined the synergistic effect of viral infection with other sensitizing agents in causing more severe acute exacerbations in the airway. This is especially true when not all exacerbation events occurred during the viral infection but may also occur well after viral clearance (Kim et al., 2008; Stolz et al., 2019) in particular the late onset of a bacterial infection (Singanayagam et al., 2018 (Singanayagam et al., , 2019a . In addition, viruses do not need to directly infect the lower airway to cause an acute exacerbation, as the nasal epithelium remains the primary site of most infections. Moreover, not all viral infections of the airway will lead to acute exacerbations, suggesting a more complex interplay between the virus and upper airway epithelium which synergize with the local airway environment in line with the "united airway" hypothesis (Kurai et al., 2013) . On the other hand, viral infections or their components persist in patients with chronic airway inflammatory disease (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Hence, their presence may further alter the local environment and contribute to current and future exacerbations. Future studies should be performed using metagenomics in addition to PCR analysis to determine the contribution of the microbiome and mycobiome to viral infections. In this review, we highlight recent data regarding viral interactions with the airway epithelium that could also contribute to, or further aggravate, acute exacerbations of chronic airway inflammatory diseases. Patients with chronic airway inflammatory diseases have impaired or reduced ability of viral clearance (Hammond et al., 2015; McKendry et al., 2016; Akbarshahi et al., 2018; Gill et al., 2018; Wang et al., 2018; Singanayagam et al., 2019b) . Their impairment stems from a type 2-skewed inflammatory response which deprives the airway of important type 1 responsive CD8 cells that are responsible for the complete clearance of virusinfected cells (Becker, 2006; McKendry et al., 2016) . This is especially evident in weak type 1 inflammation-inducing viruses such as RV and RSV (Kling et al., 2005; Wood et al., 2011; Ravi et al., 2019) . Additionally, there are also evidence of reduced type I (IFNβ) and III (IFNλ) interferon production due to type 2-skewed inflammation, which contributes to imperfect clearance of the virus resulting in persistence of viral components, or the live virus in the airway epithelium (Contoli et al., 2006; Hwang et al., 2019; Wark, 2019) . Due to the viral components remaining in the airway, antiviral genes such as type I interferons, inflammasome activating factors and cytokines remained activated resulting in prolong airway inflammation (Wood et al., 2011; Essaidi-Laziosi et al., 2018) . These factors enhance granulocyte infiltration thus prolonging the exacerbation symptoms. Such persistent inflammation may also be found within DNA viruses such as AdV, hCMV and HSV, whose infections generally persist longer (Imperiale and Jiang, 2015) , further contributing to chronic activation of inflammation when they infect the airway (Yang et al., 2008; Morimoto et al., 2009; Imperiale and Jiang, 2015; Lan et al., 2016; Tan et al., 2016; Kowalski et al., 2017) . With that note, human papilloma virus (HPV), a DNA virus highly associated with head and neck cancers and respiratory papillomatosis, is also linked with the chronic inflammation that precedes the malignancies (de Visser et al., 2005; Gillison et al., 2012; Bonomi et al., 2014; Fernandes et al., 2015) . Therefore, the role of HPV infection in causing chronic inflammation in the airway and their association to exacerbations of chronic airway inflammatory diseases, which is scarcely explored, should be investigated in the future. Furthermore, viral persistence which lead to continuous expression of antiviral genes may also lead to the development of steroid resistance, which is seen with RV, RSV, and PIV infection (Chi et al., 2011; Ford et al., 2013; Papi et al., 2013) . The use of steroid to suppress the inflammation may also cause the virus to linger longer in the airway due to the lack of antiviral clearance (Kim et al., 2008; Hammond et al., 2015; Hewitt et al., 2016; McKendry et al., 2016; Singanayagam et al., 2019b) . The concomitant development of steroid resistance together with recurring or prolong viral infection thus added considerable burden to the management of acute exacerbation, which should be the future focus of research to resolve the dual complications arising from viral infection. On the other end of the spectrum, viruses that induce strong type 1 inflammation and cell death such as IFV (Yan et al., 2016; Guibas et al., 2018) and certain CoV (including the recently emerged COVID-19 virus) (Tao et al., 2013; Yue et al., 2018; Zhu et al., 2020) , may not cause prolonged inflammation due to strong induction of antiviral clearance. These infections, however, cause massive damage and cell death to the epithelial barrier, so much so that areas of the epithelium may be completely absent post infection (Yan et al., 2016; Tan et al., 2019) . Factors such as RANTES and CXCL10, which recruit immune cells to induce apoptosis, are strongly induced from IFV infected epithelium (Ampomah et al., 2018; Tan et al., 2019) . Additionally, necroptotic factors such as RIP3 further compounds the cell deaths in IFV infected epithelium . The massive cell death induced may result in worsening of the acute exacerbation due to the release of their cellular content into the airway, further evoking an inflammatory response in the airway (Guibas et al., 2018) . Moreover, the destruction of the epithelial barrier may cause further contact with other pathogens and allergens in the airway which may then prolong exacerbations or results in new exacerbations. Epithelial destruction may also promote further epithelial remodeling during its regeneration as viral infection induces the expression of remodeling genes such as MMPs and growth factors . Infections that cause massive destruction of the epithelium, such as IFV, usually result in severe acute exacerbations with non-classical symptoms of chronic airway inflammatory diseases. Fortunately, annual vaccines are available to prevent IFV infections (Vasileiou et al., 2017; Zheng et al., 2018) ; and it is recommended that patients with chronic airway inflammatory disease receive their annual influenza vaccination as the best means to prevent severe IFV induced exacerbation. Another mechanism that viral infections may use to drive acute exacerbations is the induction of vasodilation or tight junction opening factors which may increase the rate of infiltration. Infection with a multitude of respiratory viruses causes disruption of tight junctions with the resulting increased rate of viral infiltration. This also increases the chances of allergens coming into contact with airway immune cells. For example, IFV infection was found to induce oncostatin M (OSM) which causes tight junction opening (Pothoven et al., 2015; Tian et al., 2018) . Similarly, RV and RSV infections usually cause tight junction opening which may also increase the infiltration rate of eosinophils and thus worsening of the classical symptoms of chronic airway inflammatory diseases (Sajjan et al., 2008; Kast et al., 2017; Kim et al., 2018) . In addition, the expression of vasodilating factors and fluid homeostatic factors such as angiopoietin-like 4 (ANGPTL4) and bactericidal/permeabilityincreasing fold-containing family member A1 (BPIFA1) are also associated with viral infections and pneumonia development, which may worsen inflammation in the lower airway Akram et al., 2018) . These factors may serve as targets to prevent viral-induced exacerbations during the management of acute exacerbation of chronic airway inflammatory diseases. Another recent area of interest is the relationship between asthma and COPD exacerbations and their association with the airway microbiome. The development of chronic airway inflammatory diseases is usually linked to specific bacterial species in the microbiome which may thrive in the inflamed airway environment (Diver et al., 2019) . In the event of a viral infection such as RV infection, the effect induced by the virus may destabilize the equilibrium of the microbiome present (Molyneaux et al., 2013; Kloepfer et al., 2014; Kloepfer et al., 2017; Jubinville et al., 2018; van Rijn et al., 2019) . In addition, viral infection may disrupt biofilm colonies in the upper airway (e.g., Streptococcus pneumoniae) microbiome to be release into the lower airway and worsening the inflammation (Marks et al., 2013; Chao et al., 2014) . Moreover, a viral infection may also alter the nutrient profile in the airway through release of previously inaccessible nutrients that will alter bacterial growth (Siegel et al., 2014; Mallia et al., 2018) . Furthermore, the destabilization is further compounded by impaired bacterial immune response, either from direct viral influences, or use of corticosteroids to suppress the exacerbation symptoms (Singanayagam et al., 2018 (Singanayagam et al., , 2019a Wang et al., 2018; Finney et al., 2019) . All these may gradually lead to more far reaching effect when normal flora is replaced with opportunistic pathogens, altering the inflammatory profiles (Teo et al., 2018) . These changes may in turn result in more severe and frequent acute exacerbations due to the interplay between virus and pathogenic bacteria in exacerbating chronic airway inflammatory diseases (Wark et al., 2013; Singanayagam et al., 2018) . To counteract these effects, microbiome-based therapies are in their infancy but have shown efficacy in the treatments of irritable bowel syndrome by restoring the intestinal microbiome (Bakken et al., 2011) . Further research can be done similarly for the airway microbiome to be able to restore the microbiome following disruption by a viral infection. Viral infections can cause the disruption of mucociliary function, an important component of the epithelial barrier. Ciliary proteins FIGURE 2 | Changes in the upper airway epithelium contributing to viral exacerbation in chronic airway inflammatory diseases. The upper airway epithelium is the primary contact/infection site of most respiratory viruses. Therefore, its infection by respiratory viruses may have far reaching consequences in augmenting and synergizing current and future acute exacerbations. The destruction of epithelial barrier, mucociliary function and cell death of the epithelial cells serves to increase contact between environmental triggers with the lower airway and resident immune cells. The opening of tight junction increasing the leakiness further augments the inflammation and exacerbations. In addition, viral infections are usually accompanied with oxidative stress which will further increase the local inflammation in the airway. The dysregulation of inflammation can be further compounded by modulation of miRNAs and epigenetic modification such as DNA methylation and histone modifications that promote dysregulation in inflammation. Finally, the change in the local airway environment and inflammation promotes growth of pathogenic bacteria that may replace the airway microbiome. Furthermore, the inflammatory environment may also disperse upper airway commensals into the lower airway, further causing inflammation and alteration of the lower airway environment, resulting in prolong exacerbation episodes following viral infection. Viral specific trait contributing to exacerbation mechanism (with literature evidence) Oxidative stress ROS production (RV, RSV, IFV, HSV) As RV, RSV, and IFV were the most frequently studied viruses in chronic airway inflammatory diseases, most of the viruses listed are predominantly these viruses. However, the mechanisms stated here may also be applicable to other viruses but may not be listed as they were not implicated in the context of chronic airway inflammatory diseases exacerbation (see text for abbreviations). that aid in the proper function of the motile cilia in the airways are aberrantly expressed in ciliated airway epithelial cells which are the major target for RV infection (Griggs et al., 2017) . Such form of secondary cilia dyskinesia appears to be present with chronic inflammations in the airway, but the exact mechanisms are still unknown (Peng et al., , 2019 Qiu et al., 2018) . Nevertheless, it was found that in viral infection such as IFV, there can be a change in the metabolism of the cells as well as alteration in the ciliary gene expression, mostly in the form of down-regulation of the genes such as dynein axonemal heavy chain 5 (DNAH5) and multiciliate differentiation And DNA synthesis associated cell cycle protein (MCIDAS) (Tan et al., 2018b . The recently emerged Wuhan CoV was also found to reduce ciliary beating in infected airway epithelial cell model (Zhu et al., 2020) . Furthermore, viral infections such as RSV was shown to directly destroy the cilia of the ciliated cells and almost all respiratory viruses infect the ciliated cells (Jumat et al., 2015; Yan et al., 2016; Tan et al., 2018a) . In addition, mucus overproduction may also disrupt the equilibrium of the mucociliary function following viral infection, resulting in symptoms of acute exacerbation (Zhu et al., 2009) . Hence, the disruption of the ciliary movement during viral infection may cause more foreign material and allergen to enter the airway, aggravating the symptoms of acute exacerbation and making it more difficult to manage. The mechanism of the occurrence of secondary cilia dyskinesia can also therefore be explored as a means to limit the effects of viral induced acute exacerbation. MicroRNAs (miRNAs) are short non-coding RNAs involved in post-transcriptional modulation of biological processes, and implicated in a number of diseases (Tan et al., 2014) . miRNAs are found to be induced by viral infections and may play a role in the modulation of antiviral responses and inflammation (Gutierrez et al., 2016; Deng et al., 2017; Feng et al., 2018) . In the case of chronic airway inflammatory diseases, circulating miRNA changes were found to be linked to exacerbation of the diseases (Wardzynska et al., 2020) . Therefore, it is likely that such miRNA changes originated from the infected epithelium and responding immune cells, which may serve to further dysregulate airway inflammation leading to exacerbations. Both IFV and RSV infections has been shown to increase miR-21 and augmented inflammation in experimental murine asthma models, which is reversed with a combination treatment of anti-miR-21 and corticosteroids (Kim et al., 2017) . IFV infection is also shown to increase miR-125a and b, and miR-132 in COPD epithelium which inhibits A20 and MAVS; and p300 and IRF3, respectively, resulting in increased susceptibility to viral infections (Hsu et al., 2016 (Hsu et al., , 2017 . Conversely, miR-22 was shown to be suppressed in asthmatic epithelium in IFV infection which lead to aberrant epithelial response, contributing to exacerbations (Moheimani et al., 2018) . Other than these direct evidence of miRNA changes in contributing to exacerbations, an increased number of miRNAs and other non-coding RNAs responsible for immune modulation are found to be altered following viral infections (Globinska et al., 2014; Feng et al., 2018; Hasegawa et al., 2018) . Hence non-coding RNAs also presents as targets to modulate viral induced airway changes as a means of managing exacerbation of chronic airway inflammatory diseases. Other than miRNA modulation, other epigenetic modification such as DNA methylation may also play a role in exacerbation of chronic airway inflammatory diseases. Recent epigenetic studies have indicated the association of epigenetic modification and chronic airway inflammatory diseases, and that the nasal methylome was shown to be a sensitive marker for airway inflammatory changes (Cardenas et al., 2019; Gomez, 2019) . At the same time, it was also shown that viral infections such as RV and RSV alters DNA methylation and histone modifications in the airway epithelium which may alter inflammatory responses, driving chronic airway inflammatory diseases and exacerbations (McErlean et al., 2014; Pech et al., 2018; Caixia et al., 2019) . In addition, Spalluto et al. (2017) also showed that antiviral factors such as IFNγ epigenetically modifies the viral resistance of epithelial cells. Hence, this may indicate that infections such as RV and RSV that weakly induce antiviral responses may result in an altered inflammatory state contributing to further viral persistence and exacerbation of chronic airway inflammatory diseases (Spalluto et al., 2017) . Finally, viral infection can result in enhanced production of reactive oxygen species (ROS), oxidative stress and mitochondrial dysfunction in the airway epithelium (Kim et al., 2018; Mishra et al., 2018; Wang et al., 2018) . The airway epithelium of patients with chronic airway inflammatory diseases are usually under a state of constant oxidative stress which sustains the inflammation in the airway (Barnes, 2017; van der Vliet et al., 2018) . Viral infections of the respiratory epithelium by viruses such as IFV, RV, RSV and HSV may trigger the further production of ROS as an antiviral mechanism Aizawa et al., 2018; Wang et al., 2018) . Moreover, infiltrating cells in response to the infection such as neutrophils will also trigger respiratory burst as a means of increasing the ROS in the infected region. The increased ROS and oxidative stress in the local environment may serve as a trigger to promote inflammation thereby aggravating the inflammation in the airway (Tiwari et al., 2002) . A summary of potential exacerbation mechanisms and the associated viruses is shown in Figure 2 and Table 1 . While the mechanisms underlying the development and acute exacerbation of chronic airway inflammatory disease is extensively studied for ways to manage and control the disease, a viral infection does more than just causing an acute exacerbation in these patients. A viral-induced acute exacerbation not only induced and worsens the symptoms of the disease, but also may alter the management of the disease or confer resistance toward treatments that worked before. Hence, appreciation of the mechanisms of viral-induced acute exacerbations is of clinical significance to devise strategies to correct viral induce changes that may worsen chronic airway inflammatory disease symptoms. Further studies in natural exacerbations and in viral-challenge models using RNA-sequencing (RNA-seq) or single cell RNA-seq on a range of time-points may provide important information regarding viral pathogenesis and changes induced within the airway of chronic airway inflammatory disease patients to identify novel targets and pathway for improved management of the disease. Subsequent analysis of functions may use epithelial cell models such as the air-liquid interface, in vitro airway epithelial model that has been adapted to studying viral infection and the changes it induced in the airway (Yan et al., 2016; Boda et al., 2018; Tan et al., 2018a) . Animal-based diseased models have also been developed to identify systemic mechanisms of acute exacerbation (Shin, 2016; Gubernatorova et al., 2019; Tanner and Single, 2019) . Furthermore, the humanized mouse model that possess human immune cells may also serves to unravel the immune profile of a viral infection in healthy and diseased condition (Ito et al., 2019; Li and Di Santo, 2019) . For milder viruses, controlled in vivo human infections can be performed for the best mode of verification of the associations of the virus with the proposed mechanism of viral induced acute exacerbations . With the advent of suitable diseased models, the verification of the mechanisms will then provide the necessary continuation of improving the management of viral induced acute exacerbations. In conclusion, viral-induced acute exacerbation of chronic airway inflammatory disease is a significant health and economic burden that needs to be addressed urgently. In view of the scarcity of antiviral-based preventative measures available for only a few viruses and vaccines that are only available for IFV infections, more alternative measures should be explored to improve the management of the disease. Alternative measures targeting novel viral-induced acute exacerbation mechanisms, especially in the upper airway, can serve as supplementary treatments of the currently available management strategies to augment their efficacy. New models including primary human bronchial or nasal epithelial cell cultures, organoids or precision cut lung slices from patients with airways disease rather than healthy subjects can be utilized to define exacerbation mechanisms. These mechanisms can then be validated in small clinical trials in patients with asthma or COPD. Having multiple means of treatment may also reduce the problems that arise from resistance development toward a specific treatment.
Where do the respiratory viruses primarily infect and replicate?
false
3,886
{ "text": [ "within airway epithelial cells" ], "answer_start": [ 7487 ] }
1,689
Chikungunya: A Potentially Emerging Epidemic? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860491/ SHA: f7c3160bef4169d29e2a8bdd79dd6e9056d4774c Authors: Thiboutot, Michelle M.; Kannan, Senthil; Kawalekar, Omkar U.; Shedlock, Devon J.; Khan, Amir S.; Sarangan, Gopalsamy; Srikanth, Padma; Weiner, David B.; Muthumani, Karuppiah Date: 2010-04-27 DOI: 10.1371/journal.pntd.0000623 License: cc-by Abstract: Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts. Text: Chikungunya virus (CHIKV), a mosquito-borne pathogen listed by National Institute of Allergy and Infectious Diseases (NIAID) as a Category C Priority Pathogen that causes Chikungunya fever (CHIKF), has been spreading throughout Asia, Africa, and parts of Europe in recent times [1, 2, 3] . CHIKV is an arthropod-borne virus (arbovirus) and is transmitted to humans primarily by Aedes aegypti, the infamous yellow fever propagator [4, 5] . CHIKV infection is marked by severe joint pain, contorting its victims into unusual postures [6] . The disease gets its name from the Kimakonde vernacular language of Tanzania and Mozambique, and the word chikungunya means ''that which contorts or bends up'' and translates in Swahili to ''the illness of the bended walker'' [7, 8, 9] . In Africa, CHIKV is maintained in a sylvatic cycle among forest-dwelling Aedes spp. mosquitoes, wild primates, squirrels, birds, and rodents ( Figure 1 ) [10] . In Asia, the disease is vectored by Ae. aegypti and Ae. albopictus [11] . Transmission in Asia occurs in an urban cycle whereby the mosquito spreads the disease from an infected human to an uninfected human, following an epidemiological pattern similar to dengue fever [12] . The 2005-2006 epidemic of CHIKV in La Reunion islands in the Indian Ocean, spurred the discovery of a new vector species, Ae. albopictus [5] . Wrecking over one-third of the island's population, this epidemic peaked its devastation between January and February 2006, when over 46,000 cases came into light every week, including 284 deaths [5, 13] . Ae. albopictus is common in urban areas of the United States and is already flourishing in 36 states, raising grave concerns to the immunologically naive populace of the United States [14] . Accordingly, this review elaborately details the epidemiology and global expansion of CHIKV, describes its clinical features and pathogenesis and its symptoms and complications, and finally nominates a possible vaccine approach against CHIKV infection. CHIKV has been isolated into three genotypes based on phylogenetic studies. These genotypes, based on the gene sequences of an Envelope protein (E1), are Asian, East/Central/ South African, and West African [4, 11, 15] . Using phylogenetic models, Cherian et al. estimate that the Asian genotype of CHIKV emerged between 50 and 310 y ago, and the West and East African genotypes diverged between 100 and 840 y ago [15] . Since then, CHIKV has come a long way, with several mutations incorporated, and has continued to wreak epidemics in several regions. Recent activities of CHIKV include the Indian epidemic in 2005-2006, which was followed by a sudden explosion of cases in 2007. An estimated 1.3 million people across 13 states were reported to be infected in India [12, 16] , and CHIKV was also widespread in Malaysia, Sri Lanka, and Indonesia [17] . In July-August of 2007, CHIKV was reported in Italy, probably brought in by travelers from CHIKV-prone regions of India, Africa, and Indian Ocean islands such as Mauritius, Madagascar, and Seychelles. Few of the Italian isolates were found to have evolved from the Kerala isolate, which was associated with a A226V shift in E1 gene that represents a successful evolutionary adaptation in the mosquito vector similar to the ones observed in Reunion Island [2, 18, 19] . In recent times, with an increase in global travel, the risk for spreading CHIKV to non-endemic regions has heightened [1] . Several travelers have brought CHIKV home with them after visiting areas with actively infected populations [12, 20] . Such cases have been documented in European countries, Australia, Asia, and the United States [8, 21] . The United States has already reported at least twelve cases of travel-associated CHIKV, while France has reported 850 cases, and the United Kingdom 93 [8, 14] . Beyond this, CHIKV-infected travelers have also been diagnosed in Australia, Belgium, Canada, Czech Republic, French Guiana, Germany, Hong Kong, Italy, Japan, Kenya, Malaysia, Martinique, Norway, Switzerland, and Sri Lanka [21] . Some travelers were viremic, worrying public health officials about the spread of CHIKV to new areas [1, 8] . The incubation time for CHIKV is relatively short, requiring only 2-6 d with symptoms usually appearing 4-7 d post-infection [22] . Vazeille et al. detected CHIKV in the salivary glands of Ae. albopictus only 2 d after infection [5] . Upon infection, CHIKF tends to present itself in two phases. The first stage is acute, while the second stage, experienced by most but not all, is persistent, causing disabling polyarthritis. Characteristics of the acute phase include an abrupt onset of fever, arthralgia, and in some cases, maculopapular rash [6, 23] . The acute phase causes such intense joint and muscular pain that makes movement very difficult and prostrates its victims [6, 20] . Ninety-five percent of infected adults are symptomatic after infection, and of these, most become disabled for weeks to months as a result of decreased dexterity, loss of mobility, and delayed reaction. Eighteen months after disease onset, 40% of patients are found to still have anti-CHIKV IgM [6, 18, 23, 24] . The chronic stage of CHIKF is characterized by polyarthralgia that can last from weeks to years beyond the acute stage [6] . CHIKV has been shown to attack fibroblasts, explaining the involvement of muscles, joints, and skin connective tissues. The high number of nociceptive nerve endings found within the joints and muscle connective tissues can explain pain associated with CHIKF [25, 26] . More than 50% of patients who suffer from severe CHIKF are over 65 y old, and more than 33% of them die. Most adults who suffer from severe CHIKF have underlying medical conditions [6, 24, 27] . The other group that is disproportionately affected by severe CHIKV is children. Other complications associated with CHIKV, from most common to least common, include respiratory failure, cardiovascular decompensation, meningoencephalitis, severe acute hepatitis, severe cutaneous effects, other central nervous system problems, and kidney failure [6, 18, 20, 23, 24, 26, 27] . CHIKV undertakes a complex replication cycle upon host infection (Figure 2 ), which makes its genome susceptible to mutations [28, 29] . For instance, Ae. aegypti, responsible for epidemics in Kenya, Comoros, and Seychelles, carried CHIKV with an alanine in the 226 position of the E1 gene (E1-A226) [4, 18] . However, when the virus struck La Reunion Islands, a decline in population of Ae. aegypti, due to massive dichlorodiphenyltrichloroethane usage and dearth of Ae. albopictus species' www.plosntds.org population, resulted in an ecological pressure, favoring replacement of alanine at position 226 with valine (E1-A226V) [5] . This mutation allowed CHIKV's secondary vector species, Ae. albopictus, to supplement Ae. aegypti as its primary vector [5] . Within a year, the E1-A226V mutation was present in La Reunion Island, and Ae. albopictus apparently vectored the large epidemic infecting 34% of La Reunion Island's population [5] . All of the CHIKV strains isolated from Mayotte carried the E1-A226V mutation, and the mutation was also found in Madagascar in 2007 [5] . The E1-A226V mutation was not present at the beginning of the Indian Ocean Islands outbreak (before September 2005). However, more than 90% of later viral strains found there had incorporated the mutation (December-March 2006), indicating a genotype switch during the winter season [5, 18, 20] . The E1-A226V mutation also enabled an increase in infectivity of Ae. albopictus when compared to its infectivity of Ae. aegypti [4, 11, 18, 30] , and with several factors taken together, Ae. albopictus has become the new preferred and more lethal vector for CHIKV [4, 5, 11] . In fact, Tsetsarkin et al. found that a Green Fluorescent Protein tagged E1-A226V virus was 100 times more infective to Ae. albopictus than it was to Ae. aegypti [4] . In all the Indian Ocean Islands, Ae. albopictus became the main vector for CHIKV within 1-2 y after CHIKV was introduced to the region [31] . Of note is that Ae. aegypti has most likely been established in North America for over 300 y, while Ae. albopictus has been in many areas of the US, since 1985, primarily in Florida [32] and since then has expanded its range in the country. Reiskind et al. set out to determine if Ae. aegypti and Ae. albopictus mosquitoes captured in Florida were susceptible to CHIKV infection by a La Reunion isolate [32] . Each mosquito tested was highly susceptible to infection by a full-length infectious clone of the La Réunion Island isolate, CHIKV LR2006 OPY1 strain. Even though the Ae. albopictus strains were more susceptible to infection, overall ecology and differences in human biting patterns need to be studied further Characteristically, there are two rounds of translation: (+) sense genomic RNA (49S9 = 11.7 kb) acts directly as mRNA and is partially translated (59 end) to produce non-structural proteins (nsp's). These proteins are responsible for replication and formation of a complementary (2) strand, the template for further (+) strand synthesis. Subgenomic mRNA (26 S = 4.1 kb) replication occurs through the synthesis of full-length (2) intermediate RNA, which is regulated by nsp4 and p123 precursor in early infection and later by mature nsp's. Translation of the newly synthesized sub-genomic RNA results in production of structural proteins such as Capsid and protein E2-6k-E1 (from 39 end of genome). Assembly occurs at the cell surface, and the envelope is acquired as the virus buds from the cell and release and maturation almost simultaneous occurred. Replication occurs in the cytoplasm and is very rapid (,4 h) [28, 29] . doi:10.1371/journal.pntd.0000623.g002 www.plosntds.org to gain a more accurate understanding of a potential CHIKV epidemic in the US [32] . During the 7 d preceding birth, no human mother has been reported to transmit the disease vertically. However, about 50% of newborns delivered while the mother was infected with CHIKV contracted the disease from their mother, despite the method of delivery. Furthermore, there have been instances of CHIKV transmission from mother to fetus causing congenital illness and fetal death [33] . During the 2005-2006 La Reunion Island outbreaks, Ramful et al. discovered that mothers could transmit CHIKV to their progeny during the perinatal period (Day 24 to Day +1) [33, 34] , and it is associated with a high degree of morbidity. By mean Day 4 of life, all of the neonates were symptomatic for CHIKV, exhibiting common CHIKF symptoms. Six neonates were confirmed to have contracted CHIKV and developed mengoencephalitis. Of those mothers who, during the La Reunion Island epidemic, were infected long before delivery, only three fetal deaths were reported [12, 33] . Ramful et al. theorized that motherto-child transmission most likely happens transplacentally shortly before delivery [33] . A similar study by Gerardin et al. reported nineteen cases of neonatal infection associated with intrapartum maternal viremia that progressed to develop encephalitis owing to vertical transmission from infected mothers [34] . Clinical and epidemiological similarities with dengue fever make CHIKV diagnosis difficult, which may lead physicians to misdiagnose CHIKV as dengue fever; therefore, the incidence of CHIKV may actually be higher than currently believed (Table 1 ) [6, 12, 35] . The amount of time elapsed since disease onset is the most critical parameter when choosing a diagnostic test. CHIKV can be detected and isolated by culturing with mosquito cells (C6/36), Vero cells (mammalian), or in mice [26] . However, this method can take at least a week and only achieves a high sensitivity during the viremic phase, which usually only lasts up to 48 h after the bite. Five days post-infection, the viral isolation approach has a low sensitivity but is still the preferred method for detecting the CHIKV strain [12, 26, 31, 35] . RT-PCR on the other hand is a faster and more sensitive method that can be used within the first week of disease onset [26] , and it is currently the most sensitive method for detecting and quantifying viral mRNA [4, 36] . Classic serological detection, by assays such as ELISA [37] , immunofluorescence [5, 38] , complement binding, and haemagglutination inhibition [39] , constitutes the second diagnostic tool used for biological diagnosis of CHIKV infection. These proven techniques are useful for detection of Antigen in mosquitoes during epidemiological studies. These assays detect virus-specific IgM and IgG, however the sensitivity and specificity of these assays has been poorly characterized. Viral competence, or the potential of viral infection and transmission, is an important parameter that can be quantified by ELISA, viral culture, and PCR. A study by Ng et al. showed biomarkers indicative of severe CHIKV infection [40] . They found decreased levels of RANTES and increased levels of Interleukin-6 (IL-6) and Interleukin-1b (IL-1b) that could be sued for CHIKV detection in patients as indicators of CHIKV-driven cytokine storm. Couderc et al. demonstrate another cytokine, type-I IFN, as a key player in the progression to CHIKV infection [26] . Using an IFN-a/b null mouse model, they demonstrated evidence of muscles, joints, and skin as privileged CHIKV targets, which is consistent with human pathology. Although Ng et al. concluded that RANTES levels were significantly suppressed in severe CHIKF patients [40] , interestingly, an increase in levels of RANTES has been observed in dengue infection [41] . Since the symptoms of CHIKF mimic those of dengue fever, results obtained from this study strongly suggest that RANTES could be a potential distinctive biomarker that differentiates between these two clinically similar diseases. There are no approved antiviral treatments currently available for CHIKV [1, 3, 12, 42] . Currently, CHIKF is treated symptomatically, usually with non-steroidal anti-inflammatory drugs or steroids, bed rest, and fluids. Movement and mild exercise are thought to decrease stiffness and morning arthralgia, but heavy exercise may exacerbate rheumatic symptoms. Corticosteroids may be used in cases of debilitating chronic CHIKV infection. There is a debate about the appropriateness of chloroquine as treatment for unresolved, non-steroidal anti-inflammatory drugresistant arthritis [43] . A study showed that viral production was www.plosntds.org drastically reduced at 16 h post-infection after treatment with 100 mM dec-RVKR-cmk (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone), a furine inhibitor [42, 44] . Chloroquine acted by raising the pH, blocking low pH-dependent entry of virus into the cell. It is important to note that dec-RVKR-cmk or chloroquine only inhibited viral spreading from cell to cell, not CHIKV replication once it had entered the cell [43] . However, most would agree that the best weapon against CHIKV is prevention. A live CHIKV vaccine developed by the United States reached phase II clinical trial encompassing 59 healthy volunteers [45] . Eight percent of the volunteers experienced transient arthralgia, while 98% of the volunteers had seroconversion [45] . However, live CHIKV vaccines are still questionable. One cannot discount the risk of a live vaccine possibly inducing chronic rheumatism. Also, there is the question as to whether widespread use among the public could trigger mosquito transmission or lead to chronic infection or viral reversion [1] . An alternative approach would be to produce a chimeric vaccine against CHIKV. Wang et al. developed a chimeric alphavirus vaccine that is uniformly attenuated and does not cause reactogenicity in mice [3] . Three different versions of this vaccine were made using three different backbone vectors: Venezuelan equine encephalitis virus (VEEV) attenuated vaccine strain T-83, naturally attenuated eastern equine encephalitis virus (EEEV), and attenuated Sindbis virus (SINV). In short, CHIKV structural proteins were engineered into the backbones of the aforementioned vaccines to produce the chimeras [3] . These chimeras were found to stimulate a strong humoral immunity, and even at doses of 5.3-5.8 log 10 PFU, they did not trigger reactogenicity. When vaccinated mice were challenged with CHIKV, neither adult nor neonatal mice gained weight, had fever, or displayed signs of neurological illness. Upon comparison of the chimeras with the Army181/25 vaccine, the Army vaccine resulted in higher levels of viremia and replication in the joints of neonatal mice. Because the joints are known targets of CHIKV, Wang et al. noted their vaccine might avoid the negative reactogenic side effects of the Army vaccine. After being subcutaneously vaccinated with 5.3-5.8 log 10 PFU of the chimeric vaccines, mice produced strong neutralizing antibody titers. The VEEV and EEEV chimeras yielded higher neutralizing antibody titers than the SINV chimera without being more virulent. On top of this, the VEEV and EEEV CHIKV chimeras seemed to be more immunogenic than the Army vaccine despite the chimeras' lower viremia and replication in the joints of neonatal mice [3] . Tiwari et al. [46] adopted a different strategy using formalin inactivated CHIKV in combination with alhydrogel (Aluminum Hydroxide) as an adjuvant. This study clearly suggests that this vaccine elicits both humoral and cell-mediated immune responses in mice, providing its immunogenic potential. A recent study by Couderc et al. [47] showed passive immunization as a potential treatment for CHIKV infection. Using purified immunoglobulin extracted from convalescent CHIKV patients, they demonstrated effective neutralizing activity against CHIKV infection both in vitro and in vivo. This thereby establishes a potential preventive and therapeutic approach to combat CHIKV infection. Pathogenesis studies conducted with related alpha virus, like RRV, have shown the role of macrophages in persistence on infection [48] . They also demonstrated the role of RRV-specific CD8 T cells in clearing viral load in infected patients, thereby warranting similar investigations with CHIKV and the importance of investigating a cell-mediated immune response-based vaccine against CHIKV [49] . There are always certain risks associated with live attenuated or inactivated viral vaccines [50] . One way to avoid these potential problems is to construct a consensus-based DNA vaccine. DNA based vaccines have an improved safety profile as compared to live or attenuated vaccines [51, 52] . A consequence of CHIKV's rapid evolution is difficulty in constructing a vaccine that will be able to Figure 3 . Levels of CHIKV-specific IgG in mice immunized with CHIKV vaccines. Each group of C57BL/6 mice (n = 5) was immunized with 12.5 mg of pVax1 control vector or CHIKV vaccine plasmids as indicated at 0 and 2 wk. Mice were bled 2 wk after each immunization, and each group's serum pool was diluted to 1:100 and 1:500 for reaction with specific vaccine constructs. Serum was incubated for 1 h at 37uC on 96-well plates coated with 2 mg/ml of respective CHIKV peptides, and antibody was detected using anti-mouse IgG-HRP and OD was measured at 405 nm. doi:10.1371/journal.pntd.0000623.g003 www.plosntds.org effectively protect large populations from multiple strains of the virus. One of the strengths of DNA consensus vaccines is its ability to induce cross-reactive immune responses against the three distinct phylogenetic groups of CHIKV. Also DNA-based vaccines can be produced more rapidly than protein-based vaccines. Recently, Muthumani et al. constructed a vaccine that was shown to induce both humoral and cellular immunity in vivo in 3-4-wk-old female C57/BL6 mice [49] . These mice were immunized using an in vivo electroporation method to deliver the vaccine into the quadriceps muscle. The consensus construct was designed against E1, E2, and the core protein capsid. To design the construct, they aligned 21 sequences of CHIKV isolated between 1952 and 2006, using strains from differing countries, including La Reunion Island. The most common nucleotide among the sequences was chosen at each position to be used in the consensus construct, taking care not to alter the reading frame. They conducted codon and RNA optimization, added a strong Kozak sequence, and substituted signal peptide with an immunoglobulin E leader sequence to improve vaccine efficacy. After immunizing the mice, spleens were harvested along with serum and tested to determine antibody titer. After three immunizations, consensus E1, E2, and C vaccines were shown to induce T-cell immune responses leading to strong IFN-c responses and proliferation in C57/BL6 mice. Furthermore, when compared with control mice, immunized mice had higher total IgG levels as well as higher anti-E1 specific, anti-E2 specific, and anti-C specific IgG antibodies, suggesting a strong humoral immune response ( Figure 3 ) and also specificity for the antigens encoded in the vaccine constructs ( Figure 4 ). Because of its promising results and the need for a safer vaccine, this consensus DNA vaccine deserves further investigation. Determining longevity of protective effects of the vaccine and persistence of antibody and IFN-c responses could be the next step of investigation. Challenged studies of immunized mice must also be carried out. CHIKV mosquito-borne disease has caused massive outbreaks for at least half a century but is no longer confined to the www.plosntds.org developing nations. It began to encroach into the boundaries of the developing world. As a result, the NIAID has designated CHIKV as a Category C pathogen alongside the influenza and SARS-CoV viruses [3] . Realization of the potential severity of this disease is exigent; for instance, if used as a biological weapon, the world economy could be severely crippled; if enough members of the armed forces were to become infected during a military deployment, military operations could be significantly affected. Efforts to monitor the disease will only provide minimal warning in a global society, and steps to prevent the morbidity and mortality associated with pandemic are imperative [21, 31] . Despite the gravity of its infectious potency and the fear of it being a potential biological weapon, there is currently no vaccine for CHIKV infections. Live attenuated vaccine trials were carried out in 2000, but funding for the project was discontinued. Newer approaches such as DNA vaccines appear promising over conventional strategies like live attenuated or inactivated virus and thus call for further investigation. Recent advances such electroporation delivery and incorporation of adjuvants has boosted DNA vaccine efficacy [51, 53] . Despite the low antibody response to DNA vaccines, other numerous advantages have overshadowed these minor drawbacks (Table 2) , the most important one being the ability to induce both humoral and cellular immune responses [51, 54] . Judging by recent success, such as the immunogenic construct developed by Muthumani et al., DNA vaccines could play a major role in combating CHIKV [49] . Vaccines are literally a critical component of CHIKV disease control and therefore research in this area is highly encouraged. The dramatic spread of dengue viruses (DENV) throughout tropical America since 1980 via the same vectors and human hosts underscores the risk to public health in the Americas. The adverse events associated with the current live vaccine are well documented [55] . Realizing these drawbacks, earnest efforts should be taken to develop new strategies to forestall further spread and complications.
What did the study report?
false
2,525
{ "text": [ "neonatal infection associated with intrapartum maternal viremia that progressed to develop encephalitis owing to vertical transmission from infected mothers" ], "answer_start": [ 12254 ] }
1,584
Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138590/ SHA: ef3d6cabc804e5eb587b34249b539c1b5efa4cc4 Authors: Hoffmann, Jonathan; Machado, Daniela; Terrier, Olivier; Pouzol, Stephane; Messaoudi, Mélina; Basualdo, Wilma; Espínola, Emilio E; Guillen, Rosa M.; Rosa-Calatrava, Manuel; Picot, Valentina; Bénet, Thomas; Endtz, Hubert; Russomando, Graciela; Paranhos-Baccalà, Gláucia Date: 2016-12-06 DOI: 10.1038/srep38532 License: cc-by Abstract: Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia. Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 . With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways. In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity. Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 . MiRNA-200a-3p was overexpressed after both single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤ 0.05; Fig. 2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response. Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) . These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10. analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001). Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis. This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) . The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms involved in the immunopathology of pneumonia. In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died, in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia. Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia. Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences). MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal. In silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 . THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO "Pocket book of hospital care for children" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 . a IP-10 values are expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture). Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States).
What was a severe limitation of this study?
false
5,212
{ "text": [ "unable to evaluate the levels of immunomodulators in the serum samples of a control group" ], "answer_start": [ 23550 ] }
1,594
Gemcitabine and Nucleos(t)ide Synthesis Inhibitors Are Broad-Spectrum Antiviral Drugs that Activate Innate Immunity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923505/ SHA: f1e1e2511e051195c8327a56d5c311a2dd4ab6b3 Authors: Shin, Hye Jin; Kim, Chonsaeng; Cho, Sungchan Date: 2018-04-20 DOI: 10.3390/v10040211 License: cc-by Abstract: Nucleoside analogs have been frequently identified as antiviral agents. In recent years, gemcitabine, a cytidine analog in clinical use for the treatment of many solid tumors, was also shown to have antiviral activity against a broad range of viruses. Nucleoside analogs generally interfere with cellular nucleos(t)ide synthesis pathways, resulting in the depletion or imbalance of (d)NTP pools. Intriguingly, a few recent reports have shown that some nucleoside analogs, including gemcitabine, activated innate immunity, inducing the expression of interferon-stimulated genes, through nucleos(t)ide synthesis inhibition. The precise crosstalk between these two independent processes remains to be determined. Nonetheless, we summarize the current knowledge of nucleos(t)ide synthesis inhibition-related innate immunity and propose it as a newly emerging antiviral mechanism of nucleoside analogs. Text: Nucleoside analogs have been historically used for anti-cancer chemotherapy because they inhibit cellular DNA/RNA polymerases [1] . More recently, nucleoside analogs have expanded their therapeutic applications and are being used to develop antiviral drugs against a wide range of serious and life-threatening viruses. Some nucleoside analog drugs targeting specific viral polymerases (acyclovir for herpesviruses, zidovudine for human immunodeficiency virus (HIV), and sofosbuvir for hepatitis C virus (HCV)) have been successful in clinical trials [2] [3] [4] [5] and are currently in use for the treatment of virus-infected patients. Another class of nucleoside analog drugs such as ribavirin, more broadly-acting on various viruses, has been used in conjunction with IFN-α [6] . Importantly, extensive studies on the antiviral action of ribavirin have established the underlying molecular framework of nucleoside analogs. The primary mechanism to explain the antiviral effect of nucleoside analogs is based on their direct action on viral polymerization. Nucleoside analogs are transported into the cells and phosphorylated by the consecutive action of viral or cellular kinases, eventually generating nucleotide triphosphates. Mature nucleotide analogs, which are similar to physiological nucleotides, can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( Figure 1 ). Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [7] [8] [9] [10] . By targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferon-stimulated genes (ISGs). Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report [1] . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broad-spectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. can directly incorporate into the growing viral genome during polymerization, resulting in the termination of chain reaction or the accumulation of mutations ( Figure 1 ). Alternatively, nucleotide analogs can bind to the nucleotide-binding region on viral polymerases and block the entry of incoming natural nucleotides. The other mechanism is based on the modulation of cellular nucleos(t)ide synthesis. There have been accumulating reports that nucleoside analogs act as antiviral agents by interfering with host nucleos(t)ide synthesis pathways [7] [8] [9] [10] . By targeting metabolic enzymes(s), nucleoside analogs block the natural flow of nucleos(t)ide synthesis and consequently cause the depletion or imbalance of (d)NTP pools. As viral replication is highly dependent on the availability of host nucleotides, a nucleotide-defective condition decreases the efficiency of viral replication. A more recently proposed mechanism has been based on the observations that a few nucleoside analogs activate innate immunity, especially involving the upregulation of interferonstimulated genes (ISGs). Importantly, this phenomenon is usually mediated by the inhibition of nucleotide synthesis, suggesting a potential crosstalk between nucleotide biosynthesis and innate immunity. However, the precise mechanism of this crosstalk remains to be elucidated. There is now an increasing number of nucleoside analogs with antiviral activity toward a wide range of viruses. They have been well-summarized in a previous report [1] . In the present review, we focus more on gemcitabine as a nucleoside analog, which is clinically relevant and whose broadspectrum antiviral activity has been recently reported by many groups including our group. More importantly, we summarize inhibitors of the purine/pyrimidine biosynthesis pathways that induce innate immunity and propose possible mechanisms of action for these inhibitors. Figure 1 . The mechanism of antiviral effect of nucleos(t)ide analogs. Nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [11, 12] . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV) [13] [14] [15] [16] [17] [18] . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug Figure 1 . The mechanism of antiviral effect of nucleos(t)ide analogs. Nucleos(t)ide synthesis inhibition-related innate immunity, a newly emerging antiviral mechanism of nucleoside analogs, was highlighted by yellow boxes. Gemcitabine is a cytidine analog that has been clinically used for the treatment of various cancers [11, 12] . However, in recent years, the antiviral activity of gemcitabine has also been reported against a broad range of RNA viruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), Zika virus (ZIKV), HCV, poliovirus (PV), influenza A virus (IAV), HIV, and enteroviruses (EV) [13] [14] [15] [16] [17] [18] . The antiviral activities of gemcitabine against the abovementioned viruses are summarized in Table 1 . MERS-CoV and SARS-CoV belong to the family of Coronaviridae and are causative agents of severe viral respiratory illness in humans. To efficiently select appropriate antiviral drug candidates, Dyall et al. screened 290 FDA-approved drugs in virus-infected Vero E6 cells and identified gemcitabine as one of drugs with antiviral activity against both MERS-CoV and SARS-CoV (EC 50 of 1.2 µM and 4.9 µM, respectively) [13] . More recently, gemcitabine was shown to effectively suppress ZIKV infection and replication in human retinal pigment epithelium (RPE) cells, particularly at non-cytotoxic concentrations (EC 50 of 0.01 µM vs. CC 50 of > 10 µM) [14] . ZIKV, a member of the Flaviviridae family, can infect pregnant women and cause congenital abnormalities such as microcephaly in infants, which has attracted increasing public attention as well as extensive research and development into possible treatments. Effective antiviral activities of gemcitabine were also found for the replication of HCV in Huh-7 cells and the infection of HIV in U373-MAGI-CXCR4 CEM cells, with estimated EC 50 s of 12 nM and 16.3 nM, respectively [17, 19] , which were lower concentrations than those used in cancer therapy [20] . In the case of HIV, the combination of gemcitabine with decitabine, another nucleoside analog in clinical use for cancer therapy, synergistically reduced HIV infectivity by increasing the viral mutation frequency [21] . In a follow up study, Clouser et al. further reported the antiviral effect of gemcitabine against HIV-related retrovirus, murine leukemia virus (MuLV), in vitro (EC 50 of 1.6 nM) and even in murine AIDS model [17] . A significant antiviral effect of gemcitabine on IAVs was also reported for RPE cells by Denisova et al. (EC 50 of 0.068 µM) [16] . They also tested whether gemcitabine had an antiviral effect on several other viruses of different families and found its strong inhibitory effect on Sindbis virus and herpes simplex virus-1 (HSV-1) (>2 log reduction in virus titer) but relatively weak effects on Semliki forest virus and human echovirus 6, and minimal effects on Bunyamwera virus, measles virus (MeV), and vaccinia virus [16] . The antiviral effect of gemcitabine on EVs, initially performed on Coxsackievirus B3 (CVB3), was found from screening FDA-approved drugs in CVB3 replicon-harboring Vero cells by our group (EC 50 of 0.4 µM) [18] . Its broad-spectrum antiviral activity on EVs was further identified by observing a similar inhibitory effect on enterovirus 71 (EV71) and human rhinoviruses (HRVs) (EC 50 s of 1 and 1-5 µM, respectively). In the case of HRV, the antiviral effect of gemcitabine was further confirmed in a virus-infected mouse model [22] . In this study, intranasal administration of gemcitabine significantly lowered the pulmonary viral load and inflammation by decreasing proinflammatory cytokines, including TNF-α and IL-1β, and the number of lung infiltrating lymphocytes. More recently, Zhang et al. also identified gemcitabine as the best anti-PV inhibitor from a screen of FDA-approved drugs in PV replicon-harboring HeLa cells (EC 50 of 0.3 µM) [15] . As previously mentioned, accumulating evidence has definitively demonstrated that gemcitabine is an effective broad-spectrum inhibitor of RNA viruses and has a therapeutic potential for the treatment of various virus-associated diseases. Moreover, it is possible that gemcitabine is effective for other untested RNA viruses. Because gemcitabine is a deoxycytidine analog that interferes with DNA as well as RNA synthesis, DNA viruses may not be the exception. Consistent with this possibility, there has been a report that the infection of HSV-1, which is a representative DNA virus classified into the Herpesviridae family, was strongly affected by gemcitabine [16] . Most of the abovementioned viruses have, at best, limited prophylactic or therapeutic drugs as possible treatments. This is especially true for newly emerging or re-emerged viruses involving serious illnesses, such as MERS-CoV, SARS-CoV, and ZIKV, which are major threats to public health and which urgently need an effective treatment during their early stages of infection. In this regard, repurposing of gemcitabine for the treatment of patients infected with these deadly viruses is a realistic approach. Importantly, it is noteworthy that ZIKV was the most strongly affected by gemcitabine, with a low nanomolar EC 50 , which was lower than that used in cancer therapy [14, 20] . Even for other viruses with a relatively high EC 50 , there is an option to treat patients with a combination of gemcitabine with other antiviral agents. In this manner, an effective antiviral treatment may be achieved by the synergistic action of two antivirals with much lower doses for each drug, which minimizes deleterious side effects when used clinically. As an example, the synergistic antiviral effect of gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, was reported against EVs such as CVB3 and EV71 [18] . As previously mentioned, the combination of gemcitabine with decitabine synergistically suppressed HIV infectivity both in vitro and in vivo [17, 21] . However, the actual use of gemcitabine in virus-infected patients necessitates prior in vivo animal studies and clinical trials. Even though most antiviral data have originated from in vitro studies, two recent studies have reported the antiviral effects of gemcitabine in murine models [17, 22] . More extensive analyses of gemcitabine in animal models in the near future will accelerate its therapeutic applications in clinical trials. Most studies regarding the antiviral activity of gemcitabine lack experimental evidence of the mode of action. However, our group has recently reported that gemcitabine had an anti-EV effect by targeting the salvage pathway of pyrimidine biosynthesis [23] . Moreover, gemcitabine strongly induced the expression of several ISGs including CXCL10, IRF7, IRF9, IFIT1, and DDX58, which were the major effectors in the innate immunity that defended the host against the virus infection. These results were consistent with a previous report that gemcitabine stimulated the production of IFN-β and IFN-γ in IAV-infected RPE cells [16] . Importantly, the activation of ISGs was well-correlated with the inhibition of pyrimidine biosynthesis, suggesting a link between pyrimidine biosynthesis and innate immunity. Similar phenomena in terms of ISG activation have been previously reported with a few compounds out of several purine or pyrimidine biosynthesis inhibitors that had antiviral activity, as summarized in Table 2 [6, 10, [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . Regarding purine biosynthesis inhibitors, ribavirin and mycophenolic acid (MPA) are inhibitors of inosine-5 -monophosphate (IMP) dehydrogenase (IMPDH), which is a key enzyme of the purine biosynthesis pathway. These inhibitors have been successfully used as clinical antiviral or immunosuppressant agents for decades. Both have antiviral activities against viruses such as HCV, hepatitis E virus (HEV), MERS-CoV, dengue virus, yellow fever, hepatitis B virus, West Nile virus (WNV), Chikungunya virus (CHIKV), and IAV [24] [25] [26] [27] [28] [29] [30] , majorly through the inhibition of the purine biosynthesis pathway, with the antiviral activity against HCV and HEV shown to involve the stimulation of ISGs [10, 30] . For the antiviral activity of ribavirin against HCV, ribavirin specifically induced the expression of IRF7, IRF9, and ISG15 mRNAs, which are known to be important for anti-HCV immune responses [6] . ISG activation occurred through an undefined mechanism that was different from the classical IFN signaling, intracellular dsRNA sensing pathway, Toll-like receptor and nuclear factor B pathways. More importantly, ribavirin-induced ISG activation and antiviral activity were suppressed using supplemented guanosine, a natural analog of ribavirin, suggesting IMPDH inhibition-mediated ISG activation as an alternative innate immunity pathway. Like ribavirin, MPA remarkably induced the expression of several ISGs, including IRF1, IRF9, ISG15, IFI6, IRF7, CXCL10, IFIT2, and IFITM3 mRNAs in naïve or HEV-infected Huh-7 cells, and the induction of ISGs was at least partially abrogated by the use of supplemented guanosine [10] . Mechanistically, the induction of ISGs by MPA was independent of the classical JAK/STAT system, which is similar to that observed with ribavirin [30] . Similar results were obtained with several IMPDH1 or IMPDH2 inhibitors, with various affinities, that were custom-designed and synthesized [10] . As shown in Table 2 , most pyrimidine biosynthesis inhibitors target dihydroorotate dehydrogenase (DHODH), an essential enzyme in de novo pyrimidine synthesis. Lucas-Hourani et al. identified DD264 as an interferon-sensitive response element (ISRE)-stimulating compound from high-throughput screening, and further analyses suggested that it was a DHODH inhibitor with a strong antiviral activity against various viruses including MeV, CHIKV, and WNV [37] . DD264 enhanced the expression of several ISGs, which were almost completely suppressed by the addition of supplemented uridine, indicating DHODH inhibition-mediated ISG activation. Moreover, the antiviral activity of and ISG activation by DD264 required the interferon regulatory factor 1 (IRF1) transcription factor, a master regulator of antiviral gene expression [37] , which was consistent with the observation that the anti-HCV activity of MPA was partially mediated by IRF1 [30] . In this study, similar results were shown with brequinar, another well-known DHODH inhibitor. FA-613 is also an antiviral compound, which inhibits the pyrimidine biosynthesis pathway, probably via targeting DHODH and inducing the expression of ISGs such as IFNB1, CXCL10, ISG15, and CCL5 [38] . However, whether ISG activation is mediated by pyrimidine biosynthesis inhibition remains to be determined. The mechanism of nucleotide synthesis inhibitor-induced ISG activation is still presently unclear. Nevertheless, there has been accumulating evidence showing that nucleotide synthesis inhibitor-induced ISG activation is independent of the classical JAK/STAT-mediated IFN signal [6, 10, 23] . First, Wang et al. clearly showed that ISG activation and anti-HEV activity induced by MPA or brequinar was not mediated by JAK [10] . Second, IRF7 induction by ribavirin was not affected by knockdown of STAT1, while that of IFN-α was strongly affected under the same conditions [6] . Third, our recent study with gemcitabine further confirmed IFN signal-independent ISG activation by parallel studies comparing the effects of gemcitabine and IFN-α. In our study, the phosphorylation of STAT1 at Tyr701, which was dramatically triggered by IFN-α, did not occur when treated with gemcitabine [23] . Moreover, the upregulation of DDX58 mRNAs induced by gemcitabine was not affected by IRF9 knockdown, which was contrary to the result that IFN-α-induced upregulation of DDX58 mRNAs was significantly suppressed under the same conditions. Consistent with above observations, there have been some reports that ISGs was induced in the absence of JAK1 or STAT1 activation [43, 44] . Despite limited data, we speculate the scenario of ISG activation that is independent of JAK/STAT-mediated IFN signal. Purine or pyrimidine biosynthesis inhibitors could interfere with the metabolic pathway through targeting some key enzymes such as IMPDH and DHODH, leading to the depletion or imbalance of the (d)NTP pool. Inactivation of metabolic enzyme(s) itself or consequently altered nucleos(t)ide pools might trigger a signal, which is ultimately delivered to certain cis-acting elements on the promoter of a subset of ISGs, possibly through the relay of kinases and transcription factors. Based on the previously mentioned reports, this signal is less likely to be dependent on STAT1/2-IRF9 (IFN-stimulated gene factor 3; ISGF3), at least for gemcitabine, which is the major transcriptional complex in the IFN-induced JAK/STAT pathway. It should also be considered that Thomas et al. excluded the involvement of an intracellular double-stranded RNA sensing pathway, Toll-like receptor and nuclear factor κB pathways, as well as a classical IFN signal in the activation of ISGs induced by ribavirin [6] . Despite the consensus of ISG activation, each purine/pyrimidine biosynthesis inhibitor seems to induce distinct sets of ISGs, at least with different patterns [10] . Targeting an enzyme in which pathways (purine or pyrimidine synthesis) or steps (early/late and de novo/salvage) produce different levels of intermediates and nucleos(t)ides will consequently result in diverse outcomes of ISG activations. There might be more than one signaling pathway involved. The synergistic antiviral activity of gemcitabine and ribavirin observed in our study might be explained by the possible existence of two separate signaling pathways that mediate each inhibition of nucleotide synthesis toward ISG activation. Systematic analyses of signaling kinases, IRFs, and STATs using siRNA knockdown and/or pharmacological inhibition and metabolic analyses of corresponding intermediates and nucleos(t)ides should therefore clarify the underlying molecular mechanisms of ISG activation by purine/pyrimidine biosynthesis inhibitors. As newly emerging or re-emerged viruses such as SARS-CoV, MERS-CoV, and ZIKV have become a major threat to public health, the need for broad-spectrum antiviral drug has increased. In this regard, nucleoside analogs that directly target viral RNA-dependent RNA polymerase and present a high barrier to the development of resistant viruses have been considered advantageous. Moreover, recent discovery of a new antiviral mode of nucleoside analogs acting through innate immunity strengthens the molecular basis for their therapeutic application as broad-spectrum antiviral drugs. Nucleoside analogs probably induce different subsets of ISGs, at least with a different pattern, leading to various combinations of ISGs and resulting antiviral outcomes. Moreover, according to Schoggins et al., different viruses are affected by distinct subsets of ISGs and some ISGs such as IRF1, MB21D1, HPSE, DDX58, MDA, and IFITM3 act broadly on various viruses [45] . Thus, more systematic analyses on the subsets of ISGs induced by antiviral nucleoside analogs are required for the identification of better antiviral drugs that can be used broadly or specifically. Given the clinical side effects of IFN treatment, nucleotide analogs that differ from IFN in the activation of subsets of ISGs need to be considered as alternatives. Nevertheless, nucleoside analogs interfering with the host nucleotide synthesis pathway suggest possible side effects in their clinical applications. Careful evaluation of clinical safety is required and their application for the urgent measure of patients infected with deadly viruses would be worth being primarily considered.
What nucleoside analog is the focus of the current study?
false
5,233
{ "text": [ "gemcitabine" ], "answer_start": [ 6040 ] }
2,461
Mucosal immune responses induced by oral administration recombinant Bacillus subtilis expressing the COE antigen of PEDV in newborn piglets https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418403/ SHA: 5caced13bcb8a42cca41369c5a71ae7df5381ca8 Authors: Wang, Jialu; Huang, Lulu; Mou, Chunxiao; Zhang, En; Wang, Yongheng; Cao, Yanan; Yang, Qian Date: 2019-03-15 DOI: 10.1042/bsr20182028 License: cc-by Abstract: Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets and causes substantial economic losses in the world. PED virus (PEDV) spreads by fecal–oral contact and can be prevented by oral immunization. Therefore, it is necessary to develop an effective oral vaccine against PEDV infection. Currently, Bacillus subtilis as recombinant vaccine carrier has been used for antigen delivery and proved well in immune effect and safety. The present study evaluated the immunogenicity of recombinant Bacillus subtilis (B. subtilis-RC) in piglets via oral administration. After oral immunization in piglets, B. subtilis-RC significantly increased the local mucosal immune responses. Oral administration with B. subtilis-RC significantly improved the level of specific mucosal immunoglobulin A (IgA) antibodies against PEDV infection, through enlarging the area of Peyer’s patches (PPs) and increasing the number of ileum IgA(+) secreting (SIgA) cells. In the meantime, B. subtilis-RC remarkably increased the number of intraepithelial lymphocytes (IELs). We also observed that oral administration of B. subtilis-RC significantly increased CD3(+)T lymphocytes’ numbers and up-regulated the ratio of CD4(+)/CD8(+) T cells. Furthermore, high titers of specific serum immunoglobulin G (IgG) revealed satisfactory systemic immune response against PEDV infection. In summary, our study demonstrated that oral administration of B. subtilis-RC could trigger a high level of local and systemic immune responses and would be a promising candidate vaccine against PEDV infection in piglets. Text: Porcine epidemic diarrhea (PED) characterized by highly fatal acute diarrhea in piglets, results in enormous losses in the worldwide pig industry [1] . The causative agent PED virus (PEDV) belongs to the porcine coronaviruses (CoVs). PEDV infection mainly spreads through the digestive tract [2] , and damages the host intestine mucosal surfaces by infecting the intestine epithelial cells [3] . Therfore enhancing intestinal mucosal immunity can elicit effective mucosal immune responses against PEDV infection [4] . Currently, traditional vaccines (intramuscular route or subcutaneous injection) have been developed and applied widely in the market [5] . These vaccines administered parenterally cannot effectively induce high titers of maternal antibodies and virus-specific IgA antibodies, resulting in inadequate mucosal protection to against PEDV infection [6] . Furthermore, these maternal antibodies in the milk were always degraded by gastric acid and pepsin before entering the intestinal tract. Effective PEDV vaccines must provide adequate mucosal protection in the intestinal tract. However, the effective vaccines are currently lacking [7] . As a superior way of mucosal immunization, oral administration can protect the gut and stimulate the common mucosal immune system [8] . Besides, oral immunization has several attractive features which include safety, and a straightforward, inexpensive, and needle-free approach [9] . Therefore, oral immunization often delivers large amounts of antigens to prevent the diarrheal diseases [10] . Nevertheless, there are several challenges by oral immunization, which consist of physical, chemical, and biological barriers when delivering antigens to the gastrointestinal (GI) tract (such as gastric acids, pepsin, and trypsin in the GI tract) [11] . It is a substantial problem that digestive acids and proteases can degrade antigen proteins for nutrient absorption [12] . Therefore, the vaccine delivery system has been applied to solve the problem. The system can protect antigens from the severe environment of the GI tract and deliver antigens to intestinal mucosa [13] . Currently, Bacillus subtilis (B. subtilis) is widely used as a vaccine delivery system for its unique characteristics. As a nonpathogenic Gram-positive bacterium, B. subtilis has been regarded as a novel probiotic and food additive in humans and animals [14] . The B. subtilis has adjuvant activity and can deliver heterologous antigens to the GI tract, providing additional immunity stimulation [15] . Besides, research had shown that orally administered B. subtilis could also enhance immune regulation and gut health in pigs [16] . Moreover, oral administration of B. subtilis could elicit humoral and cellular immune responses to the maintenance of gut homeostasis by dendritic cells (DCs) [17] . DCs are the most important professional antigen-presenting cells and can effectively regulate antibody titers [18] . DCs naturally exist in the gut-associated lymphoid tissue (GALT), including Peyer's patches (PPs), isolated lymphoid follicles (ILFs), mesenteric lymph nodes (MLNs), and scatter throughout the subepithelial lamina propria (LP) of the small intestine and colon [19] . Furthermore, B. subtilis is convenient for genetic manipulation and has developed a large variety of genetic tools [20] . Therefore, B. subtilis is widely used as an effective vaccine delivery system to induce mucosal immune responses and shows unique effect on the immune system. In the present report, we explored the immune effect of a recombinant B. subtilis (B. subtilis-RC) which had been successfully constructed with expressing PEDV COE protein in piglets. Our research indicated that B. subtilis-RC was beneficial to the mucosal immune system development, and could effectively generate specific antibodies against PEDV infection, suggesting a potential approach for preventing PEDV infection. The B. subtilis WB800 was kindly provided by Dr. Xuewen Gao (from the department of plant pathology, Nanjing Agricultural University) [21] . B. subtilis-RC previously constructed in our laboratory was able to express the gene COE (499-638 amino acids in S protein). Prior to oral administration, the recombinant strain was grown in LB broth at 37 • C for 12 h, and then washed twice with PBS, and suspended in PBS to reach a final concentration of 1 × 10 10 CFU/ml. The PEDV Zhejiang08 strain was provided by the Veterinary Medicine Research Centre of the Beijing Dabeinong Technology Group Co., Ltd. [22] . The virus was cultured in African green monkey kidney cells (Vero cells) and purified by using a discontinuous sucrose density gradient. The virus was UV-inactivated at UV dose of 4 J/cm 2 for 24 h to achieve a complete loss of infectivity [23] . The purified virus concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, U.S.A.). ELISA: Rabbit anti-pig IgG (horseradish peroxidase (HRP)), Goat Anti-Pig IgA (HRP) were purchased from Abcam. Second antibody: DyLight 649-conjugated goat anti-mouse IgG antibody, DyLight 488-conjugated goat anti-rabbit IgG antibody, DyLight 594-conjugated goat anti-rabbit IgG antibody were purchased from Multi-science, Hangzhou, China. ABC-based system (biotinylated goat anti-rabbit IgG antibody) was used as the secondary antibody with DAB as a chromogen was purchased from Boster, Wuhan, China. Specific pathogen-free (SPF) DLY piglets (Duroc and Landrace and Yorkshire) were kindly provided by Jiangsu Academy of Agricultural Sciences (Nanjing, China). The animal experiments had been approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health's guidelines for the performance of animal experiments. Twelve newborn piglets were randomly divided into three groups (four piglets in each group), and housed under similar conditions in different stables in order to avoid probiotic cross-contamination. The piglets were orally dosed with 100 μl of B. subtilis-RC. The control groups of piglets were orally administered with inactivated PEDV (100 μg/dose) and equal volume of PBS. The immunization protocol was performed on the piglets that were 5 days old ( Figure 1C ), and signed as 0 day. Then booster immunizations were administered on 5 days. Specimen collection was then performed every 7 days post boost immunization ( Figure 1C ). Blood samples were collected weekly from all piglets after the boost immunization and allowed to clot overnight at room temperature to collect serum. Blood samples were separated by centrifugation and stored at −20 • C in order to detect the levels of specific IgG and IgA. Three swabs were collected every week lasting for 1 month, including nasal, oral, and feces swabs for the ELISA. The piglets were sacrificed in 33 days. The same location of the small intestine and ileum tissues from each piglet were fixed with Bonn's liquid and 4% paraformaldehyde. The small intestine tissues in same location were fixed with Bouin Fixative Solution for 24 h, embedded in paraffin, and sectioned at 4-μm thickness. The sections were placed on glass slides. Hematoxylin-eosin staining was applied to the paraffin sections, then observing and taking photographs under optical microscope (OLYMPUS CX23). The number of intraepithelial lymphocytes (IELs) were counted in every 100 epithelial cells under the same multiple light microscope amongst ten pictures from each group [24] . The immunohistochemistry detection was performed with the SABC kit (Boster Bioscience). Hydrogen peroxide was used to deactivate intrinsic peroxidase. Antigen retrieval was performed in a water bath using citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2). Sections were incubated with diluted anti-IgA antibody (1:100; Abcam) overnight at 4 • C. As negative controls, immunostaining performed by incubating samples with control antiserum instead of primary antibody. The addition of biotin-labeled secondary antibody to the slides was followed by adding HRP-labeled streptavidin. After staining with DAB, the slides were recorded using a digital camera (Leica-DM4000B) [25] . The isolated intestines with PPs were transferred to ice-cold PBS. Then, remaining fat and connective tissue was removed and washed thoroughly with ice-cold PBS. Next, the intestine was cut longitudinally into 0.5-cm fragments. The fragments were incubated with 5 ml of 30 mM EDTA and placed in 5 ml digestion solution containing 4% FBS, 0.5 mg/ml each of Collagenase D (Roche) and DNase I (Sigma), and 50 U/ml Dispase (Fisher). The fragments were incubated with Dulbecco's PBS (DPBS) for 20 min at 37 • C by slow rotation (100 rpm). After incubating, the epithelial cells layer which contained the IELs were separated by intensive vortex and passed through a 70-μm cell strainer. Single cell suspension was collected and washed twice by DPBS, the solution was vortexed intensely and passed through a 40-μm cell strainer. Supernatants was washed by precooled RPMI medium 1640 (Thermo Fisher Scientific) and suspended by 10 ml of the 40% fraction of a 40:80 Percoll gradient, overlaid on 5 ml of the 80% fraction in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifuging for 20 min at 2500 rpm. LP lymphocytes (LPLs) were collected at the interphase of the Percoll gradient, then washed and suspended in FACS buffer or T cell medium. In the meantime, flow cytometry analysis was performed on BD Facscalibur (BD Biosciences) instruments and analyzed by FlowJo software. All antibodies were purchased from BD Pharmingen or eBiosciences. Isolated single-cell suspensions were stained with anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, all at 1:100 dilution for 30 min on ice, and washed with PBS twice, and analyzed by FACS [26] . Cytokines interleukin (IL) 10 (IL-10) and IL-1β (Abcam) were measured by ELISA according to the manufacturer's instructions. Data were acquired on an automated ELISA plate reader at OD 450 nm immediately. PEDV neutralizing antibodies were measured in intestine washing liquid by plaque reduction neutralization test (PRNT). The test was performed as previously described with minor modifications [27] . A total of 450 μl of intestine washing liquid was two-fold serially diluted and mixed with 50 μl viral suspension containing 10 3 TCID 50 PEDV virus for 1 h at 37 • C in 12-well flat bottomed tissue culture plates. The mixture was then inoculated for 1 h at 37 • C and 5% CO 2 . Then, the mixture was inoculated with Vero cells suspension (approximately 1.0 × 10 6 ml −1 ) for another 3-4 days. After staining with Crystal Violet, the plates were observed under a microscope for cytopathic effect. Data were obtained as the means + − S.E.M. of three replicates per test in a single experiment. GraphPad Prism V6.0 (San Diego, CA, U.S.A.) used to perform statistical analyses. Tukey's multiple comparison tests and one-way ANOVA were used to analyze the significance of the difference between means. P-values less than 0.05 (P<0.05) were considered significant and P-values less than 0.01 (P<0.01) as highly significant. PPs are a concentrate of lymphoid tissue and the primary site for immunoglobulin A (IgA) production which is crucial to regulate the homeostatic balance of intestine [28] . The area of PPs is a key immunity indicator. Oral administration with B. subtilis-RC significantly (P<0.01) increased the area of PPs compared with two control groups as shown in Figure 1A . In addition, the villi length of ileum got longer by oral administration with B. subtilis-RC (P<0.01) than the other two groups ( Figure 1B) . These primarily confirmed that B. subtilis-RC was beneficial to maintain the structure of intestine. Intestinal IELs are a large and diverse population of lymphoid cells residing within the intestinal epithelial cells (IECs), and forming the intestinal mucosal barrier [29] . IELs are important part of the gut mucosal immune system. The level of specific anti-PEDV ileum IgA + secreting (SIgA) antibody in piglets was measured by ELISA in the mouth and feces. As shown in Figure 3A ,B, antigen-specific mucosal SIgA in the above sites was clearly higher than inactivated PEDV group (P<0.05 or P<0.01). As expected, the mouth had higher levels of SIgA than other sites. After oral immunization, the level of serum anti-PEDV IgG antibody in piglets immunized with B. subtilis-RC, inactivated PEDV or PBS were determined by ELISA, as shown in Figure 3C . The results indicated that although the titers dropped during sampling period, the IgG level of B. subtilis-RC still significantly increased from 0 to 33 days than inactivated PEDV group (P<0.05 or P<0.01). CD3 + T lymphocytes are the fundamental cell surface markers of T lymphocytes, therefore, the number of CD3 + T lymphocytes could represent the quantity of T lymphocytes. Consequently, we analyzed the number of CD3 + T lymphocytes in ileum. The data indicated that both B. subtilis-RC and inactivated PEDV could dramatically (P<0.05) increase CD3 + T lymphocytes compared with PBS group ( Figure 4A ). These changes showed confident evidence that oral administration with B. subtilis-RC had a good influence on intestinal mucosal immunity in piglets. SIgA is the main immunoglobulin isotype in animals, largely secreted across the intestinal mucosal surface especially in the small intestine [30] . SIgA plays an important role in intestinal mucosal immunity and reflects on the intestinal mucosal immunity. After oral administration with B. subtilis-RC, the number of IgA secreting cells had quickly risen compared with the other two groups (P<0.05) ( Figure 4B) . These results showed that oral administration with B. subtilis-RC was conducive to intestinal mucosal immunity and could increase the number of IgA secreting cells to produce positive effects on against PEDV infection. A great deal of immune cells are scattered in the epithelial cells. IECs indirectly or directly interact with innate and adaptive immune cells by presenting antigens to lymphocytes [31] . Consequently, learning about how the lymphocytes are distributed in the small intestinal mucosa is very meaningful for mucosal immunology. Previous data had shown that CD3 + T lymphocytes significantly (P<0.05) increased ( Figure 4A ), so we further analyzed the immunological classification of CD3 + T lymphocytes. The lymphocyte of the ileum with PPs junction was isolated and the lymphocytes of CD3, CD4, and CD8 were analyzed by three colors flow cytometry ( Figure 5A ). These results showed that CD3 + CD4 + T cells have obviously (P<0.01) increased ( Figure 5B ), nevertheless the CD3 + CD8 + T cells remarkably (P<0.05) declined ( Figure 5C ). After calculation, the ratio of CD4 + /CD8 + T cells increased ( Figure 5D ). This ratio could also further measure the immunity levels of piglets. Cytokine IL-1β and IL-10 levels were determined to evaluate cellular immune responses induced by B. subtilis-RC as shown in Figure 6A ,B. As we can see from the diagram, significantly (P<0.01) higher IL-1β and IL-10 were produced after oral administration with B. subtilis-RC than the other two groups. These all revealed that B. subtilis-RC could stimulate cytokines release to mediate communication with and between cells of the immune system, improving the mucosal immune response to PEDV infection. The PEDV neutralizing antibodies were detected by PRNT assay. Oral administration with B. subtilis-RC could effectively reduce the plaque-forming ability of PEDV (P<0.01) compared with other two groups in Figure 7 . This revealed that B. subtilis-RC could stimulate high level of PEDV neutralizing antibodies against PEDV infection. Amidst the PEDV outbreak, various vaccines have been developed to control diseases and the effects are unsatisfactory. Oral vaccines can induce more robust mucosal immunity than injectable counterparts [32] . Therefore, oral immunization has appeared as an effective strategy for controlling PEDV outbreak [33] . It is now clear that effective mucosal immune response requires serum IgG and mucosal SIgA [34] . SIgA is the basis of the mucosal immune system, playing an important role in maintaining the immune homeostasis, and neutralizing the invasive pathogens. Serum IgG represents systemic immune responses. During PEDV infections, oral immunization elicits not only mucosal but also systemic immune responses very well [35] . Our data showed a strong and long-lasting anti-PEDV IgG response were detected by oral administration with B. subtilis-RC in piglets. Although as time went on, the antibody titers declined a little, it still stayed on overhead compared with control groups and with accordance to the changeable tendency of antibodies. The change of specific IgA showed similar results in mouth and feces mucosa. All these changes had contributed to fight PEDV infection. As the extra immunity boost, B. subtilis-RC reduced the ability of pathogens to cross the intestinal mucosa and the systemic spread of invasive pathogens [36] . The mucosal immune system generates immune responses through immune cells that reside in mucosal compartments. T lymphocytes residing in the mucosa play important roles in mucosal immunity [37] . We further explored the species, amounts, and distribution of T lymphocytes in the intestine mucosa. CD3 is a fundamental cell surface marker of T lymphocytes [38] . The result showed that the number of CD3 + T lymphocytes significantly increased, and these revealed that B. subtilis-RC could stimulate T-cell maturation. According to the molecules expressed on the cell surface, T lymphocytes can further divide into T helper cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells) [39] . Furthermore, we observed that the ratio of CD4 + /CD8 + T cells increased by oral administration. The CD4/CD8 ratio measures the ratio of T helper cells to cytotoxic T cells. Therefore, we could see that oral administration B. subtilis-RC could strengthen Th1 immune response by raising the ratio of CD4 + /CD8 + T cells. Small intestine morphology can directly reflect the intestinal health and plays an important role in maintaining the intestine immune system [40] . The early stage of PEDV infection is frequently accompanied by necrosis and exfoliation of infected villous epithelial cells, ultimately resulting in acute, severe villous atrophy [41] . Therefore, the effective work of maintaining intestine morphology is a good indicator for assessing the efficacy of vaccines. After oral administration with B. subtilis-RC, we found the area of PPs expanded significantly. PPs are small masses of lymphatic tissue and form an important part of the immune system by recruiting and inducting the T cells to prevent the growth of pathogens in the intestines. Furthermore, an increase in the number of IELs demonstrated the effectiveness of B. subtilis-RC. Moreover, the villi length of ileum showed some encouraging results that a well-formed intestine morphology came into being by B. subtilis-RC. The satisfactory intestine morphology was the first step on the road against PEDV infection. Several morphology results proved that B. subtilis-RC could remarkably maintain the intestine morphology and form comprehensive protection. As previously mentioned, oral administration with B. subtilis-RC could stimulate T-cell proliferation and differentiation and modulate the immune response. Moreover, cytokines are small-molecule proteins with wide biological activity, synthesized and secreted by immune cells and some non-immune cells [42] . As a cell signaling molecule, it mainly acts to regulate immune responses, participating in the differentiation and development of immune cells, mediating inflammatory responses, stimulating hematopoiesis, and participating in tissue repair. Previous studies had demonstrated that PEDV inhibited both NF-κB and pro-inflammatory cytokines [43] . Therefore, cytokines are a key indicator for evaluating the ability of a vaccine to stimulate immune responses. In this study, we had observed that IL-1β and IL-10 increased (P<0.01) remarkably. IL-1β as one of the earliest pro-inflammatory cytokines and is centrally involved in the initiation and regulation of inflammatory and innate immune responses. Research had shown that IL-1β could significantly up-regulate the local and systemic immune tissues post microbial infection [44] . In addition, IL-10 is a potent anti-inflammatory cytokine that plays an essential role in preventing inflammatory and autoimmune pathologies [45] . In summary, both data showed that oral administration with B. subtilis-RC regulated and enhanced immunity by up-regulating cytokines IL-1β and IL-10. In conclusion, the present results demonstrated that oral immunization with B. subtilis-RC could effectively induce local mucosal and systematic immune responses against PEDV infection, while enhancing and regulating the immune function by raising the ratio of CD4 + /CD8 + T cells and cytokines IL-1β and IL-10, thus pointing to a promising oral vaccine candidate for PEDV infection in piglets.
What is interleukin-1Beta?
false
613
{ "text": [ "pro-inflammatory cytokines" ], "answer_start": [ 22214 ] }
2,683
Estimating the number of infections and the impact of non- pharmaceutical interventions on COVID-19 in 11 European countries 30 March 2020 Imperial College COVID-19 Response Team Seth Flaxmani Swapnil Mishra*, Axel Gandy*, H JulietteT Unwin, Helen Coupland, Thomas A Mellan, Harrison Zhu, Tresnia Berah, Jeffrey W Eaton, Pablo N P Guzman, Nora Schmit, Lucia Cilloni, Kylie E C Ainslie, Marc Baguelin, Isobel Blake, Adhiratha Boonyasiri, Olivia Boyd, Lorenzo Cattarino, Constanze Ciavarella, Laura Cooper, Zulma Cucunuba’, Gina Cuomo—Dannenburg, Amy Dighe, Bimandra Djaafara, Ilaria Dorigatti, Sabine van Elsland, Rich FitzJohn, Han Fu, Katy Gaythorpe, Lily Geidelberg, Nicholas Grassly, Wi|| Green, Timothy Hallett, Arran Hamlet, Wes Hinsley, Ben Jeffrey, David Jorgensen, Edward Knock, Daniel Laydon, Gemma Nedjati—Gilani, Pierre Nouvellet, Kris Parag, Igor Siveroni, Hayley Thompson, Robert Verity, Erik Volz, Caroline Walters, Haowei Wang, Yuanrong Wang, Oliver Watson, Peter Winskill, Xiaoyue Xi, Charles Whittaker, Patrick GT Walker, Azra Ghani, Christl A. Donnelly, Steven Riley, Lucy C Okell, Michaela A C Vollmer, NeilM.Ferguson1and Samir Bhatt*1 Department of Infectious Disease Epidemiology, Imperial College London Department of Mathematics, Imperial College London WHO Collaborating Centre for Infectious Disease Modelling MRC Centre for Global Infectious Disease Analysis Abdul LatifJameeI Institute for Disease and Emergency Analytics, Imperial College London Department of Statistics, University of Oxford *Contributed equally 1Correspondence: nei|[email protected], [email protected] Summary Following the emergence of a novel coronavirus (SARS-CoV-Z) and its spread outside of China, Europe is now experiencing large epidemics. In response, many European countries have implemented unprecedented non-pharmaceutical interventions including case isolation, the closure of schools and universities, banning of mass gatherings and/or public events, and most recently, widescale social distancing including local and national Iockdowns. In this report, we use a semi-mechanistic Bayesian hierarchical model to attempt to infer the impact of these interventions across 11 European countries. Our methods assume that changes in the reproductive number— a measure of transmission - are an immediate response to these interventions being implemented rather than broader gradual changes in behaviour. Our model estimates these changes by calculating backwards from the deaths observed over time to estimate transmission that occurred several weeks prior, allowing for the time lag between infection and death. One of the key assumptions of the model is that each intervention has the same effect on the reproduction number across countries and over time. This allows us to leverage a greater amount of data across Europe to estimate these effects. It also means that our results are driven strongly by the data from countries with more advanced epidemics, and earlier interventions, such as Italy and Spain. We find that the slowing growth in daily reported deaths in Italy is consistent with a significant impact of interventions implemented several weeks earlier. In Italy, we estimate that the effective reproduction number, Rt, dropped to close to 1 around the time of Iockdown (11th March), although with a high level of uncertainty. Overall, we estimate that countries have managed to reduce their reproduction number. Our estimates have wide credible intervals and contain 1 for countries that have implemented a|| interventions considered in our analysis. This means that the reproduction number may be above or below this value. With current interventions remaining in place to at least the end of March, we estimate that interventions across all 11 countries will have averted 59,000 deaths up to 31 March [95% credible interval 21,000-120,000]. Many more deaths will be averted through ensuring that interventions remain in place until transmission drops to low levels. We estimate that, across all 11 countries between 7 and 43 million individuals have been infected with SARS-CoV-Z up to 28th March, representing between 1.88% and 11.43% ofthe population. The proportion of the population infected to date — the attack rate - is estimated to be highest in Spain followed by Italy and lowest in Germany and Norway, reflecting the relative stages of the epidemics. Given the lag of 2-3 weeks between when transmission changes occur and when their impact can be observed in trends in mortality, for most of the countries considered here it remains too early to be certain that recent interventions have been effective. If interventions in countries at earlier stages of their epidemic, such as Germany or the UK, are more or less effective than they were in the countries with advanced epidemics, on which our estimates are largely based, or if interventions have improved or worsened over time, then our estimates of the reproduction number and deaths averted would change accordingly. It is therefore critical that the current interventions remain in place and trends in cases and deaths are closely monitored in the coming days and weeks to provide reassurance that transmission of SARS-Cov-Z is slowing. SUGGESTED CITATION Seth Flaxman, Swapnil Mishra, Axel Gandy et 0/. Estimating the number of infections and the impact of non— pharmaceutical interventions on COVID—19 in 11 European countries. Imperial College London (2020), doi: https://doi.org/10.25561/77731 1 Introduction Following the emergence of a novel coronavirus (SARS-CoV-Z) in Wuhan, China in December 2019 and its global spread, large epidemics of the disease, caused by the virus designated COVID-19, have emerged in Europe. In response to the rising numbers of cases and deaths, and to maintain the capacity of health systems to treat as many severe cases as possible, European countries, like those in other continents, have implemented or are in the process of implementing measures to control their epidemics. These large-scale non-pharmaceutical interventions vary between countries but include social distancing (such as banning large gatherings and advising individuals not to socialize outside their households), border closures, school closures, measures to isolate symptomatic individuals and their contacts, and large-scale lockdowns of populations with all but essential internal travel banned. Understanding firstly, whether these interventions are having the desired impact of controlling the epidemic and secondly, which interventions are necessary to maintain control, is critical given their large economic and social costs. The key aim ofthese interventions is to reduce the effective reproduction number, Rt, ofthe infection, a fundamental epidemiological quantity representing the average number of infections, at time t, per infected case over the course of their infection. Ith is maintained at less than 1, the incidence of new infections decreases, ultimately resulting in control of the epidemic. If Rt is greater than 1, then infections will increase (dependent on how much greater than 1 the reproduction number is) until the epidemic peaks and eventually declines due to acquisition of herd immunity. In China, strict movement restrictions and other measures including case isolation and quarantine began to be introduced from 23rd January, which achieved a downward trend in the number of confirmed new cases during February, resulting in zero new confirmed indigenous cases in Wuhan by March 19th. Studies have estimated how Rt changed during this time in different areas ofChina from around 2-4 during the uncontrolled epidemic down to below 1, with an estimated 7-9 fold decrease in the number of daily contacts per person.1'2 Control measures such as social distancing, intensive testing, and contact tracing in other countries such as Singapore and South Korea have successfully reduced case incidence in recent weeks, although there is a riskthe virus will spread again once control measures are relaxed.3'4 The epidemic began slightly laterin Europe, from January or later in different regions.5 Countries have implemented different combinations of control measures and the level of adherence to government recommendations on social distancing is likely to vary between countries, in part due to different levels of enforcement. Estimating reproduction numbers for SARS-CoV-Z presents challenges due to the high proportion of infections not detected by health systems”7 and regular changes in testing policies, resulting in different proportions of infections being detected over time and between countries. Most countries so far only have the capacity to test a small proportion of suspected cases and tests are reserved for severely ill patients or for high-risk groups (e.g. contacts of cases). Looking at case data, therefore, gives a systematically biased view of trends. An alternative way to estimate the course of the epidemic is to back-calculate infections from observed deaths. Reported deaths are likely to be more reliable, although the early focus of most surveillance systems on cases with reported travel histories to China may mean that some early deaths will have been missed. Whilst the recent trends in deaths will therefore be informative, there is a time lag in observing the effect of interventions on deaths since there is a 2-3-week period between infection, onset of symptoms and outcome. In this report, we fit a novel Bayesian mechanistic model of the infection cycle to observed deaths in 11 European countries, inferring plausible upper and lower bounds (Bayesian credible intervals) of the total populations infected (attack rates), case detection probabilities, and the reproduction number over time (Rt). We fit the model jointly to COVID-19 data from all these countries to assess whether there is evidence that interventions have so far been successful at reducing Rt below 1, with the strong assumption that particular interventions are achieving a similar impact in different countries and that the efficacy of those interventions remains constant over time. The model is informed more strongly by countries with larger numbers of deaths and which implemented interventions earlier, therefore estimates of recent Rt in countries with more recent interventions are contingent on similar intervention impacts. Data in the coming weeks will enable estimation of country-specific Rt with greater precision. Model and data details are presented in the appendix, validation and sensitivity are also presented in the appendix, and general limitations presented below in the conclusions. 2 Results The timing of interventions should be taken in the context of when an individual country’s epidemic started to grow along with the speed with which control measures were implemented. Italy was the first to begin intervention measures, and other countries followed soon afterwards (Figure 1). Most interventions began around 12th-14th March. We analyzed data on deaths up to 28th March, giving a 2-3-week window over which to estimate the effect of interventions. Currently, most countries in our study have implemented all major non-pharmaceutical interventions. For each country, we model the number of infections, the number of deaths, and Rt, the effective reproduction number over time, with Rt changing only when an intervention is introduced (Figure 2- 12). Rt is the average number of secondary infections per infected individual, assuming that the interventions that are in place at time t stay in place throughout their entire infectious period. Every country has its own individual starting reproduction number Rt before interventions take place. Specific interventions are assumed to have the same relative impact on Rt in each country when they were introduced there and are informed by mortality data across all countries. Figure l: Intervention timings for the 11 European countries included in the analysis. For further details see Appendix 8.6. 2.1 Estimated true numbers of infections and current attack rates In all countries, we estimate there are orders of magnitude fewer infections detected (Figure 2) than true infections, mostly likely due to mild and asymptomatic infections as well as limited testing capacity. In Italy, our results suggest that, cumulatively, 5.9 [1.9-15.2] million people have been infected as of March 28th, giving an attack rate of 9.8% [3.2%-25%] of the population (Table 1). Spain has recently seen a large increase in the number of deaths, and given its smaller population, our model estimates that a higher proportion of the population, 15.0% (7.0 [18-19] million people) have been infected to date. Germany is estimated to have one of the lowest attack rates at 0.7% with 600,000 [240,000-1,500,000] people infected. Imperial College COVID-19 Response Team Table l: Posterior model estimates of percentage of total population infected as of 28th March 2020. Country % of total population infected (mean [95% credible intervall) Austria 1.1% [0.36%-3.1%] Belgium 3.7% [1.3%-9.7%] Denmark 1.1% [0.40%-3.1%] France 3.0% [1.1%-7.4%] Germany 0.72% [0.28%-1.8%] Italy 9.8% [3.2%-26%] Norway 0.41% [0.09%-1.2%] Spain 15% [3.7%-41%] Sweden 3.1% [0.85%-8.4%] Switzerland 3.2% [1.3%-7.6%] United Kingdom 2.7% [1.2%-5.4%] 2.2 Reproduction numbers and impact of interventions Averaged across all countries, we estimate initial reproduction numbers of around 3.87 [3.01-4.66], which is in line with other estimates.1'8 These estimates are informed by our choice of serial interval distribution and the initial growth rate of observed deaths. A shorter assumed serial interval results in lower starting reproduction numbers (Appendix 8.4.2, Appendix 8.4.6). The initial reproduction numbers are also uncertain due to (a) importation being the dominant source of new infections early in the epidemic, rather than local transmission (b) possible under-ascertainment in deaths particularly before testing became widespread. We estimate large changes in Rt in response to the combined non-pharmaceutical interventions. Our results, which are driven largely by countries with advanced epidemics and larger numbers of deaths (e.g. Italy, Spain), suggest that these interventions have together had a substantial impact on transmission, as measured by changes in the estimated reproduction number Rt. Across all countries we find current estimates of Rt to range from a posterior mean of 0.97 [0.14-2.14] for Norway to a posterior mean of2.64 [1.40-4.18] for Sweden, with an average of 1.43 across the 11 country posterior means, a 64% reduction compared to the pre-intervention values. We note that these estimates are contingent on intervention impact being the same in different countries and at different times. In all countries but Sweden, under the same assumptions, we estimate that the current reproduction number includes 1 in the uncertainty range. The estimated reproduction number for Sweden is higher, not because the mortality trends are significantly different from any other country, but as an artefact of our model, which assumes a smaller reduction in Rt because no full lockdown has been ordered so far. Overall, we cannot yet conclude whether current interventions are sufficient to drive Rt below 1 (posterior probability of being less than 1.0 is 44% on average across the countries). We are also unable to conclude whether interventions may be different between countries or over time. There remains a high level of uncertainty in these estimates. It is too early to detect substantial intervention impact in many countries at earlier stages of their epidemic (e.g. Germany, UK, Norway). Many interventions have occurred only recently, and their effects have not yet been fully observed due to the time lag between infection and death. This uncertainty will reduce as more data become available. For all countries, our model fits observed deaths data well (Bayesian goodness of fit tests). We also found that our model can reliably forecast daily deaths 3 days into the future, by withholding the latest 3 days of data and comparing model predictions to observed deaths (Appendix 8.3). The close spacing of interventions in time made it statistically impossible to determine which had the greatest effect (Figure 1, Figure 4). However, when doing a sensitivity analysis (Appendix 8.4.3) with uninformative prior distributions (where interventions can increase deaths) we find similar impact of Imperial College COVID-19 Response Team interventions, which shows that our choice of prior distribution is not driving the effects we see in the main analysis. Figure 2: Country-level estimates of infections, deaths and Rt. Left: daily number of infections, brown bars are reported infections, blue bands are predicted infections, dark blue 50% credible interval (CI), light blue 95% CI. The number of daily infections estimated by our model drops immediately after an intervention, as we assume that all infected people become immediately less infectious through the intervention. Afterwards, if the Rt is above 1, the number of infections will starts growing again. Middle: daily number of deaths, brown bars are reported deaths, blue bands are predicted deaths, CI as in left plot. Right: time-varying reproduction number Rt, dark green 50% CI, light green 95% CI. Icons are interventions shown at the time they occurred. Imperial College COVID-19 Response Team Table 2: Totalforecasted deaths since the beginning of the epidemic up to 31 March in our model and in a counterfactual model (assuming no intervention had taken place). Estimated averted deaths over this time period as a result of the interventions. Numbers in brackets are 95% credible intervals. 2.3 Estimated impact of interventions on deaths Table 2 shows total forecasted deaths since the beginning of the epidemic up to and including 31 March under ourfitted model and under the counterfactual model, which predicts what would have happened if no interventions were implemented (and R, = R0 i.e. the initial reproduction number estimated before interventions). Again, the assumption in these predictions is that intervention impact is the same across countries and time. The model without interventions was unable to capture recent trends in deaths in several countries, where the rate of increase had clearly slowed (Figure 3). Trends were confirmed statistically by Bayesian leave-one-out cross-validation and the widely applicable information criterion assessments —WA|C). By comparing the deaths predicted under the model with no interventions to the deaths predicted in our intervention model, we calculated the total deaths averted up to the end of March. We find that, across 11 countries, since the beginning of the epidemic, 59,000 [21,000-120,000] deaths have been averted due to interventions. In Italy and Spain, where the epidemic is advanced, 38,000 [13,000- 84,000] and 16,000 [5,400-35,000] deaths have been averted, respectively. Even in the UK, which is much earlier in its epidemic, we predict 370 [73-1,000] deaths have been averted. These numbers give only the deaths averted that would have occurred up to 31 March. lfwe were to include the deaths of currently infected individuals in both models, which might happen after 31 March, then the deaths averted would be substantially higher. Figure 3: Daily number of confirmed deaths, predictions (up to 28 March) and forecasts (after) for (a) Italy and (b) Spain from our model with interventions (blue) and from the no interventions counterfactual model (pink); credible intervals are shown one week into the future. Other countries are shown in Appendix 8.6. 03/0 25% 50% 753% 100% (no effect on transmissibility) (ends transmissibility Relative % reduction in R. Figure 4: Our model includes five covariates for governmental interventions, adjusting for whether the intervention was the first one undertaken by the government in response to COVID-19 (red) or was subsequent to other interventions (green). Mean relative percentage reduction in Rt is shown with 95% posterior credible intervals. If 100% reduction is achieved, Rt = 0 and there is no more transmission of COVID-19. No effects are significantly different from any others, probably due to the fact that many interventions occurred on the same day or within days of each other as shown in Figure l. 3 Discussion During this early phase of control measures against the novel coronavirus in Europe, we analyze trends in numbers of deaths to assess the extent to which transmission is being reduced. Representing the COVlD-19 infection process using a semi-mechanistic, joint, Bayesian hierarchical model, we can reproduce trends observed in the data on deaths and can forecast accurately over short time horizons. We estimate that there have been many more infections than are currently reported. The high level of under-ascertainment of infections that we estimate here is likely due to the focus on testing in hospital settings rather than in the community. Despite this, only a small minority of individuals in each country have been infected, with an attack rate on average of 4.9% [l.9%-ll%] with considerable variation between countries (Table 1). Our estimates imply that the populations in Europe are not close to herd immunity ("50-75% if R0 is 2-4). Further, with Rt values dropping substantially, the rate of acquisition of herd immunity will slow down rapidly. This implies that the virus will be able to spread rapidly should interventions be lifted. Such estimates of the attack rate to date urgently need to be validated by newly developed antibody tests in representative population surveys, once these become available. We estimate that major non-pharmaceutical interventions have had a substantial impact on the time- varying reproduction numbers in countries where there has been time to observe intervention effects on trends in deaths (Italy, Spain). lfadherence in those countries has changed since that initial period, then our forecast of future deaths will be affected accordingly: increasing adherence over time will have resulted in fewer deaths and decreasing adherence in more deaths. Similarly, our estimates of the impact ofinterventions in other countries should be viewed with caution if the same interventions have achieved different levels of adherence than was initially the case in Italy and Spain. Due to the implementation of interventions in rapid succession in many countries, there are not enough data to estimate the individual effect size of each intervention, and we discourage attributing associations to individual intervention. In some cases, such as Norway, where all interventions were implemented at once, these individual effects are by definition unidentifiable. Despite this, while individual impacts cannot be determined, their estimated joint impact is strongly empirically justified (see Appendix 8.4 for sensitivity analysis). While the growth in daily deaths has decreased, due to the lag between infections and deaths, continued rises in daily deaths are to be expected for some time. To understand the impact of interventions, we fit a counterfactual model without the interventions and compare this to the actual model. Consider Italy and the UK - two countries at very different stages in their epidemics. For the UK, where interventions are very recent, much of the intervention strength is borrowed from countries with older epidemics. The results suggest that interventions will have a large impact on infections and deaths despite counts of both rising. For Italy, where far more time has passed since the interventions have been implemented, it is clear that the model without interventions does not fit well to the data, and cannot explain the sub-linear (on the logarithmic scale) reduction in deaths (see Figure 10). The counterfactual model for Italy suggests that despite mounting pressure on health systems, interventions have averted a health care catastrophe where the number of new deaths would have been 3.7 times higher (38,000 deaths averted) than currently observed. Even in the UK, much earlier in its epidemic, the recent interventions are forecasted to avert 370 total deaths up to 31 of March. 4 Conclusion and Limitations Modern understanding of infectious disease with a global publicized response has meant that nationwide interventions could be implemented with widespread adherence and support. Given observed infection fatality ratios and the epidemiology of COVlD-19, major non-pharmaceutical interventions have had a substantial impact in reducing transmission in countries with more advanced epidemics. It is too early to be sure whether similar reductions will be seen in countries at earlier stages of their epidemic. While we cannot determine which set of interventions have been most successful, taken together, we can already see changes in the trends of new deaths. When forecasting 3 days and looking over the whole epidemic the number of deaths averted is substantial. We note that substantial innovation is taking place, and new more effective interventions or refinements of current interventions, alongside behavioral changes will further contribute to reductions in infections. We cannot say for certain that the current measures have controlled the epidemic in Europe; however, if current trends continue, there is reason for optimism. Our approach is semi-mechanistic. We propose a plausible structure for the infection process and then estimate parameters empirically. However, many parameters had to be given strong prior distributions or had to be fixed. For these assumptions, we have provided relevant citations to previous studies. As more data become available and better estimates arise, we will update these in weekly reports. Our choice of serial interval distribution strongly influences the prior distribution for starting R0. Our infection fatality ratio, and infection-to-onset-to-death distributions strongly influence the rate of death and hence the estimated number of true underlying cases. We also assume that the effect of interventions is the same in all countries, which may not be fully realistic. This assumption implies that countries with early interventions and more deaths since these interventions (e.g. Italy, Spain) strongly influence estimates of intervention impact in countries at earlier stages of their epidemic with fewer deaths (e.g. Germany, UK). We have tried to create consistent definitions of all interventions and document details of this in Appendix 8.6. However, invariably there will be differences from country to country in the strength of their intervention — for example, most countries have banned gatherings of more than 2 people when implementing a lockdown, whereas in Sweden the government only banned gatherings of more than 10 people. These differences can skew impacts in countries with very little data. We believe that our uncertainty to some degree can cover these differences, and as more data become available, coefficients should become more reliable. However, despite these strong assumptions, there is sufficient signal in the data to estimate changes in R, (see the sensitivity analysis reported in Appendix 8.4.3) and this signal will stand to increase with time. In our Bayesian hierarchical framework, we robustly quantify the uncertainty in our parameter estimates and posterior predictions. This can be seen in the very wide credible intervals in more recent days, where little or no death data are available to inform the estimates. Furthermore, we predict intervention impact at country-level, but different trends may be in place in different parts of each country. For example, the epidemic in northern Italy was subject to controls earlier than the rest of the country. 5 Data Our model utilizes daily real-time death data from the ECDC (European Centre of Disease Control), where we catalogue case data for 11 European countries currently experiencing the epidemic: Austria, Belgium, Denmark, France, Germany, Italy, Norway, Spain, Sweden, Switzerland and the United Kingdom. The ECDC provides information on confirmed cases and deaths attributable to COVID-19. However, the case data are highly unrepresentative of the incidence of infections due to underreporting as well as systematic and country-specific changes in testing. We, therefore, use only deaths attributable to COVID-19 in our model; we do not use the ECDC case estimates at all. While the observed deaths still have some degree of unreliability, again due to changes in reporting and testing, we believe the data are ofsufficient fidelity to model. For population counts, we use UNPOP age-stratified counts.10 We also catalogue data on the nature and type of major non-pharmaceutical interventions. We looked at the government webpages from each country as well as their official public health division/information webpages to identify the latest advice/laws being issued by the government and public health authorities. We collected the following: School closure ordered: This intervention refers to nationwide extraordinary school closures which in most cases refer to both primary and secondary schools closing (for most countries this also includes the closure of otherforms of higher education or the advice to teach remotely). In the case of Denmark and Sweden, we allowed partial school closures of only secondary schools. The date of the school closure is taken to be the effective date when the schools started to be closed (ifthis was on a Monday, the date used was the one of the previous Saturdays as pupils and students effectively stayed at home from that date onwards). Case-based measures: This intervention comprises strong recommendations or laws to the general public and primary care about self—isolation when showing COVID-19-like symptoms. These also include nationwide testing programs where individuals can be tested and subsequently self—isolated. Our definition is restricted to nationwide government advice to all individuals (e.g. UK) or to all primary care and excludes regional only advice. These do not include containment phase interventions such as isolation if travelling back from an epidemic country such as China. Public events banned: This refers to banning all public events of more than 100 participants such as sports events. Social distancing encouraged: As one of the first interventions against the spread of the COVID-19 pandemic, many governments have published advice on social distancing including the recommendation to work from home wherever possible, reducing use ofpublictransport and all other non-essential contact. The dates used are those when social distancing has officially been recommended by the government; the advice may include maintaining a recommended physical distance from others. Lockdown decreed: There are several different scenarios that the media refers to as lockdown. As an overall definition, we consider regulations/legislations regarding strict face-to-face social interaction: including the banning of any non-essential public gatherings, closure of educational and public/cultural institutions, ordering people to stay home apart from exercise and essential tasks. We include special cases where these are not explicitly mentioned on government websites but are enforced by the police (e.g. France). The dates used are the effective dates when these legislations have been implemented. We note that lockdown encompasses other interventions previously implemented. First intervention: As Figure 1 shows, European governments have escalated interventions rapidly, and in some examples (Norway/Denmark) have implemented these interventions all on a single day. Therefore, given the temporal autocorrelation inherent in government intervention, we include a binary covariate for the first intervention, which can be interpreted as a government decision to take major action to control COVID-19. A full list of the timing of these interventions and the sources we have used can be found in Appendix 8.6. 6 Methods Summary A Visual summary of our model is presented in Figure 5 (details in Appendix 8.1 and 8.2). Replication code is available at https://github.com/|mperia|CollegeLondon/covid19model/releases/tag/vl.0 We fit our model to observed deaths according to ECDC data from 11 European countries. The modelled deaths are informed by an infection-to-onset distribution (time from infection to the onset of symptoms), an onset-to-death distribution (time from the onset of symptoms to death), and the population-averaged infection fatality ratio (adjusted for the age structure and contact patterns of each country, see Appendix). Given these distributions and ratios, modelled deaths are a function of the number of infections. The modelled number of infections is informed by the serial interval distribution (the average time from infection of one person to the time at which they infect another) and the time-varying reproduction number. Finally, the time-varying reproduction number is a function of the initial reproduction number before interventions and the effect sizes from interventions. Figure 5: Summary of model components. Following the hierarchy from bottom to top gives us a full framework to see how interventions affect infections, which can result in deaths. We use Bayesian inference to ensure our modelled deaths can reproduce the observed deaths as closely as possible. From bottom to top in Figure 5, there is an implicit lag in time that means the effect of very recent interventions manifest weakly in current deaths (and get stronger as time progresses). To maximise the ability to observe intervention impact on deaths, we fit our model jointly for all 11 European countries, which results in a large data set. Our model jointly estimates the effect sizes of interventions. We have evaluated the effect ofour Bayesian prior distribution choices and evaluate our Bayesian posterior calibration to ensure our results are statistically robust (Appendix 8.4). 7 Acknowledgements Initial research on covariates in Appendix 8.6 was crowdsourced; we thank a number of people across the world for help with this. This work was supported by Centre funding from the UK Medical Research Council under a concordat with the UK Department for International Development, the NIHR Health Protection Research Unit in Modelling Methodology and CommunityJameel. 8 Appendix: Model Specifics, Validation and Sensitivity Analysis 8.1 Death model We observe daily deaths Dam for days t E 1, ...,n and countries m E 1, ...,p. These daily deaths are modelled using a positive real-Valued function dam = E(Dam) that represents the expected number of deaths attributed to COVID-19. Dam is assumed to follow a negative binomial distribution with The expected number of deaths (1 in a given country on a given day is a function of the number of infections C occurring in previous days. At the beginning of the epidemic, the observed deaths in a country can be dominated by deaths that result from infection that are not locally acquired. To avoid biasing our model by this, we only include observed deaths from the day after a country has cumulatively observed 10 deaths in our model. To mechanistically link ourfunction for deaths to infected cases, we use a previously estimated COVID- 19 infection-fatality-ratio ifr (probability of death given infection)9 together with a distribution oftimes from infection to death TE. The ifr is derived from estimates presented in Verity et al11 which assumed homogeneous attack rates across age-groups. To better match estimates of attack rates by age generated using more detailed information on country and age-specific mixing patterns, we scale these estimates (the unadjusted ifr, referred to here as ifr’) in the following way as in previous work.4 Let Ca be the number of infections generated in age-group a, Na the underlying size of the population in that age group and AR“ 2 Ca/Na the age-group-specific attack rate. The adjusted ifr is then given by: ifra = fififié, where AR50_59 is the predicted attack-rate in the 50-59 year age-group after incorporating country-specific patterns of contact and mixing. This age-group was chosen as the reference as it had the lowest predicted level of underreporting in previous analyses of data from the Chinese epidemic“. We obtained country-specific estimates of attack rate by age, AR“, for the 11 European countries in our analysis from a previous study which incorporates information on contact between individuals of different ages in countries across Europe.12 We then obtained overall ifr estimates for each country adjusting for both demography and age-specific attack rates. Using estimated epidemiological information from previous studies,“'11 we assume TE to be the sum of two independent random times: the incubation period (infection to onset of symptoms or infection- to-onset) distribution and the time between onset of symptoms and death (onset-to-death). The infection-to-onset distribution is Gamma distributed with mean 5.1 days and coefficient of variation 0.86. The onset-to-death distribution is also Gamma distributed with a mean of 18.8 days and a coefficient of va riation 0.45. ifrm is population averaged over the age structure of a given country. The infection-to-death distribution is therefore given by: um ~ ifrm ~ (Gamma(5.1,0.86) + Gamma(18.8,0.45)) Figure 6 shows the infection-to-death distribution and the resulting survival function that integrates to the infection fatality ratio. Figure 6: Left, infection-to-death distribution (mean 23.9 days). Right, survival probability of infected individuals per day given the infection fatality ratio (1%) and the infection-to-death distribution on the left. Using the probability of death distribution, the expected number of deaths dam, on a given day t, for country, m, is given by the following discrete sum: The number of deaths today is the sum of the past infections weighted by their probability of death, where the probability of death depends on the number of days since infection. 8.2 Infection model The true number of infected individuals, C, is modelled using a discrete renewal process. This approach has been used in numerous previous studies13'16 and has a strong theoretical basis in stochastic individual-based counting processes such as Hawkes process and the Bellman-Harris process.”18 The renewal model is related to the Susceptible-Infected-Recovered model, except the renewal is not expressed in differential form. To model the number ofinfections over time we need to specify a serial interval distribution g with density g(T), (the time between when a person gets infected and when they subsequently infect another other people), which we choose to be Gamma distributed: g ~ Gamma (6.50.62). The serial interval distribution is shown below in Figure 7 and is assumed to be the same for all countries. Figure 7: Serial interval distribution g with a mean of 6.5 days. Given the serial interval distribution, the number of infections Eamon a given day t, and country, m, is given by the following discrete convolution function: _ t—1 Cam — Ram ZT=0 Cr,mgt—‘r r where, similarto the probability ofdeath function, the daily serial interval is discretized by fs+0.5 1.5 gs = T=s—0.Sg(T)dT fors = 2,3, and 91 = fT=Og(T)dT. Infections today depend on the number of infections in the previous days, weighted by the discretized serial interval distribution. This weighting is then scaled by the country-specific time-Varying reproduction number, Ram, that models the average number of secondary infections at a given time. The functional form for the time-Varying reproduction number was chosen to be as simple as possible to minimize the impact of strong prior assumptions: we use a piecewise constant function that scales Ram from a baseline prior R0,m and is driven by known major non-pharmaceutical interventions occurring in different countries and times. We included 6 interventions, one of which is constructed from the other 5 interventions, which are timings of school and university closures (k=l), self—isolating if ill (k=2), banning of public events (k=3), any government intervention in place (k=4), implementing a partial or complete lockdown (k=5) and encouraging social distancing and isolation (k=6). We denote the indicator variable for intervention k E 1,2,3,4,5,6 by IkI’m, which is 1 if intervention k is in place in country m at time t and 0 otherwise. The covariate ”any government intervention” (k=4) indicates if any of the other 5 interventions are in effect,i.e.14’t’m equals 1 at time t if any of the interventions k E 1,2,3,4,5 are in effect in country m at time t and equals 0 otherwise. Covariate 4 has the interpretation of indicating the onset of major government intervention. The effect of each intervention is assumed to be multiplicative. Ram is therefore a function ofthe intervention indicators Ik’t’m in place at time t in country m: Ram : R0,m eXp(— 212:1 O(Rheum)- The exponential form was used to ensure positivity of the reproduction number, with R0,m constrained to be positive as it appears outside the exponential. The impact of each intervention on Ram is characterised by a set of parameters 0(1, ...,OL6, with independent prior distributions chosen to be ock ~ Gamma(. 5,1). The impacts ock are shared between all m countries and therefore they are informed by all available data. The prior distribution for R0 was chosen to be R0,m ~ Normal(2.4, IKI) with K ~ Normal(0,0.5), Once again, K is the same among all countries to share information. We assume that seeding of new infections begins 30 days before the day after a country has cumulatively observed 10 deaths. From this date, we seed our model with 6 sequential days of infections drawn from cl’m,...,66’m~EXponential(T), where T~Exponential(0.03). These seed infections are inferred in our Bayesian posterior distribution. We estimated parameters jointly for all 11 countries in a single hierarchical model. Fitting was done in the probabilistic programming language Stan,19 using an adaptive Hamiltonian Monte Carlo (HMC) sampler. We ran 8 chains for 4000 iterations with 2000 iterations of warmup and a thinning factor 4 to obtain 2000 posterior samples. Posterior convergence was assessed using the Rhat statistic and by diagnosing divergent transitions of the HMC sampler. Prior-posterior calibrations were also performed (see below). 8.3 Validation We validate accuracy of point estimates of our model using cross-Validation. In our cross-validation scheme, we leave out 3 days of known death data (non-cumulative) and fit our model. We forecast what the model predicts for these three days. We present the individual forecasts for each day, as well as the average forecast for those three days. The cross-validation results are shown in the Figure 8. Figure 8: Cross-Validation results for 3-day and 3-day aggregatedforecasts Figure 8 provides strong empirical justification for our model specification and mechanism. Our accurate forecast over a three-day time horizon suggests that our fitted estimates for Rt are appropriate and plausible. Along with from point estimates we all evaluate our posterior credible intervals using the Rhat statistic. The Rhat statistic measures whether our Markov Chain Monte Carlo (MCMC) chains have converged to the equilibrium distribution (the correct posterior distribution). Figure 9 shows the Rhat statistics for all of our parameters Figure 9: Rhat statistics - values close to 1 indicate MCMC convergence. Figure 9 indicates that our MCMC have converged. In fitting we also ensured that the MCMC sampler experienced no divergent transitions - suggesting non pathological posterior topologies. 8.4 SensitivityAnalysis 8.4.1 Forecasting on log-linear scale to assess signal in the data As we have highlighted throughout in this report, the lag between deaths and infections means that it ta kes time for information to propagate backwa rds from deaths to infections, and ultimately to Rt. A conclusion of this report is the prediction of a slowing of Rt in response to major interventions. To gain intuition that this is data driven and not simply a consequence of highly constrained model assumptions, we show death forecasts on a log-linear scale. On this scale a line which curves below a linear trend is indicative of slowing in the growth of the epidemic. Figure 10 to Figure 12 show these forecasts for Italy, Spain and the UK. They show this slowing down in the daily number of deaths. Our model suggests that Italy, a country that has the highest death toll of COVID-19, will see a slowing in the increase in daily deaths over the coming week compared to the early stages of the epidemic. We investigated the sensitivity of our estimates of starting and final Rt to our assumed serial interval distribution. For this we considered several scenarios, in which we changed the serial interval distribution mean, from a value of 6.5 days, to have values of 5, 6, 7 and 8 days. In Figure 13, we show our estimates of R0, the starting reproduction number before interventions, for each of these scenarios. The relative ordering of the Rt=0 in the countries is consistent in all settings. However, as expected, the scale of Rt=0 is considerably affected by this change — a longer serial interval results in a higher estimated Rt=0. This is because to reach the currently observed size of the epidemics, a longer assumed serial interval is compensated by a higher estimated R0. Additionally, in Figure 14, we show our estimates of Rt at the most recent model time point, again for each ofthese scenarios. The serial interval mean can influence Rt substantially, however, the posterior credible intervals of Rt are broadly overlapping. Figure 13: Initial reproduction number R0 for different serial interval (SI) distributions (means between 5 and 8 days). We use 6.5 days in our main analysis. Figure 14: Rt on 28 March 2020 estimated for all countries, with serial interval (SI) distribution means between 5 and 8 days. We use 6.5 days in our main analysis. 8.4.3 Uninformative prior sensitivity on or We ran our model using implausible uninformative prior distributions on the intervention effects, allowing the effect of an intervention to increase or decrease Rt. To avoid collinearity, we ran 6 separate models, with effects summarized below (compare with the main analysis in Figure 4). In this series of univariate analyses, we find (Figure 15) that all effects on their own serve to decrease Rt. This gives us confidence that our choice of prior distribution is not driving the effects we see in the main analysis. Lockdown has a very large effect, most likely due to the fact that it occurs after other interventions in our dataset. The relatively large effect sizes for the other interventions are most likely due to the coincidence of the interventions in time, such that one intervention is a proxy for a few others. Figure 15: Effects of different interventions when used as the only covariate in the model. 8.4.4 To assess prior assumptions on our piecewise constant functional form for Rt we test using a nonparametric function with a Gaussian process prior distribution. We fit a model with a Gaussian process prior distribution to data from Italy where there is the largest signal in death data. We find that the Gaussian process has a very similartrend to the piecewise constant model and reverts to the mean in regions of no data. The correspondence of a completely nonparametric function and our piecewise constant function suggests a suitable parametric specification of Rt. Nonparametric fitting of Rf using a Gaussian process: 8.4.5 Leave country out analysis Due to the different lengths of each European countries’ epidemic, some countries, such as Italy have much more data than others (such as the UK). To ensure that we are not leveraging too much information from any one country we perform a ”leave one country out” sensitivity analysis, where we rerun the model without a different country each time. Figure 16 and Figure 17 are examples for results for the UK, leaving out Italy and Spain. In general, for all countries, we observed no significant dependence on any one country. Figure 16: Model results for the UK, when not using data from Italy for fitting the model. See the Figure 17: Model results for the UK, when not using data from Spain for fitting the model. See caption of Figure 2 for an explanation of the plots. 8.4.6 Starting reproduction numbers vs theoretical predictions To validate our starting reproduction numbers, we compare our fitted values to those theoretically expected from a simpler model assuming exponential growth rate, and a serial interval distribution mean. We fit a linear model with a Poisson likelihood and log link function and extracting the daily growth rate r. For well-known theoretical results from the renewal equation, given a serial interval distribution g(r) with mean m and standard deviation 5, given a = mZ/S2 and b = m/SZ, and a subsequently R0 = (1 + %) .Figure 18 shows theoretically derived R0 along with our fitted estimates of Rt=0 from our Bayesian hierarchical model. As shown in Figure 18 there is large correspondence between our estimated starting reproduction number and the basic reproduction number implied by the growth rate r. R0 (red) vs R(FO) (black) Figure 18: Our estimated R0 (black) versus theoretically derived Ru(red) from a log-linear regression fit. 8.5 Counterfactual analysis — interventions vs no interventions Figure 19: Daily number of confirmed deaths, predictions (up to 28 March) and forecasts (after) for all countries except Italy and Spain from our model with interventions (blue) and from the no interventions counterfactual model (pink); credible intervals are shown one week into the future. DOI: https://doi.org/10.25561/77731 Page 28 of 35 30 March 2020 Imperial College COVID-19 Response Team 8.6 Data sources and Timeline of Interventions Figure 1 and Table 3 display the interventions by the 11 countries in our study and the dates these interventions became effective. Table 3: Timeline of Interventions. Country Type Event Date effective School closure ordered Nationwide school closures.20 14/3/2020 Public events banned Banning of gatherings of more than 5 people.21 10/3/2020 Banning all access to public spaces and gatherings Lockdown of more than 5 people. Advice to maintain 1m ordered distance.22 16/3/2020 Social distancing encouraged Recommendation to maintain a distance of 1m.22 16/3/2020 Case-based Austria measures Implemented at lockdown.22 16/3/2020 School closure ordered Nationwide school closures.23 14/3/2020 Public events All recreational activities cancelled regardless of banned size.23 12/3/2020 Citizens are required to stay at home except for Lockdown work and essential journeys. Going outdoors only ordered with household members or 1 friend.24 18/3/2020 Public transport recommended only for essential Social distancing journeys, work from home encouraged, all public encouraged places e.g. restaurants closed.23 14/3/2020 Case-based Everyone should stay at home if experiencing a Belgium measures cough or fever.25 10/3/2020 School closure Secondary schools shut and universities (primary ordered schools also shut on 16th).26 13/3/2020 Public events Bans of events >100 people, closed cultural banned institutions, leisure facilities etc.27 12/3/2020 Lockdown Bans of gatherings of >10 people in public and all ordered public places were shut.27 18/3/2020 Limited use of public transport. All cultural Social distancing institutions shut and recommend keeping encouraged appropriate distance.28 13/3/2020 Case-based Everyone should stay at home if experiencing a Denmark measures cough or fever.29 12/3/2020 School closure ordered Nationwide school closures.30 14/3/2020 Public events banned Bans of events >100 people.31 13/3/2020 Lockdown Everybody has to stay at home. Need a self- ordered authorisation form to leave home.32 17/3/2020 Social distancing encouraged Advice at the time of lockdown.32 16/3/2020 Case-based France measures Advice at the time of lockdown.32 16/03/2020 School closure ordered Nationwide school closures.33 14/3/2020 Public events No gatherings of >1000 people. Otherwise banned regional restrictions only until lockdown.34 22/3/2020 Lockdown Gatherings of > 2 people banned, 1.5 m ordered distance.35 22/3/2020 Social distancing Avoid social interaction wherever possible encouraged recommended by Merkel.36 12/3/2020 Advice for everyone experiencing symptoms to Case-based contact a health care agency to get tested and Germany measures then self—isolate.37 6/3/2020 School closure ordered Nationwide school closures.38 5/3/2020 Public events banned The government bans all public events.39 9/3/2020 Lockdown The government closes all public places. People ordered have to stay at home except for essential travel.40 11/3/2020 A distance of more than 1m has to be kept and Social distancing any other form of alternative aggregation is to be encouraged excluded.40 9/3/2020 Case-based Advice to self—isolate if experiencing symptoms Italy measures and quarantine if tested positive.41 9/3/2020 Norwegian Directorate of Health closes all School closure educational institutions. Including childcare ordered facilities and all schools.42 13/3/2020 Public events The Directorate of Health bans all non-necessary banned social contact.42 12/3/2020 Lockdown Only people living together are allowed outside ordered together. Everyone has to keep a 2m distance.43 24/3/2020 Social distancing The Directorate of Health advises against all encouraged travelling and non-necessary social contacts.42 16/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a Norway measures cough or fever symptoms.44 15/3/2020 ordered Nationwide school closures.45 13/3/2020 Public events banned Banning of all public events by lockdown.46 14/3/2020 Lockdown ordered Nationwide lockdown.43 14/3/2020 Social distancing Advice on social distancing and working remotely encouraged from home.47 9/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a Spain measures cough or fever symptoms.47 17/3/2020 School closure ordered Colleges and upper secondary schools shut.48 18/3/2020 Public events banned The government bans events >500 people.49 12/3/2020 Lockdown ordered No lockdown occurred. NA People even with mild symptoms are told to limit Social distancing social contact, encouragement to work from encouraged home.50 16/3/2020 Case-based Advice to self—isolate if experiencing a cough or Sweden measures fever symptoms.51 10/3/2020 School closure ordered No in person teaching until 4th of April.52 14/3/2020 Public events banned The government bans events >100 people.52 13/3/2020 Lockdown ordered Gatherings of more than 5 people are banned.53 2020-03-20 Advice on keeping distance. All businesses where Social distancing this cannot be realised have been closed in all encouraged states (kantons).54 16/3/2020 Case-based Advice to self—isolate if experiencing a cough or Switzerland measures fever symptoms.55 2/3/2020 Nationwide school closure. Childminders, School closure nurseries and sixth forms are told to follow the ordered guidance.56 21/3/2020 Public events banned Implemented with lockdown.57 24/3/2020 Gatherings of more than 2 people not from the Lockdown same household are banned and police ordered enforceable.57 24/3/2020 Social distancing Advice to avoid pubs, clubs, theatres and other encouraged public institutions.58 16/3/2020 Case-based Advice to self—isolate for 7 days if experiencing a UK measures cough or fever symptoms.59 12/3/2020 9 References 1. Li, R. et al. Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV2). Science (2020) doi:10.1126/science.abb3221. 2. Zhang, J. et al. Patterns of human social contact and contact with animals in Shanghai, China. 5cLRep.9,1—11(2019) 3. Worldometers.info. Hong Kong: coronavirus cases. https://www.wo rldometers.info/co ronavirus/country/china-hong-kong-sar/. 4. Ferguson, N. et al. Impact of non-pharmaceutical interventions (NPIs) to reduce COVID-19 mortality and healthcare demand (Report 9). https://www.imperial.ac.uk/mrc-global-infectious- disease-analysis/news--wuhan-coronavirus/. 5. Cereda, D. et al. The early phase of the COVID-19 outbreak in Lombardy, Italy. arXiv (2020). 6. Zhao, A. J. et al. Title: Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 Brief Title : Antibody responses in COVID-19 patients. (2020). 7. Jombart, T. et al. Inferring the number of COVID-19 cases from recently reported deaths. medRXiV 2020.03.10.20033761(2020)doi:10.1101/2020.03.10.20033761. 8. Zhang, J. et al. Age profile of susceptibility, mixing, and social distancing shape the dynamics of the novel coronavirus disease 2019 outbreak in China. (2020) doi:10.1101/2020.03.19.20039107. 9. Lourenco, J. et al. Fundamental principles of epidemic spread highlight the immediate need for large-scale serological surveys to assess the stage of the SARS-CoV-2 epidemic. doi:10.1101/2020.03.24.20042291 10. United Nations, Department of Economic and Social Affairs, Population Division. World Population Prospects 2019: Data Booket. ST/ESA/SER.A/424. (2019). 11. Verity, R. et al. Estimates ofthe severity of COVID-19 disease. Lancet Infect Dis in press, (2020). 12. Walker, P. G. T. et al. Report 12: The Global Impact of COVID-19 and Strategies for Mitigation and Suppression. 13. Fraser, C. Estimating Individual and Household Reproduction Numbers in an Emerging Epidemic. PL05 ONE 2, e758 (2007). 14. Cori, A., Ferguson, N. M., Fraser, C. & Cauchemez, S. A New Framework and Software to Estimate Time-Varying Reproduction Numbers During Epidemics. Am. J. Epidemiol. 178, 1505—1512 (20131 15. Nouvellet, P. et al. A simple approach to measure transmissibility and forecast incidence. Epidemics 22, 29—35 (2018). 16. Cauchemez, 8., Valleron, A. J., Boelle, P. Y., Flahault, A. & Ferguson, N. M. Estimating the impact of school closure on influenza transmission from Sentinel data. Nature 452, 750—754 (2008). 17. Bellman, R. & Harris, T. On Age-Dependent Binary Branching Processes. Ann. Math. 55, 280— 295(19521 18. Bellman, R. & Harris, T. E. On the Theory of Age-Dependent Stochastic Branching Processes. Proc. Natl. Acad. Sci. 34, 601—604 (1948). 19. Stan Development Team. 2018. The Stan Core Library, Version 2.18.0. http://mc-stan.org. 20. Bundesministerium. Coronavirus (COVID-19): Status quo — Schulen, Hochschulen, Universitaten und Forschungsinstitutionen. https://www.bmbwf.gv.at/Ministerium/Informationspflicht/corona/corona_status.html. 21. Henley, J. Coronavirus: EU states enact tough measures to stem spread. The Guardian https://www.theguardian.com/world/2020/mar/10/coronavirus-several-eu-states-ban-mass-events- after-italian-lockdown (2020). 22. Bundesministerium. Coronavirus - Aktuelle MaBnahmen. https://www.sozialministerium.at/Informationen-zum-Coronavirus/Coronavirus—Aktuelle- MaBnahmen.html (2020). 23. Federal Public Service. Coronavirus : Phase 2 maintained, transition to the federal phase and additional measures. https://www.info-coronavirus.be/en/2020/03/12/phase-2-maintained- transition-to-the-federal-phase-and-additional-measures/ (2020). 24. Belgium.be. Coronavirus: reinforced measures | Belgium.be. https://www.belgium.be/en/news/2020/coronavirus_reinforced_measures (2020). 25. Federal Public Service. Protect yourself and protect the others. https://www.info- coronavirus.be/en/2020/03/10/protect-yourself—and-protect-the-others/ (2020). 26. Wikipedia. 2020 coronavirus pandemic in Denmark. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_Denmark. 27. Stephensen, Emma K|inker; Hansen, T. S. Danmark lukker ned: Her er regeringens nye tiltag. TV2 https://nyheder.tv2.dk/samfund/2020-03-11-danmark-lukker-ned-her-er-regeringens-nye-tiltag (20201 28. Politi. Nye tiltag mod covid-19. Politi https://politi.dk/coronavirus-i-danmark/seneste-nyt-fra- myndighederne/nye-tiltag-mod-covid-19 (2020). 29. Styrelsen for Patientsikkerhed. Indberetning om covid-19zlnformation om mulighed for p\aabud til enkeltpersoner (coronavirus/covid-19). https://stps.dk/da/ansvar-og- retningslinjer/vejledning/indberetning-om-covid-19/#. 30. Wikipedia. 2020 coronavirus pandemic in France. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_France. 31. The Local. France bans gatherings of more than 100 people as coronavirus death toll rises - The Local. The Local https://www.thelocal.fr/20200313/france-bans-gatherings-of—over-100-people- to-fight-coronavirus-pandemic (2020). 32. Henley, Jon; Willsher, Kim; Kassam, A. Coronavirus: France imposes lockdown as EU calls for 30-day travel ban. The Guardian https://www.theguardian.com/world/2020/mar/16/coronavirus- spain-takes-over-private-healthcare-amid-more-european-lockdowns (2020). 33. Wikipedia. 2020 coronavirus pandemic in Germany. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_Germany. 34. BMI. Coronavirus: Fragen und Antworten. Bundesministerium des Innern,fur Bau und Heimat https://web.archive.org/web/20200317073042/https://www.bmi.bund.de/SharedDocs/faqs/DE/the men/bevoelkerungsschutz/coronavirus/coronavirus-faqs.htmI#doc13738352bodyText7. 35. BBC News. Coronavirus: Germany tightens curbs and bans meetings of more than two. BBC News https://www.bbc.co.uk/news/world-europe-51999080 (2020). 36. Bundesregierung. Kanzlerin trifft Regierungschefs der Lander Sozialkontakte vermeiden, Ausbreitung verlangsamen. https://www.bundesregierung.de/breg-de/themen/coronavirus/mpk- 1730186(2020) 37. Robert Koch Institut. Antworten auf haufig gestellte Fragen zum Coronavirus SARS-CoV-2. Robert Koch Institut https://web.archive.org/web/20200312004624/https://www.rki.de/SharedDocs/FAQ/NCOV2019/F AQ_Liste.html (2020). 38. Ministero della Salute. Governo annuncia sospensione dell’attivita didattica dal 5 a|15 marzo. Ministero della Salute http://www.salute.gov.it/portale/nuovocoronavirus/dettaglioVideoNuovoCoronavirus.jsp?lingua=ita liano&menu=multimedia&p=video&id=2052 (2020). 39. CNN. Italy prohibits travel and cancels all public events in its northern region. CNN https://edition.cnn.com/2020/03/08/europe/italy-coronavirus-lockdown-europe-intl/index.html (2020). 40. Attualita. Coronavirus: stop a pub, cinema, teatro e discoteche anche a Roma. Ecco cosa prevede il nuovo decreto. Roma Today https://www.romatoday.it/attualita/coronavirus-pub-cinema- teatri-locali-chiusi-nuovo-decreto.html (2020). 41. Gazzetta Ufficiale. DECRETO DEL PRESIDENTE DEL CONSIGLIO DEl MINISTRI. Gazzetta Ufflclale https://www.gazzettaufficiale.it/eli/id/2020/03/08/20A01522/sg (2020). 42. Helsedirektoratet. The Norwegian Directorate of Health has issued a decision to close schools and other educational institutions. Helsedirektoratet https://www.helsedirektoratet.no/nyheter/the- norwegian-directorate-of—health-has-issued-a-decision-to-close-schools-and-other-educationa|- institutions (2020). 43. Krostensen, Mette; Hellem-Hansen, Viktoria L.; Tandstad, B. Folkehelseinstituttet mener 23.000 kan vaere smittet. NRK https://www.nrk.no/norge/folkehelseinstituttet-mener-23.000-kan- vaere-smittet-1.14958149 (2020). 44. Norweigen Government. The Government is establishing clear quarantine and isolation rules. regjeringen.no https://www.regjeringen.no/en/aktuelt/the-government-is-establishing-clear- quarantine-and-isolation-rules/id2693647/ (2020). 45. Wikipedia. 2020 coronavirus pandemic in Spain. Wikimedia Foundation https://en.wikipedia.org/wiki/2020_coronavirus_pandemic_in_Spain. 46. Gabinete de Prensa. El Gobierno anuncia nuevas medidas para evitar la extension del nuevo coronavirus COVID-19. Gobierno de Espana https://www.mscbs.gob.es/gabinete/notasPrensa.do?id=4807 (2020). 47. Gabinete de Prensa. El Consejo Interterritorial del SNS acuerda medidas concretas para zonas con transmision comunitaria significativa de coronavirus. Gobierno de Espana https://www.mscbs.gob.es/gabinete/notasPrensa.do?id=4806 (2020). 48. Folkhalsomyndigheten. Larosaten och gymnasieskolor uppmanas nu att bedriva distansundervisning. Folkhdlsomyndigheten https://www.folkhalsomyndigheten.se/nyheter-och- press/nyhetsarkiv/2020/mars/larosaten-och-gymnasieskolor-uppmanas-nu-att-bedriva- distansundervisning(2020). 49. The Local. Sweden bans large events to halt coronavirus spread. The Local https://www.theloca|.se/20200311/sweden-to-ban-large-public-gatherings-over-coronavirus (2020). 50. Radosevich. Stockholmers urged to work from home as COVID-19 community spread confirmed. Sveriges Radio https://sverigesradio.se/sida/artikel.aspx?programid=2054&artikel=7430511(2020). 51. Folkhalsomyndigheten. Flera tecken p\aa samhallsspridning av covid-19 i Sverige. Folkhdlsomyndigheten https://www.folkhalsomyndigheten.se/nyheter-och- press/nyhetsarkiv/2020/mars/flera-tecken-pa-samhallsspridning-av-covid-19-i-sverige/ (2020). 52. Bundesamt fur Gesendheit BAG. Bundesrat verscharft Massnahmen gegen das Coronavirus zum Schutz der Gesundheit und unterstUtzt betroffene Branchen. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das-bag/aktuell/medienmitteilungen.msg-id-78437.html (20201 53. Bundesamt fur Gesundheit BAG. Coronavirus: Bundesrat verbietet Ansammlungen von mehr als fUnf Personen. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das- bag/aktuell/medienmitteilungen.msg-id-78513.html (2020). 54. Bundesamt fur Gesundheit BAG. Coronavirus: Bundesrat erklart die «ausserordentliche Lage» und verscharft die Massnahmen. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das-bag/aktuell/medienmitteilungen.msg-id-78454.html (20201 55. Bundesamt fur Gesundheit BAG. Neue Hygiene- und Verhaltensregeln zum Schutz gegen das neue Coronavirus. Schweizerische Eidgenossenschaft https://www.bag.admin.ch/bag/de/home/das- bag/a ktuell/medienmitteilungen.msg-id-78304.html (2020). 56. UK Government, D. for E. Schools, colleges and early years settings to close. UK Government https://www.gov.uk/government/news/schools-colleges-and-early-years-settings-to-close (2020). 57. UK Government. PM address to the nation on coronavirus: 23 March 2020. UK Government https://www.gov.uk/government/speeches/pm-address-to-the-nation-on-coronavirus-23-march- 2020(20201 58. Boycott-Owen, Mason; Bowman, Verity; Kelly-Linden, Jordan; Gartner, A. G. H. S. T. Coronavirus: Boris Johnson puts UK in lockdown as death tolls reaches 55. The Telegraph https://www.telegraph.co.uk/global-health/science-and-disease/coronavirus-news-uk-latest- update-covid-19-death-toll-cases/ (2020). 59. BBC News. Coronavirus: People with fever or ’continuous’ cough told to self—isolate. BBC News https://www.bbc.co.uk/news/uk-51857856 (2020).
How can a semi-mechanistic Bayesian hierarchical model estimate changes to the reproductive number?
false
801
{ "text": [ "calculating backwards from the deaths observed over time to estimate transmission that\noccurred several weeks prior, allowing for the time lag between infection and death." ], "answer_start": [ 2468 ] }
1,561
Acute Hemorrhagic Encephalitis Responding to Combined Decompressive Craniectomy, Intravenous Immunoglobulin, and Corticosteroid Therapies: Association with Novel RANBP2 Variant https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5857578/ SHA: ef6638accc1ef599ad1aafd47b3a86f2b904cc76 Authors: Alawadhi, Abdulla; Saint-Martin, Christine; Bhanji, Farhan; Srour, Myriam; Atkinson, Jeffrey; Sébire, Guillaume Date: 2018-03-12 DOI: 10.3389/fneur.2018.00130 License: cc-by Abstract: BACKGROUND: Acute hemorrhagic encephalomyelitis (AHEM) is considered as a rare form of acute disseminated encephalomyelitis characterized by fulminant encephalopathy with hemorrhagic necrosis and most often fatal outcome. OBJECTIVE: To report the association with Ran Binding Protein (RANBP2) gene variant and the response to decompressive craniectomy and high-dose intravenous methylprednisolone (IVMP) in life-threatening AHEM. DESIGN: Single case study. CASE REPORT: A 6-year-old girl known to have sickle cell disease (SCD) presented an acquired demyelinating syndrome (ADS) with diplopia due to sudden unilateral fourth nerve palsy. She received five pulses of IVMP (30 mg/kg/day). Two weeks after steroid weaning, she developed right hemiplegia and coma. Brain magnetic resonance imaging showed a left frontal necrotico-hemorrhagic lesion and new multifocal areas of demyelination. She underwent decompressive craniotomy and evacuation of an ongoing left frontoparietal hemorrhage. Comprehensive investigations ruled out vascular and infectious process. The neurological deterioration stopped concomitantly with combined neurosurgical drainage of the hematoma, decompressive craniotomy, IVMP, and intravenous immunoglobulins (IVIG). She developed during the following months Crohn disease and sclerosing cholangitis. After 2-year follow-up, there was no new neurological manifestation. The patient still suffered right hemiplegia and aphasia, but was able to walk. Cognitive/behavioral abilities significantly recovered. A heterozygous novel rare missense variant (c.4993A>G, p.Lys1665Glu) was identified in RANBP2, a gene associated with acute necrotizing encephalopathy. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells. CONCLUSION: In any ADS occurring in the context of SCD and/or autoimmune condition, we recommend to slowly wean steroids and to closely monitor the patient after weaning to quickly treat any recurrence of neurological symptom with IVMP. This case report, in addition to others, stresses the likely efficacy of combined craniotomy, IVIG, and IVMP treatments in AHEM. RANBP2 mutations may sensitize the brain to inflammation and predispose to AHEM. Text: Acute hemorrhagic encephalomyelitis (AHEM) or acute hemorrhagic leukoencephalitis is considered a rare and extremely severe form of acute disseminated encephalomyelitis (ADEM). AHEM is characterized by an acute and rapidly progressive encephalopathy including hemorrhagic necrosis of the parenchyma of the central nervous system. It is usually fatal (1) (2) (3) . Many treatment options have been used including intravenous (IV) steroids, intravenous immunoglobulins (IVIG), and plasmapheresis (4) . There have been few reports of survival following early intervention with high-dose corticosteroid therapy and/or decompressive craniotomy (5) (6) (7) (8) (9) . RANBP2, a nuclear pore protein, has numerous roles in the cell cycle. RANBP2 is associated with microtubules and mitochondria suggesting roles in intracellular protein trafficking or energy maintenance and homeostasis of neuronal cells. RANBP2 mutations have been reported in acute necrotizing encephalopathy (ANE) which could present with coma, convulsions, and encephalopathy. The hallmark of ANE is multiple, symmetric brain lesions located in the thalami bilaterally, putamina, deep periventricular white matter, cerebellum, and brainstem. It could be triggered by a viral infection in previously healthy children (10) . We report a new case of AHEM associated to a Ran Binding Protein (RANBP)-2 variant and responsive to combined craniectomy, intravenous methylprednisolone (IVMP), and IVIG as inaugural manifestation of multisystemic autoimmunity in a girl with sickle cell disease (SCD). A 6-year-old girl known for SCD treated on folic acid and hydroxyurea was admitted for new-onset diplopia [day 0 (D0): refers to the start of the diplopia] 6 weeks after respiratory tract infection due to rhinovirus. She was diagnosed with a fourth nerve palsy secondary to an acquired demyelinating syndrome. The initial brain magnetic resonance imaging (MRI) performed at D5 after onset of neurological symptom showed left midbrain and pontine edema with expansion of the brainstem, right caudate nucleus, and scattered supratentorial white matter foci of high T2/FLAIR signal (Figure 1 ). Brain MR angiography (MRA) showed a normal appearing circle of Willis. The cerebrospinal fluid (CSF) obtained by lumber puncture was normal (WBC 1 cells/μl, RBC 0 cells/μl, glucose 2.9 mmol/L, protein 0.18 g/L, and absent oligoclonal bands). The infectious workup including blood bacterial culture, CSF bacterial and viral cultures, nasopharyngeal aspirate (tested for Influenza A, Influenza B, Parainfluenza 1-2-3, Respiratory Syncytial Virus, Adenovirus, Coronavirus 229E, Coronavirus OC43, Metapneumovirus, Enterovirus, and Rhinovirus), and serologies for Epstein-Barr virus, Mycoplasma pneumoniae, HTLV I, HTLV II, HIV1, and Lyme disease were negative. Bartonella Henselae IgG was positive (1:1,280) reflecting a previously acquired common and self-limited infection in our area. Antinuclear antibodies (ANA) were positive (1:160). B12 and folate levels were normal. Smooth muscle antibodies were negative. Anti-mitochondrial antibodies were positive. Sedimentation rate was 65 mm/h. She was treated with five doses of IVMP (30 mg/kg/day) followed by 9 days of oral prednisone (1 mg/kg/day). At discharge, her neurological exam was significant only for vertical diplopia. She presented 1 month later with 5 days of upper respiratory tract infection symptoms, fever, headache, and a rapidly progressive right-hand weakness (D30) with normal alertness. She had normal blood pressure (120/81 mmHg). She was started on cefotaxime, vancomycin, and acyclovir. White cell count was 13.4 × 10 9 /L, hemoglobin was 7.8 g/L, and platelets were 239 × 10 9 /L. While in the MRI machine (D30) she deteriorated with vomiting and reduced level of consciousness (Glasgow Coma Scale dropped from 15 to 8 over 30 min). Brain MRI showed a rapid progression over a few sequences of an active bleed involving both superficial and deep gray matter as well as subcortical white matter of the left hemisphere anterior quadrant. Brain MRA was normal (Figures 2A-F) . The patient was immediately brought out of the magnet and her physical exam demonstrated unequal dilated pupils. She received IV mannitol and hypertonic saline for the management of acute intracranial hypertension/ herniation and was taken for surgery. She underwent left frontotemporoparietal decompressive craniotomy, evacuation of left frontoparietal intracerebral hemorrhage, and insertion of an external ventricular drain (EVD). Upon opening the skull, there was significant dural tension, and on opening the dura mater, there was a large amount of bleeding, in addition to brain swelling and necrosis. Estimated blood loss was 3.5 L. She received 8 units of packed red blood cells, 3 units of cryoprecipitate, 6 units of fresh frozen plasma, and 3 units of platelets. Coagulation profile showed international normalization ratio = 3.38, prothrombin time = 51.2 s, and partial thromboplastin time = 122 s. An intraventricular pressure monitor was inserted. She returned with stable vitals to PICU. At D31, the CT scan showed extensive multi-compartmental bleed involving the left frontoparietal lobes, the interhemispheric fissure, and the left hemispheric arachnoid spaces. New white matter lesions were detected in the left posterior parietal and occipital lobes and in the left caudate head. MRI at D33 showed interval worsening with disseminated gray and white matter non-hemorrhagic lesions in the right cerebral and both cerebellar hemispheres, bilateral deep gray nuclei, as well as new necrotic non-hemorrhagic lesions in the left hemisphere (Figures 2G-I) . She was started on IVMP (30 mg/kg/ day for 5 days) and IVIG (1 g/kg/day for 2 days). Repeat MRI at D9 showed no new parenchymal hemorrhage and partial resolution of the non-hemorrhagic lesions (Figure 3) . Prednisolone was tapered course over 6 weeks. At discharge (D71), she was able to say a few words and had better power of her right side. Brain MRI performed 3 months later showed complete resolution of the non-hemorrhagic non-necrotic lesions, mainly seen in the right cerebral hemisphere and the cerebellum. Brain biopsy of the hematoma, some small vessels, cortex, and white matter showed necrotic area, reactive and non-specific findings which could be entirely explained by compressive changes adjacent to a hematoma. There was diffuse microglial activation and signs of early microinfarcts. Blood, CSF and urine culture, and PCR (HSV1/2) were negative for bacteria and for viruses. CSF obtained through craniotomy and EVD performed at D32 showed elevated proteins 2.56 g/L, glucose 3.6 mmol/L, white blood cells 9 cells/μL, and red blood cells 1,341 cells/μL. ANA and anti-DNA antibody were negative. Anti-extractable nuclear antigens (SSA-RO, SSB-LA, smith, RNP) were negative. Serum autoimmune antibodies panel (NMO, NMDAR, AMPA I/II, GAB, MAG, VGCC, MOG, YO, HU, RI) were negative but GAD antibody was slightly positive, possibly due to the IVIG infusion. EBV showed no signs of recent infection. After discharge, the patient was started on regular transfusion exchange. Six months later, the patient was diagnosed to have Crohn's disease and primary sclerosing cholangitis. Two years later, the patient still suffers right hemiparesis but is able to walk without support. She presents an expressive aphasia. Her intellectual abilities are average, or below the mean but in the normal range, except for the speed of information processing, verbal working memory, and some elaborated executive functions. A gene panel ( Table 1 ) targeting inflammatory disorders and post-infectious necrotic encephalopathies found a heterozygous RANBP2 missense mutation (NM_006267.4, c.4993A>G, p.Lys1665Glu). This mutation has not been previously reported in the HGMD database. This variant has been observed at a frequency of <0.01% across the entire Broad ExAC dataset of individuals without severe childhood onset disease (6/117,118 alleles). Analysis of amino acid conservation indicates that the wild-type amino acid Lys1665 is conserved in 59 of 60 mammals examined, including 12 of 12 primates, and in 25 of 34 nonmammalian vertebrates increasing the likelihood that a change at this position might not be tolerated. In silico tools predict that this variant is damaging (SIFT and Align GVGD). Several differential diagnoses of acute encephalopathy in a patient with sickle cell anemia can be considered. An infectious encephalitis, including herpes encephalitis, was ruled out by blood and CSF bacterial and viral cultures and negative HSV I/ II PCR. Nasopharyngeal aspirate was negative for viruses. Some infections have been previously associated with necrotizing encephalitis such as Influenza A (11) . SCD patients are prone to ischemic or hemorrhagic strokes (12) . Primary hemorrhagic stroke is uncommon in pediatric SCD. Most cases were from adults and have been described in the context of previous ischemic stroke, aneurysms, low hemoglobin, acute chest syndrome, and hypertransfusions. Moreover, although hemorrhagic stroke has been described in SCD patients receiving transfusion or corticosteroids, it was in the context of elevated blood pressure which was not present in our case (13) . This was ruled out as the MRI findings were not consistent with a specific vascular territory and normal arterial and venous flows were shown on vascular imaging. Another differential is posterior reversible encephalopathy syndrome which has been reported in SCD patients (13) (14) (15) (16) . However, it is unlikely in our case due to the severity of the brain injury and the absence of classic precipitating factors of posterior reversible encephalopathy syndrome such as high blood pressure. Macrophage activation syndrome could also lead to acute necrotic brain injury. However, it is associated to high ferritin and low triglycerides at the time of the encephalopathy, other multisystemic injuries, typical neuropathological findings, and recurrence over time, which were not noted in our patient (17) . Parvovirus B19 has been described to cause encephalopathy in sickle cell patients. It is associated with aplastic anemia. It caused punctate areas of hemorrhages in the basal ganglia, periventricular white matter, and mainly along the posterior parietal cortex. This was attributed to parvovirus B19-induced vasculitis (18) . In our patient, there was no sign of aplasia or any neuroradiological finding of parvovirus B19 infection. Finally, acute encephalitis has been observed in SCD patients in the context of arterial hypoxemia from fat embolism, pulmonary embolism, sudden anemia, or acute chest syndrome due to pneumonia (19) . This was ruled out as the patient did not have clinical or radiological signs of acute chest syndrome or embolism and there was no arterial hypoxemia. Acute hemorrhagic encephalomyelitis has been described in pediatric patients following ADEM or ADEM-like episodes (20, 21) . AHEM is the most plausible diagnosis in our patients based on the clinical and radiological presentation, the preceding ADEM-like episode, and the exclusion of other etiologies of acute encephalopathy. Other patients with AHEM have been described in the SCD context (7, 19) . Many treatment options have been used to treat AHEM; of these, IV steroids have been associated with survival following aggressive, high-dose corticosteroid therapy (5) (6) (7) (8) (9) (22) (23) (24) (25) . Autosomal dominant mutations (with incomplete penetrance) in RANBP2 have been associated with susceptibility to infectioninduced necrotizing encephalopathy (26, 27) . Previously healthy patients with pathogenic mutations in RANBP2 can present acutely with encephalopathy and convulsions in the context of an infection, with brain imaging revealing involvement of the brainstem, thalami, putamina, cerebellum and external capsules, and claustrum (10) . Our patient has a similar presentation and imaging features as infection-induced necrotizing encephalopathy, including bilateral thalamic involvement. The rare heterozygous previously unreported variant we identified in RANBP2 affects a very conserved aminoacid and is predicted deleterious using in silico tools (a prediction tool performing a fast bioinformatics analysis which can predict the pathogenicity of a variant based on the change to an amino acid). It is possible that this variant is pathogenic and responsible for the clinical phenotype. There is an overlap between the diagnostic criteria of AHEM and those of acute hemorrhagic encephalopathy (25, 26) making possible that both entities might be part of the same pathophysiological continuum. RANBP2 is a protein playing an important role in the energy homeostasis of neuronal cells (28) . Hence, RANBP2 dysfunction might make neuronal cells much vulnerable to energy failure and necrosis when exposed to inflammatory or other stresses, such as those implicated in AHEM. This study was carried out in accordance with the recommendations of our institutional ethic committee. Written informed consent was obtained from all the participants for the publication. All authors participated in gathering the data, designing the article, and discussing and editing the manuscript. aCKNoWleDgMeNts We thank Dr. S. Abish, Dr. N. Ahmed, and Mrs. C. Guiraut for their help. We are grateful to the Hoppenheim Fund from the Montreal Children Hospital Foundation. The first author of this article received a scholarship from the Hoppenheim Fund, Montreal Children Hospital Foundation (2016). This work was supported by grants from Heart and Stroke Foundation of Canada (grant number: G-14-0005756), and Foundation of Stars.
What are the salient findings in Acute hemorrhagic encephalomyelitis?
false
3,033
{ "text": [ "fulminant encephalopathy with hemorrhagic necrosis" ], "answer_start": [ 615 ] }
1,660
Hantaviruses in the Americas and Their Role as Emerging Pathogens https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/ SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101 Authors: Hjelle, Brian; Torres-Pérez, Fernando Date: 2010-11-25 DOI: 10.3390/v2122559 License: cc-by Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] . The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖). Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: (1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] . (2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] . (3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil). Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] . Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] . Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease.
What can some of the other activities of N have, be linked to?
false
4,499
{ "text": [ "to the interference with an array of the intracellular processes of the normal cell" ], "answer_start": [ 10910 ] }
1,660
Hantaviruses in the Americas and Their Role as Emerging Pathogens https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185593/ SHA: efe13a8d42b60ef9f7387ea539a1b2eeb5f80101 Authors: Hjelle, Brian; Torres-Pérez, Fernando Date: 2010-11-25 DOI: 10.3390/v2122559 License: cc-by Abstract: The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses (family Bunyaviridae) and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome (HCPS), with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales [1, 2] . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections [3] [4] [5] [6] . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress (SARS), coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems [7] . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season [8] . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus [9] [10] [11] . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 [12] . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS (or hantavirus pulmonary syndrome), was linked to infection by the newly-discovered Sin Nombre virus (SNV), and the rodent Peromyscus maniculatus (deer mouse) was identified as the reservoir [13] . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome (HFRS), the syndrome caused by Old World hantaviruses [14] . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements [15] . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted [16] . It took more than two decades until the etiologic agent, Hantaan virus (HTNV), was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice [17, 18] . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus [19] . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus [19, 20] . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn [24] . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment (ER or aggresome); they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi [25, [27] [28] [29] . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF [30, 31] ; with gC1qR/p32 also identified as another potential entry receptor [32] . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies [33, 34] . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus (PHV) to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease (e.g., lethargy, fever, proteinuria, and azotemia) associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus (PUUV) [35, 36] . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity [37] [38] [39] . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host [40] [41] [42] . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form [43, 44] . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 (ISG-56) and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a (more) potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection [42, 45] . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell [44] . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein [46] . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability [47, 48] . The shorter (50-kD) nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity [49, 50] , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein [51] , an activity that could play a role in its presumed, but not yet demonstrated function as matrix [52] . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences [51] . N is also reported to interact with actin microfilaments, and the SUMO-1 protein [53, 54] . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B (NF-B). The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS [55] . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs [56] . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures [57] . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure [56, 58] . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase (-cap snatching‖). Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi [27] . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments [52] . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex (ERGIC), also known as vesicular-tubular cluster [52] . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments [59] . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV [60] . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression ( Table 2 ). The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate [61] . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively [63, 64] . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis [65] [66] [67] . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient (~ 1-5 days) capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections [62, 63] . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines [64] . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome [64, 65] . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: (1) Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns (PAMP) with the pattern recognition receptors (PRR) of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 [43] . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells [66] and differentiation of monocytes toward dendritic cells [67] . (2) Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient [68] . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element [69] . (3) Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed [70] [71] [72] [73] . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS [65, 74] . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death [75] . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically [76] . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site [77] . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads [63, [78] [79] [80] . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N [77, [81] [82] [83] . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. (1999) revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis [64] . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well [84, 85] . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% [86] [87] [88] . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation (ECMO), which is believed to have saved many patients from a lethal outcome [89] . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 [90] , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus (CHOV) arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir [91] . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate [92] . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies [93] [94] [95] [96] , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases [97] . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela [98] and Perú [99] . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 [100] . Since 1995, more than 1,000 HCPS cases have been reported in Argentina [101] ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 [102] . Case-fatality ratios in those three countries have been similar, ranging from 30% (Argentina), 36% (Chile) and 39% (Brazil). Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women [103] , while in Chile the ratio is more biased to males (71%) [104] . In the Paraguayan Chaco the male-female ratio approaches 50% [105] . In North America, by December 2009 63% of case-patients were males [90] . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups (as indigenous and non-indigenous) are more likely correlated with the type habitat where the population resides (e.g., rural versus urban areas). In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk [92, [105] [106] [107] [108] [109] [110] [111] , although the importance of peridomestic settings as a major area of exposure has also been emphasized [112, 113] . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva [114, 115] . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation (ENSO) events in some regions [116, 117] , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence [118] [119] [120] [121] . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled (i.e., cleaning, shaking rugs, dusting) are frequently identified among patients admitted for HCPS [11, 122] . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission [123] . An alternative but uncommon route of virus transmission is by rodent bites [124] [125] [126] . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight [127] . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV [128] , which re-emphasizes the use of personal protective equipment during field work activities [129] . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus [130] [131] [132] [133] [134] [135] . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently [130, 135] . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine [136] , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions [88, 137] . Hantaviruses in the Americas are naturally hosted by rodents (Muridae and Cricetidae) as well as shrews (Soricidae) and moles (Talpidae) (Figure 1) . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown [22, 138, 139] . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos (CDSV) or Hu39694 only identified after human infections (Figure 1 ). Hantaviruses typically show high species-specificity and no intermediate host [140] . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro (OROV) and Catacamas (CATV) identified in Oryzomys couesi [141, 142] , or Maporal (MAPV) and Choclo (CHOV) hosted by O. fulvescens [91, 143] . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus [144, 145] . Also, one hantavirus genotype (e.g., Juquitiba-like virus) may be carried by more than one rodent species (O. nigripes, Oxymycterus judex, Akodon montesis). Another example is Laguna Negra virus (LANV) which after being identified in Calomys laucha [146] has also been reported in C. callosus [147] . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened [95] . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses [148, 149] , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses [6, 150] . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed [151] [152] [153] [154] [155] [156] . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents [157, 158] . In North America, spillover of Bayou virus (BAYV) may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori [159] [160] [161] . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions [151, 162] , and cross-species transmission would presumably have greater chances of success if the host species are closely related [163] . An interesting exception is found between Oxbow virus (OXBV) and Asama virus (ASAV) in which a host-switch process seemed to have occurred between mammals belonging to two families (Talpidae and Soricidae), likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time [138] . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods (unlike other viruses of the family Bunyaviridae). Spillover infection to nonhuman mammals usually results in no onward (or -dead-end‖) transmission, but if humans are infected may result in high morbidity and mortality [122, 164] . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe [12, 121] . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities [165, 166] . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody (ELISA) or were antigen (IHC) positive out of 52 suspected cases [167] . Major outbreaks due to ANDV occurred in 1996 in southern Argentina [131, 134] ; in southern Chile clusters of patients presented with hantavirus illness in 1997 [158] . In Brazil, the first outbreak was identified in the Brazilian Amazon (Maranhão State) in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested (398 total residents) [168] . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk [169] [170] [171] [172] . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features [173, 174] . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found [175] . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection [176] . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus [177] , in Central America for O. fulvescens (reservoir of Choclo virus) and Zygodontomys brevicauda (reservoir of Calabazo virus) [178] , and in South America for Akodon montensis (reservoir of Jabora virus) [162] . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat [179] . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years [169] . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama [118, 119] . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus [180] . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus [107, 181] . Rodent population dynamics are affected by seasonal changes of weather and climate [182, 183] . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload [116, 121, 184] . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño [185] . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted [186] . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections [187] . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS [102, 162] , even with seemingly slight ecological disturbance to the virus-host system [188] . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease.
What have confocal microscopy and biochemical-inhibitor studies shown?
false
4,503
{ "text": [ "that N tracks along microtubules" ], "answer_start": [ 13763 ] }
1,583
A super-spreading ewe infects hundreds with Q fever at a farmers' market in Germany https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618839/ SHA: ee1b5a9618dcc4080ed100486cedd0969e80fa4d Authors: Porten, Klaudia; Rissland, Jürgen; Tigges, Almira; Broll, Susanne; Hopp, Wilfried; Lunemann, Mechthild; van Treeck, Ulrich; Kimmig, Peter; Brockmann, Stefan O; Wagner-Wiening, Christiane; Hellenbrand, Wiebke; Buchholz, Udo Date: 2006-10-06 DOI: 10.1186/1471-2334-6-147 License: cc-by Abstract: BACKGROUND: In May 2003 the Soest County Health Department was informed of an unusually large number of patients hospitalized with atypical pneumonia. METHODS: In exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. Serologic testing confirmed the diagnosis of Q fever. We asked local health departments in Germany to identiy notified Q fever patients who had visited the farmers market. To investigate risk factors for infection we conducted a case control study (cases were Q fever patients, controls were randomly selected Soest citizens) and a cohort study among vendors at the market. The sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of Soest were tested for Coxiella burnetii (C. burnetii). RESULTS: A total of 299 reported Q fever cases was linked to this outbreak. The mean incubation period was 21 days, with an interquartile range of 16–24 days. The case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. Vendors within approximately 6 meters of the sheep's pen were at increased risk for disease compared to those located farther away. Wind played no significant role. The clinical attack rate of adults and children was estimated as 20% and 3%, respectively, 25% of cases were hospitalized. The ewe that had lambed as well as 25% of its herd tested positive for C. burnetii antibodies. CONCLUSION: Due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. As a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the 3(rd )trimester and to test animals in petting zoos regularly for C. burnetii. Text: Q fever is a worldwide zoonosis caused by Coxiella burnetii (C. burnetii), a small, gram-negative obligate intracellular bacterium. C. burnetii displays antigenic variation with an infectious phase I and less infectious phase II. The primary reservoir from which human infection occurs consists of sheep, goat and cattle. Although C. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats [1] . High concentrations of C. burnetii can be found in birth products of infected mammals [2] . Humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool [1] . Because the infectious dose is very low [3] and C. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances [4] [5] [6] . C. burnetii infection in humans is asymptomatic in approximately 50% of cases. Approximately 5% of cases are hospitalized, and fatal cases are rare [1] . The clinical presentation of acute Q fever is variable and can resemble many other infectious diseases [2] . However, the most frequent clinical manifestation of acute Q fever is a self-limited febrile illness associated with severe headache. Atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. Acute Q fever may be complicated by meningoencephalitis or myocarditis. Rarely a chronic form of Q fever develops months after the acute illness, most commonly in the form of endocarditis [1] . Children develop clinical disease less frequently [7, 8] . Because of its non-specific presentation Q fever can only be suspected on clinical grounds and requires serologic confirmation. While the indirect immunofluorescence assay (IFA) is considered to be the reference method, complement fixation (CF), ELISA and microagglutination (MA) can also be used [9] . Acute infections are diagnosed by elevated IgG and/or IgM anti-phase II antibodies, while raised anti-phase I IgG antibodies are characteristic for chronic infections [1] . In Germany, acute Q fever is a notifiable disease. Between 1991 and 2000 the annual number of cases varied from 46 to 273 cases per year [10] . In 2001 and 2002, 293 and 191 cases were notified, respectively [11, 12] . On May 26, 2003 the health department of Soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. Some patients reported having visited a farmers' market that took place on May 3 and 4, 2003 in a spa town near Soest. Since the etiology was unclear, pathogens such as SARS coronavirus were considered and strict infection control measures implemented until the diagnosis of Q fever was confirmed. An outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the National Consulting Laboratory (NCL) for Coxiellae and the Robert Koch-Institute (RKI), the federal public health institute. Because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of Q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. On May 26 and 27, 2003 we conducted exploratory interviews with patients in Soest hospitalized due to atypical pneumonia. Attending physicians were requested to test serum of patients with atypical pneumonia for Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Coxiella burnetii, Influenza A and B, Parainfluenza 1-3, Adenovirus and Enterovirus. Throat swabs were tested for Influenza virus, Adenovirus and SARS-Coronavirus. Laboratory confirmation of an acute Q fever infection was defined as the presence of IgM antibodies against phase II C. burnetii antigens (ELISA or IFA), a 4-fold increase in anti-phase II IgG antibody titer (ELISA or IFA) or in anti phase II antibody titer by CF between acute and convalescent sera. A chronic infection was confirmed when both anti-phase I IgG and anti-phase II IgG antibody titers were raised. Because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection [13, 14] we alerted internists and gynaecologists through the journal of the German Medical Association and asked them to send serum samples to the NCL if they identified patients from these risk groups who had been at the farmers' market during the outbreak. The objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. We conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within 1 km distance of one of 6 sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. For the purpose of CCS1 we defined a case (CCS1 case) as an adult resident of the town of Soest notified to the statutory sur-veillance system with Q fever, having symptom onset between May 4 and June 3, 2003. Exclusion criterion was a negative IgM-titer against phase II antigens. Two controls per case were recruited from Soest inhabitants by random digit dialing. We calculated the attributable fraction of cases exposed to the farmers' market on May 4 (AFE) as (OR-1)/OR and the attributable fraction for all cases due to this exposure as: The farmers' market was held in a spa town near Soest with many visitors from other areas of the state and even the entire country. To determine the outbreak size we therefore asked local public health departments in Germany to ascertain a possible link to the farmers' market in Soest for all patients notified with Q-fever. A case in this context ("notified case") was defined as any person with a clinical diagnosis compatible with Q fever with or without laboratory confirmation and history of exposure to the farmers' market. Local health departments also reported whether a notified case was hospitalized. To obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition (as used in the vendors' study (s.b.)) identified through random sampling of the Soest population (within CCS2 (s.b.)) as well as in two cohorts (vendors' study and the 9 sailor friends (see below)). The objective of CCS2 was to identify risk factors associated with attendance of the farmers' market on the second day. We used the same case definition as in CCS1, but included only persons that had visited the farmers' market on May 4, the second day of the market. We selected controls again randomly from the telephone registry of Soest and included only those persons who had visited the farmers' market on May 4 and had not been ill with fever afterwards. Potential controls who became ill were excluded for analysis in CCS2, but were still fully interviewed. This permitted calculation of the attack rate among visitors to the market (see below "Estimation of the overall attack rate") and gave an estimate of the proportion of clinically ill cases that were hospitalized (s.a.). In the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of C. burnetii by wind were relevant risk factors for acquiring Q fever. We obtained a list of all vendors including the approximate location of the stands from the organizer. In addition we asked the local weather station for the predominant wind direction on May 4, 2003. Telephone interviews were performed using standardized questionnaires. A case was defined as a person with onset of fever between May 4 and June 3, 2003 and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than 2 kg, fatigue, nausea or vomiting. The relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. Each stand was considered to be one stand unit (approximately 3 meters). Larger stands were counted as 2 units. The direction of the wind in relation to the sheep pen was defined by dividing the wind rose (360°) in 4 equal parts of 90°. The predominant wind direction during the market was south-south-east ( Figure 1 ). For the purpose of the analysis we divided the market area into 4 sections with the sheep pen at its center. In section 1 the wind was blowing towards the sheep pen (plus minus 45°). Section 4 was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections 2 and 3 were east and west with respect to the wind direction, respectively. Location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen (in stand units or meters) and in reference to the wind direction. We identified a small cohort of 9 sailor friends who visited the farmers' market on May 4, 2003. All of these were serologically tested independently of symptoms. We could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition (as defined in the cohort study on vendors). The overall attack rate among adults was estimated based on the following sources: (1) Interviews undertaken for recruitment of controls for CCS2 allowed the proportion of adults that acquired symptomatic Q fever among those who visited the farmers' market on the second day; Attributable fraction AFE Number of cases exposed All cases = * (2) Interviews of cases and controls in CCS2 yielded information about accompanying adults and how many of these became later "ill with fever"; (3) Results of the small cohort of 9 sailor friends (s.a.); (4) Results from the cohort study on vendors. Local health departments that identified outbreak cases of Q fever (s.a. "determination of outbreak size and descriptive epidemiology") interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. However, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. To count cases in (1), (3) and (4) we used the clinical case definition as defined in the cohort study on vendors. For the calculation of the attack rate among children elicited in CCS2 was the same for all visitors. The number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. We then estimated the number of symptomatic children (numerator). For this we assumed that the proportion of children with Q fever that were seen by physicians and were consequently notified was the same as that of adults. It was calculated as: Thus the true number of children with Q fever was estimated by the number of reported children divided by the estimated proportion reported. Then the attack rate among children could be estimated as follows: Because this calculation was based on several assumptions (number of visitors, proportion of adult visitors and clinical attack rate among adults) we performed a sensitivity analysis where the values of these variables varied. Serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks (70 animals). Samples of 25 sheep from five other flocks in the Soest area were also tested for C. burnetii. Tests were performed by ELISA with a phase I and phase II antigen mixture. We conducted statistical analysis with Epi Info, version 6.04 (CDC, Atlanta, USA). Dichotomous variables in the case control and cohort studies were compared using the Chi-Square test and numerical variables using the Kruskal-Wallis test. P-values smaller than 0.05 were considered statistically significant. The outbreak investigation was conducted within the framework of the Communicable Diseases Law Reform Act of Germany. Mandatory regulations were observed. Patients at the local hospital in Soest reported that a farmers' market had taken place on May 3 and 4, 2003 in a spa town close to the town of Soest. It was located in a park along the main promenade, spanning a distance of approximately 500 meters. The market attracted mainly three groups of people: locals, inhabitants of the greater Soest region, patients from the spa sanatoria and their visiting family or friends. Initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of May 4, 2003 . The ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. Overall 171 (65%) of 263 serum samples submitted to the NCL were positive for IgM anti-phase II antibodies by ELISA. Results of throat swabs and serum were negative for other infectious agents. (Figure 2 ). If we assume that symptom onset in cases was normally distributed with a mean of 21 days, 95% of cases (mean +/-2 standard deviations) had their onset between day 10 and 31. The two notified cases with early onset on May 6 and 8, respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. Of the 298 cases with known gender, 158 (53%) were male and 140 (47%) were female. Of the notified cases, 189 (63%) were from the county of Soest, 104 (35%) were Porportion reported number of notified adults number of vis = i iting adults attack rate among adults * Attack rate among children estimated true number of childr = e en with Q fever estimated number of children at the market from other counties in the same federal state (Northrhine Westphalia) and 6 (2%) were from five other federal states in Germany (Figure 3 ). Only eight (3%) cases were less than 18 years of age, the mean and median age was 54 and 56 years, respectively ( Figure 4 ). 75 (25%) of 297 notified cases were hospitalized, none died. Calculation of the proportion of cases hospitalized through other information sources revealed that 4 of 19 (21%; 95% CI = 6-46%; (1/5 (CCS2), 2/11 (vendors study) and 1/3 (sailor friends)) clinically ill cases were hospitalized. Laboratory confirmation was reported in 167 (56%) outbreak cases; 66 (22%) were confirmed by an increase in anti-phase II antibody titer (CF), 89 (30%) had IgM antibodies against phase II antigens, 11 (4%) were positive in both tests and one was confirmed by culture. No information was available as to whether the 132 (44%) cases without laboratory confirmation were laboratory tested. 18 patients with valvular heart defects and eleven pregnant women were examined. None of them had clinical signs of Q fever. Two (11%) of 18 cardiological patients and four (36%) of 11 pregnant women had an acute Q fever infection. During childbirth strict hygienic measures were implemented. Lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman (case 3; Table 1 ). Serological follow-up of the mothers detected chronic infection in the same woman (case 3) 12 weeks after delivery. One year follow-up of two newborn children (of cases 1 and 3) identified neither acute nor chronic Q fever infections. We recruited 20 cases and 36 controls who visited the farmers' market on May 4 for the second case control study. They did not differ significantly in age and gender (OR for male sex = 1.7; 95%CI = 0.5-5.3; p = 0.26; p-value for age = 0.23). Seventeen (85%) of 20 cases indicated that they had seen the cow (that also was on display at the market next to the sheep) compared to 7 (32%) of Geographical location of Q fever outbreak cases notified to the statutory surveillance system Figure 3 Geographical location of Q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to 8 (32%) of 25 controls (OR = 5.0; 95%CI = 1.2-22.3; p = 0.03). Touching the sheep was also significantly more common among cases (5/20 (25%) CCS2 cases vs. 0/22 (0%) controls; OR undefined; lower 95% CI = 1.1; p = 0.02). 17 (85%) of 20 CCS2 cases, but only 6 (25%) of 24 controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" (OR = 17.0; 95%CI = 3.0-112.5; p < 0.01). Among CCS2 cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. We were able to contact and interview 75 (86%) of 87 vendors, and received second hand information about 7 more (overall response rate: 94%). Fourty-five (56%) were male and 35 (44%) were female. 13 (16%) met the clinical case definition. Of the 11 vendors who worked within two stand units of the sheep pen, 6 (55%) became cases compared to only 7 (10%) of 70 persons who worked in a stand at a greater distance (relative risk (RR) = 5.5 (95%CI = 2.3-13.2; p = 0.002); Figure 1 ). Of these 7 vendors, 4 had spent time within 5 meters of the pen on May 4, one had been near the pen, but at a distance of more than 5 meters, and no information on this variable was available for the remaining 2. In the section of the market facing the wind coming from the pen (section 4, Figure 1 ), 4 (9%) of 44 vendors became cases, compared to 2 (13%) of 15 persons who worked in section 1 (p = 0.6). Among 22 persons who worked in stands that were perpendicular to the wind direction, 7 (32%) became cases. (Table 3 ). In all scenarios the AR among adults was significantly higher than that among children ( Figure 5 ). In total, 5 lambs and 5 ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at 6:30 a.m. on May 4, 2003 . Of these, 3 ewes including the one that had lambed tested positive for C. burnetii. The animals came from a flock of 67 ewes, of which 66 had given birth between February and June. The majority of the births (57 (86%)) had occurred in February and March, usually inside a stable or on a meadow located away from the town. Six ewes aborted, had stillbirths or abnormally weak lambs. Among all ewes, 17/67 (25%) tested positive for C. burnetii. The percentage of sheep that tested positive in the other 5 sheep flocks in the region ranged from 8% to 24% (8%; 12%; 12%; 16%; 24%). We have described one of the largest Q fever outbreaks in Germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. In 1954, more than 500 cases of Q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market [15] . More recently a large outbreak occurred in Jena (Thuringia) in 2005 with 322 reported cases [16] associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. The first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. The fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth Persons accompanying notified cases (source 5) were a mixture of adults and children and are therefore listed separately. in the early morning hours of May 4, 2005 . This strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with Q fever and gave a history of having visited the farmers' market. The point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged 21 days and ranged from 2 to 48 days with an interquartile range of 16 to 24 days. This is compatible with the literature [1] . An additional interview with the two cases with early onset (2 and 4 days after attending the market on May 4, Attack rates among adults and children in a most likely scenario and 8 other scenarios Figure 5 Attack rates among adults and children in a most likely scenario and 8 other scenarios. Most likely scenario: 3000 visitors, 83% adult visitors and 20% clinical attack rate among adults. Scenarios 1-8 varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" (see Table 3 ). Displayed are attack rates and 95% confidence intervals. respectively) could not identify any other source of infection. A short incubation period was recently observed in another Q fever outbreak in which the infectious dose was likely very high [17] . The second case control study among persons who visited the market on May 4 demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. This finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to (within 6 meters) the sheep pen were at significantly higher risk of acquiring Q fever. The study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. However, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. According to statutory surveillance system data, the proportion of clinical cases hospitalized was 25%, similar to the proportion of 21% found in persons pooled from the other studies conducted. Several publications report lower proportions than that found in this investigation: 4% (8/ 191) [7] , 5% [1] and 10% (4/39) [5] ), and there was at least one study with a much higher proportion (63% (10/ 16)) [18] . It is unlikely that hospitals reported cases with Q fever more frequently than private physicians because the proportion hospitalized among Q fever patients identified through random telephone calls in the Soest population or those in the two cohorts was similar to that of notified cases. Thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. Alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. The estimated attack rate among adults in the four studies varied between 16% and 33%. The estimate of 23% based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. The estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. On the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. Nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. In comparison, in a different outbreak in Germany, in which inhabitants of a village were exposed to a large herd of sheep (n = 1000-2000) [5, 7] the attack rate was estimated as 16%. In a similar outbreak in Switzerland several villages were exposed to approximately 900 sheep [19] . In the most severely affected village, the clinical attack rate was 16% (estimated from the data provided) [19] . It is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. Our estimate of the proportion of serologically confirmed cases that became symptomatic (50% (3/6)) is based on a very small sample, but consistent with the international literature. In the above mentioned Swiss outbreak, 46% of serologically positive patients developed clinical disease [7] . Only approximately half of all symptomatic cases were reported to the statutory surveillance system. Patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. Our estimated 3% attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. Nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. While it has been suggested that children are at lower risk than adults for developing symptomatic illness [7, 8] few data have been published regarding attack rates of children in comparison to adults. The estimated C. burnetii seroprevalence in the sheep flocks in the area varied from 8% to 24%. The 25% seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in June 2003 suggested a recent infection of the flock [20] . Seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. The median seroprevalence in a number of relevant studies performed in the context of human outbreaks [7, 20, 21] , was 40% compared to 1% in sheep flocks not linked to human outbreaks [20] . This outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that (in such a setting) can turn into a super-spreader upon lambing. There is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. During the past decades urbanization of rural areas and changes in animal husbandry have occurred [20] , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. Petting zoos, family farm vacations or the display of (farm) animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents [20, [22] [23] [24] . While not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. This outbreak led to the amendment and extension of existing recommendations [25] which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for C. burnetii in petting zoos, where there is close contact between humans and animals. Due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of Q fever. It also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. Abbreviations AFE = attributable fraction of cases exposed The author(s) declare that they have no competing interests.
What is Coxiella burnetii?
false
5,208
{ "text": [ "small, gram-negative obligate intracellular bacterium" ], "answer_start": [ 2413 ] }
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 BAL?
false
4,268
{ "text": [ "bronchoalveolar lavage" ], "answer_start": [ 22289 ] }
1,563
Obesity and risk of respiratory tract infections: results of an infection-diary based cohort study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819164/ SHA: ee0c318d282c0089cca94f0b2ea4d90db2ab9f8a Authors: Maccioni, Livia; Weber, Susanne; Elgizouli, Magdeldin; Stoehlker, Anne-Sophie; Geist, Ilona; Peter, Hans-Hartmut; Vach, Werner; Nieters, Alexandra Date: 2018-02-20 DOI: 10.1186/s12889-018-5172-8 License: cc-by Abstract: BACKGROUND: Respiratory tract infections (RTIs) are a major morbidity factor contributing largely to health care costs and individual quality of life. The aim of the study was to test whether obesity (BMI ≥ 30 kg/m(2)) is one of the risk factors underlying frequent RTIs in the German adult population. METHODS: We recruited 1455 individuals between 18 to 70 years from a cross-sectional survey on airway infections in Germany and invited them to self-report in diaries incident RTIs experienced during three consecutive winter/spring seasons. RTIs reported in these 18 months and summary measures adding-up individual RTIs were the outcomes of interest. RESULTS: Compared to individuals with normal weight, obese individuals reported a consistently higher frequency of upper and lower RTIs and predominantly fell in the upper 10% group of a diary sumscore adding-up 10 different RTI symptoms over time. Obesity was associated both with lower RTIs ((adjusted)OR = 2.02, 95%CI = 1.36–3.00) and upper RTIs ((adjusted)OR = 1.55, 95%CI = 1.22–1.96). Adjusting for demographic and lifestyle variables did only marginally affect ORs. Stratified analyses suggested a stronger association for women and effect modifications by sports activity and dietary habits. CONCLUSIONS: We confirm the association of obesity with infection burden and present evidence for putative interaction with sports activity and dietary patterns. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12889-018-5172-8) contains supplementary material, which is available to authorized users. Text: Frequent and severe respiratory tract infections (RTIs) constitute an important morbidity factor in our society and a considerable cost burden in terms of medical treatment and time of work-loss [1, 2] . RTIs are divided into upper RTIs (URTIs) including common cold, pharyngitis, otitis, sinusitis, laryngotracheitis, epiglottitis and lower RTIs (LRTIs) including bronchitis, pneumonia and bronchiolitis [3] . Individual exposure to infectious agents and host factors such as smoking [4, 5] and vitamin D status [6, 7] are believed to contribute to observed differences in RTI risk. In addition, the role of overweight (body mass index (BMI) = 25.0-29.9 kg/m 2 ) and in particular obesity (BMI ≥ 30 kg/m 2 ) in predisposition to RTIs is increasingly discussed [8] [9] [10] [11] [12] [13] . This growing interest is driven by the rising number of overweight and obese individuals worldwide [14] and the emerging knowledge of notable immunological imbalances in association with obesity [15] . Most of the studies targeting adults explored the association of obesity with specific RTIs and their outcomes. Thus, obesity was associated with non-allergic rhinitis [8] and influenza like-illness [9] . Moreover, two population-based studies which investigated the role of obesity as risk factor for community acquired pneumonia (CAP) in the general population resulted in controversial findings [10, 11] . Two recent Danish population-based studies reported an excess of a large spectrum of RTIs including pneumonia among obese people [12, 13] . The overall aim of our study targeting the adult population in South Baden, Germany, is to identify risk factors for the susceptibility to RTIs. Here we present data on the role of obesity as contributing factor to a high RTI burden in the German society and explore effect modification by gender, sports activity and nutritional patterns. Study participants (n = 1455) were recruited from the airway infection susceptibility (AWIS) cross sectional study querying RTI burden in an adult population in South-Baden, Germany [16] . The study protocol was approved by community officials and the Ethics Committee of the University of Freiburg (Ref. No. 258/11_120365). Based on the RTI history-score individuals of putative low, medium and high risk of future RTIs were invited to the actual sub-cohort. The RTI history score is summarizing information on the frequency and severity of RTIs and antibiotics use over the past two years, selfassessed RTI susceptibility, and occurrence of selected severe infections [16] . Study participants were requested to fill-in an additional questionnaire (baseline questionnaire) on lifestyle factors and co-morbidities and to complete monthly diaries registering the monthly occurrence and the duration (< 2 weeks, > 2 weeks) of RTIs, namely sinusitis, rhinitis, otitis media, pharyngitis/laryngitis, tonsillitis, influenza-like illness, bronchitis, pneumonia, pleurisy and other acute RTIs, from the beginning of November to the end of April of three seasons: 2012/13, 2013/14 and 2014/15. Furthermore, the intake of antibiotics, doctor visits, hospitalisation for RTIs and the impact of RTI symptoms on their daily activities were queried. Further recruitment details into the AWIS study and the present sub-cohort are presented under Additional files 1 and 2. Informed consent was obtained from all individual participants included in the study. In order to describe the association between obesity and RTIs, different outcome indicators were considered: outcomes at the level of each month ["any RTI", "any URTI" (sinusitis, rhinitis, otitis media, pharyngitis/laryngitis and tonsillitis), "any LRTI" (bronchitis, pneumonia and pleurisy), "≥3 RTIs", "any long lasting infection" (> 2 weeks)]; at the level of each winter season ("≥4 months with infections", "≥3 long lasting infections"); and at the individual level (i.e. are defined once per individual and covering the overall study period). The ten specific RTI symptom categories were considered with the binary symptom indicators "infection reported" or "no infection reported" for each month. When counting the episodes for the outcome indicator "≥3 long lasting infections", different infection symptoms were counted as separate episodes, even if they overlapped in time. However, within one symptom category at least one month without this specific infection was required to call it a new episode. We also calculated a monthly diary RTI score, averaging the ten RTI symptom categories with the coding "0" for "no infection reported", "1" for "reported infection with duration < 2 weeks", and "2" for "reported infection present with duration >2 weeks". Missing values for individual infection items were treated as zero. If an individual RTI symptom was reported, but information on duration was missing, it was counted as "reported infection with duration < 2 weeks". If all items were missing, no diary score was computed. The diary RTI score at the monthly level was expanded to a score at the seasonal level by averaging over the six months (November-April) of each season, and to an overall score at the individual level by averaging over all available months. The respective upper 10% of these diary scores within each month, season and overall served as additional outcome indicators. Further variables considered in the study were age, gender, self-reported weight and height for BMI calculation (BMI was categorized as < 30 (non-obese), 25 ≤ BMI < 30 (overweight) and ≥30 (obese)), educational level, contact with children, comorbidities, removed immunological organs, smoking status, sports activity and dietary intake patterns. Details on these variables are described in the Additional file 1 and supplementary information on dietary intake patterns is presented in Additional file 3. Statistical analysis was performed using Stata (version 14 STATSCorp, USA). Descriptive statistics: Monthly prevalences of individual RTI symptoms were computed by taking the average over all subjects available at each month and then averaging over all 18 months covered. Prevalences at the seasonal level were computed accordingly averaging over all three seasons covered. The corresponding confidence intervals (CIs) and p-values are based on a generalised linear model with identity link and binomial type variance together with robust variance estimates. The frequency of long lasting infections among all months with infections was analysed accordingly. However, due to the limited number of cases for tonsillitis and otitis media we determined the monthly frequency of long-lasting infections by pooling the data over all seasons and for pneumonia by pooling all indicated months. At the monthly level ORs were computed using a logistic regression model with a random intercept applied to the individual data for each month taking the 18 months as a categorical covariate into account in addition to the obesity status indicator. Due to its small prevalence, pleurisy was not considered as single outcome in these analyses. Outcomes at the seasonal level were analysed accordingly with the individual data for each winter season and taking into account the three seasons as a categorical covariate. Outcomes at the individual level were analysed using a logistic regression model. Results are ORs and 95% CIs. Adjusted ORs are based on including age groups and education as simultaneous categorical covariates. Furthermore, in order to study the stability of the obesity-RTI association with respect to potential confounders, ORs were adjusted by respective variables. Subjects with incomplete covariate data were excluded from multivariate analyses. Effect modification by a binary variable was assessed by fitting an overall model with the corresponding interactions parametrized so that we could directly read off the two subgroup-specific ORs. Effect modification by sports activity and nutrition patterns was explored among those representing the lower and upper third of respective scores. The study population comprised 1455 individuals (931 female and 524 male) with a median age of 51.08 years. Based on BMI calculated from self-reported weight and height, 2.1% of the population was underweight (BMI < 18.5 kg/m 2 ), 54% had a normal weight (18.5 kg/m 2 ≤ BMI < 25 kg/m 2 ), 31.1% was overweight, and 12.8% was considered obese (Table 1 ). In women, the distribution was 2.8%, 60.21%, 25.0%, and 12.1% and in men 0.76%, 43.1%, 41.8%, and 14.3%, respectively. The study participants were mainly of medium and high educational level, non-or ex-smokers, moderately affected by selected co-morbidities and they reported rather infrequent contact to small children. Further information on the study population and completed diaries is reported in Table 1 and Additional file 4. Missing rates of single items in the returned diaries were limited and ranged from 1.2% for rhinitis and pharyngitis/laryngitis to 2.6% for other acute respiratory infections. Study participants reported most frequently rhinitis (26.6%), followed by influenza-like illness (11.4%) and pharyngitis/laryngitis (10.5%), whereas pleurisy (0.10%) was rarely experienced. Any URTI (31.5%) was more frequent than any LRTI (7.9%). Apart from the LRTIs bronchitis, pneumonia and pleurisy, which more men than women reported, all other RTIs were more prevalent among women (Table 2 ). Seasonal patterns of reported infections show a February peak for two of the three assessed infection seasons (2012/13 and 2014/15, see Additional file 5). Respiratory infections with a high fraction of long duration were almost exclusively LRTIs, namely pneumonia (59%), followed by bronchitis (48.2%). Men were overrepresented among those with long-lasting RTIs ( Table 2) . Compared to normal weight individuals, overweight and obese people consistently had a higher prevalence (Table 3) for the single RTIs, URTIs, LRTIs, as well as the other outcome parameters we looked at with other acute infections and pneumonia as the exceptions. For pneumonia, only obese subjects had a higher prevalence. The overweight group was typically falling in between the groups with normal weight and obesity ( Table 3 ). The strongest association was seen for pneumonia and bronchitis, and accordingly, any LRTI was more strongly associated with obesity than any URTI. Long-lasting RTIs, frequent RTIs and high diary scores were also more strongly associated with obesity than the individual symptoms. Adjustments by age and education did only marginally change these estimates. Among subjects with an infection, long lasting infections were again associated with obesity, reaching significance for any RTI, rhinitis, pharyngitis/laryngitis, influenza-like illness, and bronchitis ( Table 3) . For a better understanding of the robustness of the relationship between RTI burden and obesity, the effect of adjusting for putative confounders was explored (Additional file 6). The studied demographic and lifestyle variables (age, gender, education level, smoking status, contact to children, asthma, sports activity, dietary patterns and previous removal of immune organs) did only marginally affect ORs. However, adjustment for asthma, chronic obstructive pulmonary disease (COPD) or a summary score covering all queried co-morbidities weakened the relationship between obesity and all outcomes considerably. Adjustment for vitamin D levels among those for which serum was available (n = 508), had only a slight effect on the magnitude of the association between obesity and RTI outcomes. The association between obesity and RTI outcomes was more prominent for women than for men and reached statistical significance only for the former (Table 4 ). For most outcomes this interaction was not significant, with the individual level diary score as an exception. When looking at sports activity, for most outcomes the association with obesity was confined to those physically more active and not seen for those reporting little sports activity (Table 5 ). For all outcomes the association was less pronounced in the latter group (compare the ratios of ORs in Table 5 ), a difference that reached significance for all outcomes except those with low prevalence. Typically the prevalence of an outcome was only increased in the small group of people with obesity and higher sports activity whereas all other groups presented rather similar patterns. Similarly, the prevalence of outcomes was increased among people with obesity and a more favourable nutritional pattern, but comparable among the other groups ( Table 6 ). The interaction reaches significance for the majority of outcomes. RTIs constitute an important morbidity factor considering the high health care costs, the time lost from work, and the impaired quality of life among those recurrently affected [1, 2, 17] . Obesity belongs to one of the host risk factors for RTI and has possibly an emerging role due to the dramatically increasing prevalence of obesity worldwide. In the present study, we report on the association of obesity with individual RTIs as well as with a diary score summarising different incident RTI symptoms over a period of 18 months. Our investigation could demonstrate an association between obesity and RTIs confirming previous findings on influenza-like illness [9] , bronchitis [18] and pneumonia [10, 12] . We also saw an association between obesity and rhinitis, sinusitis and pharyngitis/laryngitis. An elevated risk for sinusitis among obese was also reported in a populationbased cohort of Danish women [13] . None of the two Danish population-based studies [12, 13] used ORs of monthly prevalence, but hazard ratios (HRs), as they could identify events on a daily basis. The HR of 1.6 [12] for the association with RTIs and the HR of 1.48 [13] for the association with URTIs are, however, of similar magnitude to the risk estimates which we observed. Mechanistically, excess adiposity might weigh down host defence as several mouse as well as human studies have suggested [19, 20] . The here observed associations were more prominent for LRTIs compared to URTIs, but evident for both, and more pronounced when considering long lasting or frequent RTIs compared to single symptoms. Based on the infection diary data, we generated a RTI diary score summing-up all ten symptoms and allowing to average per month, per whole season or over the whole period of three years. Considering the upper ten percentile of the distribution of such scores as an outcome, associations were typically stronger than when considering single symptoms, and interactions were more pronounced. Moreover, the results of the seasonal score were very similar or even stronger than those of the three-years score, arguing for the adequacy to query six months infectious events in future studies to identify the infection-prone sub-group of the population. Lifestyle habits seem to contribute to an individual's risk for RTI. Among them, cigarette smoking has been reported as a major environmental risk factor for recurrent and severe RTIs [4, 5] . Frequent contact to small children [21, 22] , vitamin D deficiency [23, 24] , and lack of physical activity [25, 26] constitute other exposures associated with heightened RTI risks. Moreover, higher levels of education were associated with a lower risk of CAP [27] . Based on those previous findings we investigated their role as possible confounders. The association between obesity and RTIs remained nearly unchanged after adjustment for age, gender, educational status, contact to children, smoking status, sports activity and nutrition scores, suggesting that the association is not markedly confounded by the effects of these factors on both BMI and the risk of infections. Also additional adjustment by measured serum vitamin D in a subgroup for which measurements were available did not change the risk estimates considerably. This supports arguments that the observed associations between obesity and RTI burden are due to physiological differences in the immune responsiveness between obese and non-obese individuals rather than lifestyle differences. In addition, some chronic diseases, foremost asthma and COPD, are associated with both an increased risk of RTIs and obesity [28] [29] [30] [31] [32] . Considering these associations we investigated the effect of asthma, COPD and a comorbidity scoresummarizing the other chronic conditionson the relationship between obesity and individual RTIs and the RTI diary score. Adjusting for these conditions individually and even more so in a combined fashion resulted in a considerable attenuation of the association between obesity and considered RTI outcomes. Hence part of the association between infections and obesity might be explainable by associations of co-morbidities with both. We see a gender difference in the observed associations with more noticeable findings for women. A significantly increased risk for combined RTIs was also restricted to women in a Danish blood donor cohort [12] . Several lines of research support this notion: Szabova et al. and Ilavska et al. reported gender-dependent effects of obesity on the immune system [33, 34] . The effect of BMI on a variety of immune parameters including those with relevance for immune defence was much more apparent in women than in men [34] . NK cells (CD3-/CD16+/CD56+), represent first-line cells for the clearing of virus-infected cells. Reduced levels of these cells reported for obese women, but not for respective men, might underlie the gender effect seen in our study. We also investigated a potential effect modification by sports activity and nutrition. Interestingly, an association between obesity and RTIs was evident only for those obese individuals who reported a higher level of sports activity. Thus, only the group of obese people who engaged in more intensive sports activity reported RTIs more frequently whereas obese people with low sports activity and non-obese with low or high sports activity showed comparable lower prevalences for most outcomes. We hypothesize that oxidative stress induced by vigorous aerobic as well as anaerobic sports activity is exacerbated in people with obesity, but not in normal weight individuals. Evidence supporting this has been previously published [35] . An imbalanced oxidative stress status may have negative consequences on mounting an appropriate immune response towards respiratory pathogens. Excessive reactive oxygen species (ROS) was shown to hinder T cell responses to viral infection [36] and ROS accumulation was detected in autophagy-deficient effector T cells rendering them incapable of controlling viral infections [37] . A similar surprising result was found when studying the effect modification by dietary patterns. Here we queried the participants' dietary habits and classified them as adhering to a more favourable or more unfavourable dietary pattern according to Winkler et al. [38] . Aware of the limitations of a one-time assessment of a habitual diet, we found a more pronounced relationship between obesity and infections among obese people who reported an apparent healthier diet. Thus, again only the group of obese individuals who presumably eat a healthier diet showed an increased risk of RTIs. The question arises as to whether misreporting of dietary habits among these individuals with and without RTIs may explain the puzzle. One can imagine that obese individuals may have an increased perception of RTI related symptoms experiencing the contradiction between living a healthy lifestyle and being affected by excess weight and frequent infections. On the other hand the inconspicuous results from the non-obese population with respect to favourable and unfavourable diet pattern would somewhat argue against this explanation. Alternatively, among the group of people with obesity a genetically defined subgroup may exist predisposing to both, excess body weight and proneness to infections. As strengths of our study we count 1) its sample size, allowing for the analysis of effect modification, 2) its prospective design involving 18 months infection diaries for the exploration of the relationship between BMI and subsequent RTI frequency and severity, 3) the comprehensive information on lifestyle and co-morbidities allowing to study the interplay of such factors on their effect on infections, and 4) the wide range of outcome indicators considered. The uniformity of the results with respect to these outcomes also suggests that in the field of airway infection morbidity, studies may be comparable despite the fact that they often concentrate on different RTI outcomes. In line with the majority of epidemiological studies in this area of research, our study suffers from some limitations, including the reliance on self-reported outcomes and exposure data with the risk of misclassification. However, we found -for instance -a good agreement between BMI derived from self-reported weight and height data and BMI calculated from measured values available for a sub-cohort (n = 508). Moreover, differential misclassification which would substantially bias the relationship between obesity and RTIs is rather unexpected in this setting. The disproportional selection of women into the study may negatively impact the generalizability of some of our results.
What conditions are considered lower respiratory tract infections?
false
329
{ "text": [ "bronchitis, pneumonia and bronchiolitis" ], "answer_start": [ 2392 ] }
776
It is Unlikely That Influenza Viruses Will Cause a Pandemic Again Like What Happened in 1918 and 1919 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019839/ Song, Liting 2014-05-07 DOI:10.3389/fpubh.2014.00039 License:cc-by Abstract: nan Text: Influenza and influenza viruses are wellknown popular topics to medical professionals and the general public. Influenza viruses had caused a pandemic globally during 1918 and 1919, and that influenza pandemic had taken away more than 20 million people's lives in the world. However, in my opinion, it is unlikely that influenza viruses will again cause a pandemic on a level (both of the morbidity rate and the mortality rate) comparable to what happened in 1918 and 1919. Influenza viruses very easily reassort, recombine, and point mutate in nature due to their segmented RNA genome structures, however, unlike highly pathogenic (virulent) viruses like rabies virus, Lassa fever virus, smallpox virus, eastern equine encephalitis virus, Ebola virus, Marburg virus, and human immunodeficiency virus 1 (HIV-1); most influenza viruses (wild types and mutants) are moderately pathogenic. The case fatality rates of some highly virulent viruses and related references are listed in Table 1 . On November 11, 1918 , the fighting of World War I was stopped, and World War I was officially ended on June 28, 1919 with the signing of the Versailles Treaty. It is estimated that around 8.5-10 million soldiers lost their lives in World War I due to battle. The war also directly caused more than 6 million civilian deaths. Millions of people suffered from hunger and malnutrition during the war. Malnutrition weakened the human immune system and made a person more vulnerable to infectious diseases like tuberculosis and influenza, therefore, hunger and malnutrition were indirectly responsible for millions of deaths in the world in that period of time. For example, about 700,000 Germans died from malnutrition-related diseases in the years of 1914-1918. During the 1918-1919 influenza pandemic, between 21 and 25 million people died of influenza worldwide. Those people were killed both directly and indirectly by influenza virus infections. Many families were too poor to buy food and coal, and to afford health care expenses when their family members were ill. Influenza virus could infect all members of a family, and this could result in no one left to feed the fires, and to prepare food for the whole family, even if they had firewood, coal, and food left in their homes. Sadly, a large number of people died of influenza virus infections along with starvation, cold, and poor living conditions (8) . In recent years, while hunger and malnutrition are not major and serious problems in some developed countries anymore, they are still very difficult to overcome in many developing countries. In these less-developed countries, there were approximately 925 million people who suffered from hunger; 125 million children were underweight; and 195 million children were stunted each year (9) . Nevertheless, in comparison to 1918 and 1919, currently, we have much better social and economic conditions and public health systems globally; and generally speaking, the majority of people in the world have better nutritional and educational statuses; better living and working conditions; therefore, better general health and immunity. Furthermore, in 1918 and 1919, physicians and nurses almost had nothing in their hands to help individuals who were infected by influenza viruses. Today, although we still do not have very effective, powerful, and practical anti-influenza drugs available, we at least have some improved, useful, and helpful anti-viral drugs like zanamivir, and effective, convenient anti-cold medicines like Tylenol or Advil. We do not have a universal vaccine to prevent all influenza virus infections, but we can make effective vaccines to a specific influenza virus strain in a short time. Actually, in the United States of America, the influenza classed mortality rate declined from 10.2/100,000 in the 1940s to 0.56/100,000 in the 1990s; and the classed mortality rates of 1957-1958 and 1968-1969 influenza pandemics were not remarkably different from the non-pandemic seasons (10) . Because of the above reasons, we can optimistically assume that even the same strain of influenza virus, which caused pandemic in 1918 and 1919, would not be able to kill millions of people and cause a pandemic comparable to the 1918-1919 pandemic again in the future. Additionally, a significant number of viruses can cause influenza-like syndromes, such as rhinovirus, parainfluenza virus, adenovirus, coronavirus, respiratory syncytial virus, Coxsackie B virus, echovirus, and metapneumovirus (11, 12) . Some of the above-mentioned viruses like adenovirus and mutated coronavirus could cause problems that are comparable to influenza viruses (13, 14) . The World Health Organization (WHO) mistakenly raised the level of influenza pandemic alert from phase 5 to the highest phase 6 on June 11, 2009 (15) . However, the truth was that most cases of H1N1 influenza A virus infections were mild, the symptomatic case fatality rate was only 0.005% in New Zealand (16) ; and in New York City, the case fatality rate was 0.0094-0.0147% for persons ≥65 years old, and for those of 0-17 years old, the case fatality rate was 0.0008-0.0012% (17) . Some researchers argued that it should not have been called an influenza pandemic in the first place if the clinical severity was considered (15, (18) (19) (20) . I believe it was unwise that we had paid too much www.frontiersin.org 23) . Not surprisingly, every year there would be some influenza patients and a few of them would die from the infections, as it is almost impossible to eliminate influenza viruses from the natural environment in many years. The severity of a viral infection is determined by both of the viral virulence (pathogenicity) and the host immunity. Some researchers' opinions on H7N9 avian influenza virus were incorrect and/or inadequate. They mainly focused on influenza viruses and worried about viral mutations, viral pathogenicity, viral adaptation, and transmission. They overestimated the negative part of socio-economic factors of the present east China: overcrowded population in the epidemic region; very busy national and international transportation and travel; a large number of live poultry markets . . . but they underestimated the currently changed, developed, and improved positive part of socio-economic factors in China. The following factors might be used to explain why that H7N9 influenza A virus epidemic was limited and controlled in China, and only a few immunocompromised patients were killed by H7N9 influenza A virus. First, China has a relatively organized and effective public health system, there are four levels of (national, provincial, prefectural-level city, and county) centers for disease control and prevention all over China (24) . Second, physicians and nurses in China were prepared and knowledgeable of influenza virus infections. Third, samples from patients with suspected influenza virus infections were collected and sent to the local and national centers for disease control and prevention promptly. H7N9 influenza A viruses were isolated and identified very quickly. Thereby, they were able to diagnose, confirm, and report three cases of H7N9 influenza patients in the early stage of the epidemic (24, 25) . Fourth, health care and public health workers were protected properly. Consequently, none of the health professionals was infected by H7N9 influenza A virus in 2013. However, a surgeon died of H7N9 influenza in Shanghai, China in January of 2014 (26) . Fifth, they detected H7N9 influenza A viruses from the samples of chickens, pigeons, and the environment of live poultry markets in Shanghai (27) ; and closed the live poultry markets of the involved epidemic region quickly. Sixth, patients were isolated and treated timely in hospitals, 74% (1251/1689) of those close contacts of H7N9 influenza patients were monitored and observed. Thus, H7N9 influenza A virus could not spread to a bigger population (24) . Last but not least, we are connected to the Internet now, and it seems that our planet is much smaller today than the earlier days when we did not have the Internet, because communication and information exchange have become so fast, easy, and convenient presently. During that avian influenza epidemic, some influenza experts in the world shared/exchanged H7N9 influenza A virus information and provided professional consultations and suggestions efficiently and rapidly. All these public health routine practices and measures resulted in that H7N9 influenza epidemic being controlled and stopped in China (24) . I have to point out that the cases of diagnosed H7N9 avian influenza A virus infection might only be the tip of the iceberg. Aside from one laboratory confirmed asymptotic case of H7N9 influenza A virus infection in Beijing (22), there were probably many undetected mild or asymptotic cases of influenza A H7N9 infection. The reason is that most people usually think a common cold is a very common and normal occurrence, and they don't take flu-like illnesses seriously. In most situations, they would just stay home and take some medicines. Only those who have very severe flu-like symptoms would see doctors, and thereby be detected and diagnosed, accordingly the real case fatality rate should be much lower than the detected 32.14% (45/140, one case from Taiwan, and one case from Hong Kong) (22, 23). Nowadays, we travel faster, and we travel more frequently and globally, and we have more complicated social activities and lifestyles, thereby increasing the chances of viral mutation; and we realize that influenza viruses are even easier to reassort, recombine, and mutate in nature than many other RNA viruses. However, we are now living in a technologically, economically, and socially much better and advanced society. I believe influenza virus infections are controllable and preventable, with the increased population health and immunity, with the WHO Global Influenza Surveillance and Response System, and with standard/routine epidemiological practices, and with new effective anti-viral agents and vaccines in production in the future. Now, I first predict that influenza viruses will unlikely again cause a pandemic on a level comparable to what happened in 1918 and 1919. Hopefully, one day we could consider a strategy to produce a universal vaccine that can prevent people from infections of all influenza virus strains, or we could produce some very effective anti-influenza virus drugs; then influenza would not be a problem anymore. We should learn lessons from the mistakes we made in the past. It is reasonable and necessary to be cautious about influenza viruses, but overreactions or catastrophic reactions should be avoided in the future. My opinion is anti-traditional; the purpose of this article is to influence public health policy, and to save some of the limited resources and money for more important diseases like heart diseases, cancer, diabetes, AIDS, hepatitises, and tuberculosis (15) . Liting Song: conception of manuscript, drafting of manuscript, critical revision of manuscript, and final approval of manuscript. The author would like to recognize the contributions of the reviewers and editors of this manuscript for their corrections and editing, and Dr. Emanuel Goldman for correcting errors related to grammar and syntax of the final manuscript.
Approximately how many people died during the 1918-1919 influenza pandemic?
false
259
{ "text": [ "During the 1918-1919 influenza pandemic, between 21 and 25 million people died of influenza worldwide." ], "answer_start": [ 1998 ] }
2,669
Frontiers in antiviral therapy and immunotherapy https://doi.org/10.1002/cti2.1115 SHA: facbfdfa7189ca9ff83dc30e5d241ab22e962dbf Authors: Heaton, Steven M Date: 2020 DOI: 10.1002/cti2.1115 License: cc-by Abstract: nan Text: Globally, recent decades have witnessed a growing disjunction, a 'Valley of Death' 1,2 no less, between broadening strides in fundamental biomedical research and their incommensurate reach into the clinic. Plumbing work on research funding and development pipelines through recent changes in the structure of government funding, 2 new public and private joint ventures and specialist undergraduate and postgraduate courses now aim to incorporate pathways to translation at the earliest stages. Reflecting this shift, the number of biomedical research publications targeting 'translational' concepts has increased exponentially, up 1800% between 2003 and 2014 3 and continuing to rise rapidly up to the present day. Fuelled by the availability of new research technologies, as well as changing disease, cost and other pressing issues of our time, further growth in this exciting space will undoubtedly continue. Despite recent advances in the therapeutic control of immune function and viral infection, current therapies are often challenging to develop, expensive to deploy and readily select for resistance-conferring mutants. Shaped by the hostvirus immunological 'arms race' and tempered in the forge of deep time, the biodiversity of our world is increasingly being harnessed for new biotechnologies and therapeutics. Simultaneously, a shift towards host-oriented antiviral therapies is currently underway. In this Clinical & Translational Immunology Special Feature, I illustrate a strategic vision integrating these themes to create new, effective, economical and robust antiviral therapies and immunotherapies, with both the realities and the opportunities afforded to researchers working in our changing world squarely in mind. Opening this CTI Special Feature, I outline ways these issues may be solved by creatively leveraging the so-called 'strengths' of viruses. Viral RNA polymerisation and reverse transcription enable resistance to treatment by conferring extraordinary genetic diversity. However, these exact processes ultimately restrict viral infectivity by strongly limiting virus genome sizes and their incorporation of new information. I coin this evolutionary dilemma the 'information economy paradox'. Many viruses attempt to resolve this by manipulating multifunctional or multitasking host cell proteins (MMHPs), thereby maximising host subversion and viral infectivity at minimal informational cost. 4 I argue this exposes an 'Achilles Heel' that may be safely targeted via host-oriented therapies to impose devastating informational and fitness barriers on escape mutant selection. Furthermore, since MMHPs are often conserved targets within and between virus families, MMHP-targeting therapies may exhibit both robust and broadspectrum antiviral efficacy. Achieving this through drug repurposing will break the vicious cycle of escalating therapeutic development costs and trivial escape mutant selection, both quickly and in multiple places. I also discuss alternative posttranslational and RNA-based antiviral approaches, designer vaccines, immunotherapy and the emerging field of neo-virology. 4 I anticipate international efforts in these areas over the coming decade will enable the tapping of useful new biological functions and processes, methods for controlling infection, and the deployment of symbiotic or subclinical viruses in new therapies and biotechnologies that are so crucially needed. Upon infection, pathogens stimulate expression of numerous host inflammatory factors that support recruitment and activation of immune cells. On the flip side, this same process also causes immunopathology when prolonged or deregulated. 5 In their contribution to this Special Feature, Yoshinaga and Takeuchi review endogenous RNA-binding proteins (RBPs) that post-transcriptionally control expression of crucial inflammatory factors in various tissues and their potential therapeutic applications. 6 These RBPs include tristetraprolin and AUF1, which promote degradation of AU-rich element (ARE)-containing mRNA; members of the Roquin and Regnase families, which respectively promote or effect degradation of mRNAs harbouring stem-loop structures; and the increasingly apparent role of the RNA methylation machinery in controlling inflammatory mRNA stability. These activities take place in various subcellular compartments and are differentially regulated during infection. In this way, mRNA-destabilising RBPs constitute a 'brake' on the immune system, which may ultimately be toggled therapeutically. I anticipate continued efforts in this area will lead to new methods of regaining control over inflammation in autoimmunity, selectively enhancing immunity in immunotherapy, and modulating RNA synthesis and virus replication during infection. Another mRNA under post-transcriptional regulation by Regnase-1 and Roquin is Furin, which encodes a conserved proprotein convertase crucial in human health and disease. Furin, along with other PCSK family members, is widely implicated in immune regulation, cancer and the entry, maturation or release of a broad array of evolutionarily diverse viruses including human papillomavirus (HPV), influenza (IAV), Ebola (EboV), dengue (DenV) and human immunodeficiency virus (HIV). Here, Braun and Sauter review the roles of furin in these processes, as well as the history and future of furin-targeting therapeutics. 7 They also discuss their recent work revealing how two IFN-cinducible factors exhibit broad-spectrum inhibition of IAV, measles (MV), zika (ZikV) and HIV by suppressing furin activity. 8 Over the coming decade, I expect to see an ever-finer spatiotemporal resolution of host-oriented therapies to achieve safe, effective and broad-spectrum yet costeffective therapies for clinical use. The increasing abundance of affordable, sensitive, high-throughput genome sequencing technologies has led to a recent boom in metagenomics and the cataloguing of the microbiome of our world. The MinION nanopore sequencer is one of the latest innovations in this space, enabling direct sequencing in a miniature form factor with only minimal sample preparation and a consumer-grade laptop computer. Nakagawa and colleagues here report on their latest experiments using this system, further improving its performance for use in resource-poor contexts for meningitis diagnoses. 9 While direct sequencing of viral genomic RNA is challenging, this system was recently used to directly sequence an RNA virus genome (IAV) for the first time. 10 I anticipate further improvements in the performance of such devices over the coming decade will transform virus surveillance efforts, the importance of which was underscored by the recent EboV and novel coronavirus (nCoV / COVID-19) outbreaks, enabling rapid deployment of antiviral treatments that take resistance-conferring mutations into account. Decades of basic immunology research have provided a near-complete picture of the main armaments in the human antiviral arsenal. Nevertheless, this focus on mammalian defences and pathologies has sidelined examination of the types and roles of viruses and antiviral defences that exist throughout our biosphere. One case in point is the CRISPR/Cas antiviral immune system of prokaryotes, which is now repurposed as a revolutionary gene-editing biotechnology in plants and animals. 11 Another is the ancient lineage of nucleocytosolic large DNA viruses (NCLDVs), which are emerging human pathogens that possess enormous genomes of up to several megabases in size encoding hundreds of proteins with unique and unknown functions. 12 Moreover, hundreds of human-and avian-infective viruses such as IAV strain H5N1 are known, but recent efforts indicate the true number may be in the millions and many harbour zoonotic potential. 13 It is increasingly clear that host-virus interactions have generated truly vast yet poorly understood and untapped biodiversity. Closing this Special Feature, Watanabe and Kawaoka elaborate on neo-virology, an emerging field engaged in cataloguing and characterising this biodiversity through a global consortium. 14 I predict these efforts will unlock a vast wealth of currently unexplored biodiversity, leading to biotechnologies and treatments that leverage the host-virus interactions developed throughout evolution. When biomedical innovations fall into the 'Valley of Death', patients who are therefore not reached all too often fall with them. Being entrusted with the resources and expectation to conceive, deliver and communicate dividends to society is both cherished and eagerly pursued at every stage of our careers. Nevertheless, the road to research translation is winding and is built on a foundation of basic research. Supporting industry-academia collaboration and nurturing talent and skills in the Indo-Pacific region are two of the four pillars of the National Innovation and Science Agenda. 2 These frame Australia's Medical Research and Innovation Priorities, which include antimicrobial resistance, global health and health security, drug repurposing and translational research infrastructure, 15 capturing many of the key elements of this CTI Special Feature. Establishing durable international relationships that integrate diverse expertise is essential to delivering these outcomes. To this end, NHMRC has recently taken steps under the International Engagement Strategy 16 to increase cooperation with its counterparts overseas. These include the Japan Agency for Medical Research and Development (AMED), tasked with translating the biomedical research output of that country. Given the reciprocal efforts at accelerating bilateral engagement currently underway, 17 the prospects for new areas of international cooperation and mobility have never been more exciting nor urgent. With the above in mind, all contributions to this CTI Special Feature I have selected from research presented by fellow invitees to the 2018 Awaji International Forum on Infection and Immunity (AIFII) and 2017 Consortium of Biological Sciences (ConBio) conferences in Japan. Both Australia and Japan have strong traditions in immunology and related disciplines, and I predict that the quantity, quality and importance of our bilateral cooperation will accelerate rapidly over the short to medium term. By expanding and cooperatively leveraging our respective research strengths, our efforts may yet solve the many pressing disease, cost and other sustainability issues of our time.
What is the increasingly apparent role of RNA methylation machinery ?
false
4,146
{ "text": [ "in controlling inflammatory mRNA stability." ], "answer_start": [ 4479 ] }
1,689
Chikungunya: A Potentially Emerging Epidemic? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860491/ SHA: f7c3160bef4169d29e2a8bdd79dd6e9056d4774c Authors: Thiboutot, Michelle M.; Kannan, Senthil; Kawalekar, Omkar U.; Shedlock, Devon J.; Khan, Amir S.; Sarangan, Gopalsamy; Srikanth, Padma; Weiner, David B.; Muthumani, Karuppiah Date: 2010-04-27 DOI: 10.1371/journal.pntd.0000623 License: cc-by Abstract: Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005–07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts. Text: Chikungunya virus (CHIKV), a mosquito-borne pathogen listed by National Institute of Allergy and Infectious Diseases (NIAID) as a Category C Priority Pathogen that causes Chikungunya fever (CHIKF), has been spreading throughout Asia, Africa, and parts of Europe in recent times [1, 2, 3] . CHIKV is an arthropod-borne virus (arbovirus) and is transmitted to humans primarily by Aedes aegypti, the infamous yellow fever propagator [4, 5] . CHIKV infection is marked by severe joint pain, contorting its victims into unusual postures [6] . The disease gets its name from the Kimakonde vernacular language of Tanzania and Mozambique, and the word chikungunya means ''that which contorts or bends up'' and translates in Swahili to ''the illness of the bended walker'' [7, 8, 9] . In Africa, CHIKV is maintained in a sylvatic cycle among forest-dwelling Aedes spp. mosquitoes, wild primates, squirrels, birds, and rodents ( Figure 1 ) [10] . In Asia, the disease is vectored by Ae. aegypti and Ae. albopictus [11] . Transmission in Asia occurs in an urban cycle whereby the mosquito spreads the disease from an infected human to an uninfected human, following an epidemiological pattern similar to dengue fever [12] . The 2005-2006 epidemic of CHIKV in La Reunion islands in the Indian Ocean, spurred the discovery of a new vector species, Ae. albopictus [5] . Wrecking over one-third of the island's population, this epidemic peaked its devastation between January and February 2006, when over 46,000 cases came into light every week, including 284 deaths [5, 13] . Ae. albopictus is common in urban areas of the United States and is already flourishing in 36 states, raising grave concerns to the immunologically naive populace of the United States [14] . Accordingly, this review elaborately details the epidemiology and global expansion of CHIKV, describes its clinical features and pathogenesis and its symptoms and complications, and finally nominates a possible vaccine approach against CHIKV infection. CHIKV has been isolated into three genotypes based on phylogenetic studies. These genotypes, based on the gene sequences of an Envelope protein (E1), are Asian, East/Central/ South African, and West African [4, 11, 15] . Using phylogenetic models, Cherian et al. estimate that the Asian genotype of CHIKV emerged between 50 and 310 y ago, and the West and East African genotypes diverged between 100 and 840 y ago [15] . Since then, CHIKV has come a long way, with several mutations incorporated, and has continued to wreak epidemics in several regions. Recent activities of CHIKV include the Indian epidemic in 2005-2006, which was followed by a sudden explosion of cases in 2007. An estimated 1.3 million people across 13 states were reported to be infected in India [12, 16] , and CHIKV was also widespread in Malaysia, Sri Lanka, and Indonesia [17] . In July-August of 2007, CHIKV was reported in Italy, probably brought in by travelers from CHIKV-prone regions of India, Africa, and Indian Ocean islands such as Mauritius, Madagascar, and Seychelles. Few of the Italian isolates were found to have evolved from the Kerala isolate, which was associated with a A226V shift in E1 gene that represents a successful evolutionary adaptation in the mosquito vector similar to the ones observed in Reunion Island [2, 18, 19] . In recent times, with an increase in global travel, the risk for spreading CHIKV to non-endemic regions has heightened [1] . Several travelers have brought CHIKV home with them after visiting areas with actively infected populations [12, 20] . Such cases have been documented in European countries, Australia, Asia, and the United States [8, 21] . The United States has already reported at least twelve cases of travel-associated CHIKV, while France has reported 850 cases, and the United Kingdom 93 [8, 14] . Beyond this, CHIKV-infected travelers have also been diagnosed in Australia, Belgium, Canada, Czech Republic, French Guiana, Germany, Hong Kong, Italy, Japan, Kenya, Malaysia, Martinique, Norway, Switzerland, and Sri Lanka [21] . Some travelers were viremic, worrying public health officials about the spread of CHIKV to new areas [1, 8] . The incubation time for CHIKV is relatively short, requiring only 2-6 d with symptoms usually appearing 4-7 d post-infection [22] . Vazeille et al. detected CHIKV in the salivary glands of Ae. albopictus only 2 d after infection [5] . Upon infection, CHIKF tends to present itself in two phases. The first stage is acute, while the second stage, experienced by most but not all, is persistent, causing disabling polyarthritis. Characteristics of the acute phase include an abrupt onset of fever, arthralgia, and in some cases, maculopapular rash [6, 23] . The acute phase causes such intense joint and muscular pain that makes movement very difficult and prostrates its victims [6, 20] . Ninety-five percent of infected adults are symptomatic after infection, and of these, most become disabled for weeks to months as a result of decreased dexterity, loss of mobility, and delayed reaction. Eighteen months after disease onset, 40% of patients are found to still have anti-CHIKV IgM [6, 18, 23, 24] . The chronic stage of CHIKF is characterized by polyarthralgia that can last from weeks to years beyond the acute stage [6] . CHIKV has been shown to attack fibroblasts, explaining the involvement of muscles, joints, and skin connective tissues. The high number of nociceptive nerve endings found within the joints and muscle connective tissues can explain pain associated with CHIKF [25, 26] . More than 50% of patients who suffer from severe CHIKF are over 65 y old, and more than 33% of them die. Most adults who suffer from severe CHIKF have underlying medical conditions [6, 24, 27] . The other group that is disproportionately affected by severe CHIKV is children. Other complications associated with CHIKV, from most common to least common, include respiratory failure, cardiovascular decompensation, meningoencephalitis, severe acute hepatitis, severe cutaneous effects, other central nervous system problems, and kidney failure [6, 18, 20, 23, 24, 26, 27] . CHIKV undertakes a complex replication cycle upon host infection (Figure 2 ), which makes its genome susceptible to mutations [28, 29] . For instance, Ae. aegypti, responsible for epidemics in Kenya, Comoros, and Seychelles, carried CHIKV with an alanine in the 226 position of the E1 gene (E1-A226) [4, 18] . However, when the virus struck La Reunion Islands, a decline in population of Ae. aegypti, due to massive dichlorodiphenyltrichloroethane usage and dearth of Ae. albopictus species' www.plosntds.org population, resulted in an ecological pressure, favoring replacement of alanine at position 226 with valine (E1-A226V) [5] . This mutation allowed CHIKV's secondary vector species, Ae. albopictus, to supplement Ae. aegypti as its primary vector [5] . Within a year, the E1-A226V mutation was present in La Reunion Island, and Ae. albopictus apparently vectored the large epidemic infecting 34% of La Reunion Island's population [5] . All of the CHIKV strains isolated from Mayotte carried the E1-A226V mutation, and the mutation was also found in Madagascar in 2007 [5] . The E1-A226V mutation was not present at the beginning of the Indian Ocean Islands outbreak (before September 2005). However, more than 90% of later viral strains found there had incorporated the mutation (December-March 2006), indicating a genotype switch during the winter season [5, 18, 20] . The E1-A226V mutation also enabled an increase in infectivity of Ae. albopictus when compared to its infectivity of Ae. aegypti [4, 11, 18, 30] , and with several factors taken together, Ae. albopictus has become the new preferred and more lethal vector for CHIKV [4, 5, 11] . In fact, Tsetsarkin et al. found that a Green Fluorescent Protein tagged E1-A226V virus was 100 times more infective to Ae. albopictus than it was to Ae. aegypti [4] . In all the Indian Ocean Islands, Ae. albopictus became the main vector for CHIKV within 1-2 y after CHIKV was introduced to the region [31] . Of note is that Ae. aegypti has most likely been established in North America for over 300 y, while Ae. albopictus has been in many areas of the US, since 1985, primarily in Florida [32] and since then has expanded its range in the country. Reiskind et al. set out to determine if Ae. aegypti and Ae. albopictus mosquitoes captured in Florida were susceptible to CHIKV infection by a La Reunion isolate [32] . Each mosquito tested was highly susceptible to infection by a full-length infectious clone of the La Réunion Island isolate, CHIKV LR2006 OPY1 strain. Even though the Ae. albopictus strains were more susceptible to infection, overall ecology and differences in human biting patterns need to be studied further Characteristically, there are two rounds of translation: (+) sense genomic RNA (49S9 = 11.7 kb) acts directly as mRNA and is partially translated (59 end) to produce non-structural proteins (nsp's). These proteins are responsible for replication and formation of a complementary (2) strand, the template for further (+) strand synthesis. Subgenomic mRNA (26 S = 4.1 kb) replication occurs through the synthesis of full-length (2) intermediate RNA, which is regulated by nsp4 and p123 precursor in early infection and later by mature nsp's. Translation of the newly synthesized sub-genomic RNA results in production of structural proteins such as Capsid and protein E2-6k-E1 (from 39 end of genome). Assembly occurs at the cell surface, and the envelope is acquired as the virus buds from the cell and release and maturation almost simultaneous occurred. Replication occurs in the cytoplasm and is very rapid (,4 h) [28, 29] . doi:10.1371/journal.pntd.0000623.g002 www.plosntds.org to gain a more accurate understanding of a potential CHIKV epidemic in the US [32] . During the 7 d preceding birth, no human mother has been reported to transmit the disease vertically. However, about 50% of newborns delivered while the mother was infected with CHIKV contracted the disease from their mother, despite the method of delivery. Furthermore, there have been instances of CHIKV transmission from mother to fetus causing congenital illness and fetal death [33] . During the 2005-2006 La Reunion Island outbreaks, Ramful et al. discovered that mothers could transmit CHIKV to their progeny during the perinatal period (Day 24 to Day +1) [33, 34] , and it is associated with a high degree of morbidity. By mean Day 4 of life, all of the neonates were symptomatic for CHIKV, exhibiting common CHIKF symptoms. Six neonates were confirmed to have contracted CHIKV and developed mengoencephalitis. Of those mothers who, during the La Reunion Island epidemic, were infected long before delivery, only three fetal deaths were reported [12, 33] . Ramful et al. theorized that motherto-child transmission most likely happens transplacentally shortly before delivery [33] . A similar study by Gerardin et al. reported nineteen cases of neonatal infection associated with intrapartum maternal viremia that progressed to develop encephalitis owing to vertical transmission from infected mothers [34] . Clinical and epidemiological similarities with dengue fever make CHIKV diagnosis difficult, which may lead physicians to misdiagnose CHIKV as dengue fever; therefore, the incidence of CHIKV may actually be higher than currently believed (Table 1 ) [6, 12, 35] . The amount of time elapsed since disease onset is the most critical parameter when choosing a diagnostic test. CHIKV can be detected and isolated by culturing with mosquito cells (C6/36), Vero cells (mammalian), or in mice [26] . However, this method can take at least a week and only achieves a high sensitivity during the viremic phase, which usually only lasts up to 48 h after the bite. Five days post-infection, the viral isolation approach has a low sensitivity but is still the preferred method for detecting the CHIKV strain [12, 26, 31, 35] . RT-PCR on the other hand is a faster and more sensitive method that can be used within the first week of disease onset [26] , and it is currently the most sensitive method for detecting and quantifying viral mRNA [4, 36] . Classic serological detection, by assays such as ELISA [37] , immunofluorescence [5, 38] , complement binding, and haemagglutination inhibition [39] , constitutes the second diagnostic tool used for biological diagnosis of CHIKV infection. These proven techniques are useful for detection of Antigen in mosquitoes during epidemiological studies. These assays detect virus-specific IgM and IgG, however the sensitivity and specificity of these assays has been poorly characterized. Viral competence, or the potential of viral infection and transmission, is an important parameter that can be quantified by ELISA, viral culture, and PCR. A study by Ng et al. showed biomarkers indicative of severe CHIKV infection [40] . They found decreased levels of RANTES and increased levels of Interleukin-6 (IL-6) and Interleukin-1b (IL-1b) that could be sued for CHIKV detection in patients as indicators of CHIKV-driven cytokine storm. Couderc et al. demonstrate another cytokine, type-I IFN, as a key player in the progression to CHIKV infection [26] . Using an IFN-a/b null mouse model, they demonstrated evidence of muscles, joints, and skin as privileged CHIKV targets, which is consistent with human pathology. Although Ng et al. concluded that RANTES levels were significantly suppressed in severe CHIKF patients [40] , interestingly, an increase in levels of RANTES has been observed in dengue infection [41] . Since the symptoms of CHIKF mimic those of dengue fever, results obtained from this study strongly suggest that RANTES could be a potential distinctive biomarker that differentiates between these two clinically similar diseases. There are no approved antiviral treatments currently available for CHIKV [1, 3, 12, 42] . Currently, CHIKF is treated symptomatically, usually with non-steroidal anti-inflammatory drugs or steroids, bed rest, and fluids. Movement and mild exercise are thought to decrease stiffness and morning arthralgia, but heavy exercise may exacerbate rheumatic symptoms. Corticosteroids may be used in cases of debilitating chronic CHIKV infection. There is a debate about the appropriateness of chloroquine as treatment for unresolved, non-steroidal anti-inflammatory drugresistant arthritis [43] . A study showed that viral production was www.plosntds.org drastically reduced at 16 h post-infection after treatment with 100 mM dec-RVKR-cmk (Decanoyl-Arg-Val-Lys-Arg-chloromethylketone), a furine inhibitor [42, 44] . Chloroquine acted by raising the pH, blocking low pH-dependent entry of virus into the cell. It is important to note that dec-RVKR-cmk or chloroquine only inhibited viral spreading from cell to cell, not CHIKV replication once it had entered the cell [43] . However, most would agree that the best weapon against CHIKV is prevention. A live CHIKV vaccine developed by the United States reached phase II clinical trial encompassing 59 healthy volunteers [45] . Eight percent of the volunteers experienced transient arthralgia, while 98% of the volunteers had seroconversion [45] . However, live CHIKV vaccines are still questionable. One cannot discount the risk of a live vaccine possibly inducing chronic rheumatism. Also, there is the question as to whether widespread use among the public could trigger mosquito transmission or lead to chronic infection or viral reversion [1] . An alternative approach would be to produce a chimeric vaccine against CHIKV. Wang et al. developed a chimeric alphavirus vaccine that is uniformly attenuated and does not cause reactogenicity in mice [3] . Three different versions of this vaccine were made using three different backbone vectors: Venezuelan equine encephalitis virus (VEEV) attenuated vaccine strain T-83, naturally attenuated eastern equine encephalitis virus (EEEV), and attenuated Sindbis virus (SINV). In short, CHIKV structural proteins were engineered into the backbones of the aforementioned vaccines to produce the chimeras [3] . These chimeras were found to stimulate a strong humoral immunity, and even at doses of 5.3-5.8 log 10 PFU, they did not trigger reactogenicity. When vaccinated mice were challenged with CHIKV, neither adult nor neonatal mice gained weight, had fever, or displayed signs of neurological illness. Upon comparison of the chimeras with the Army181/25 vaccine, the Army vaccine resulted in higher levels of viremia and replication in the joints of neonatal mice. Because the joints are known targets of CHIKV, Wang et al. noted their vaccine might avoid the negative reactogenic side effects of the Army vaccine. After being subcutaneously vaccinated with 5.3-5.8 log 10 PFU of the chimeric vaccines, mice produced strong neutralizing antibody titers. The VEEV and EEEV chimeras yielded higher neutralizing antibody titers than the SINV chimera without being more virulent. On top of this, the VEEV and EEEV CHIKV chimeras seemed to be more immunogenic than the Army vaccine despite the chimeras' lower viremia and replication in the joints of neonatal mice [3] . Tiwari et al. [46] adopted a different strategy using formalin inactivated CHIKV in combination with alhydrogel (Aluminum Hydroxide) as an adjuvant. This study clearly suggests that this vaccine elicits both humoral and cell-mediated immune responses in mice, providing its immunogenic potential. A recent study by Couderc et al. [47] showed passive immunization as a potential treatment for CHIKV infection. Using purified immunoglobulin extracted from convalescent CHIKV patients, they demonstrated effective neutralizing activity against CHIKV infection both in vitro and in vivo. This thereby establishes a potential preventive and therapeutic approach to combat CHIKV infection. Pathogenesis studies conducted with related alpha virus, like RRV, have shown the role of macrophages in persistence on infection [48] . They also demonstrated the role of RRV-specific CD8 T cells in clearing viral load in infected patients, thereby warranting similar investigations with CHIKV and the importance of investigating a cell-mediated immune response-based vaccine against CHIKV [49] . There are always certain risks associated with live attenuated or inactivated viral vaccines [50] . One way to avoid these potential problems is to construct a consensus-based DNA vaccine. DNA based vaccines have an improved safety profile as compared to live or attenuated vaccines [51, 52] . A consequence of CHIKV's rapid evolution is difficulty in constructing a vaccine that will be able to Figure 3 . Levels of CHIKV-specific IgG in mice immunized with CHIKV vaccines. Each group of C57BL/6 mice (n = 5) was immunized with 12.5 mg of pVax1 control vector or CHIKV vaccine plasmids as indicated at 0 and 2 wk. Mice were bled 2 wk after each immunization, and each group's serum pool was diluted to 1:100 and 1:500 for reaction with specific vaccine constructs. Serum was incubated for 1 h at 37uC on 96-well plates coated with 2 mg/ml of respective CHIKV peptides, and antibody was detected using anti-mouse IgG-HRP and OD was measured at 405 nm. doi:10.1371/journal.pntd.0000623.g003 www.plosntds.org effectively protect large populations from multiple strains of the virus. One of the strengths of DNA consensus vaccines is its ability to induce cross-reactive immune responses against the three distinct phylogenetic groups of CHIKV. Also DNA-based vaccines can be produced more rapidly than protein-based vaccines. Recently, Muthumani et al. constructed a vaccine that was shown to induce both humoral and cellular immunity in vivo in 3-4-wk-old female C57/BL6 mice [49] . These mice were immunized using an in vivo electroporation method to deliver the vaccine into the quadriceps muscle. The consensus construct was designed against E1, E2, and the core protein capsid. To design the construct, they aligned 21 sequences of CHIKV isolated between 1952 and 2006, using strains from differing countries, including La Reunion Island. The most common nucleotide among the sequences was chosen at each position to be used in the consensus construct, taking care not to alter the reading frame. They conducted codon and RNA optimization, added a strong Kozak sequence, and substituted signal peptide with an immunoglobulin E leader sequence to improve vaccine efficacy. After immunizing the mice, spleens were harvested along with serum and tested to determine antibody titer. After three immunizations, consensus E1, E2, and C vaccines were shown to induce T-cell immune responses leading to strong IFN-c responses and proliferation in C57/BL6 mice. Furthermore, when compared with control mice, immunized mice had higher total IgG levels as well as higher anti-E1 specific, anti-E2 specific, and anti-C specific IgG antibodies, suggesting a strong humoral immune response ( Figure 3 ) and also specificity for the antigens encoded in the vaccine constructs ( Figure 4 ). Because of its promising results and the need for a safer vaccine, this consensus DNA vaccine deserves further investigation. Determining longevity of protective effects of the vaccine and persistence of antibody and IFN-c responses could be the next step of investigation. Challenged studies of immunized mice must also be carried out. CHIKV mosquito-borne disease has caused massive outbreaks for at least half a century but is no longer confined to the www.plosntds.org developing nations. It began to encroach into the boundaries of the developing world. As a result, the NIAID has designated CHIKV as a Category C pathogen alongside the influenza and SARS-CoV viruses [3] . Realization of the potential severity of this disease is exigent; for instance, if used as a biological weapon, the world economy could be severely crippled; if enough members of the armed forces were to become infected during a military deployment, military operations could be significantly affected. Efforts to monitor the disease will only provide minimal warning in a global society, and steps to prevent the morbidity and mortality associated with pandemic are imperative [21, 31] . Despite the gravity of its infectious potency and the fear of it being a potential biological weapon, there is currently no vaccine for CHIKV infections. Live attenuated vaccine trials were carried out in 2000, but funding for the project was discontinued. Newer approaches such as DNA vaccines appear promising over conventional strategies like live attenuated or inactivated virus and thus call for further investigation. Recent advances such electroporation delivery and incorporation of adjuvants has boosted DNA vaccine efficacy [51, 53] . Despite the low antibody response to DNA vaccines, other numerous advantages have overshadowed these minor drawbacks (Table 2) , the most important one being the ability to induce both humoral and cellular immune responses [51, 54] . Judging by recent success, such as the immunogenic construct developed by Muthumani et al., DNA vaccines could play a major role in combating CHIKV [49] . Vaccines are literally a critical component of CHIKV disease control and therefore research in this area is highly encouraged. The dramatic spread of dengue viruses (DENV) throughout tropical America since 1980 via the same vectors and human hosts underscores the risk to public health in the Americas. The adverse events associated with the current live vaccine are well documented [55] . Realizing these drawbacks, earnest efforts should be taken to develop new strategies to forestall further spread and complications.
What is the NIAID designation of CHIKV?
false
2,528
{ "text": [ "as a Category C pathogen alongside the influenza and SARS-CoV viruses" ], "answer_start": [ 22921 ] }
1,656
Improved Pharmacological and Structural Properties of HIV Fusion Inhibitor AP3 over Enfuvirtide: Highlighting Advantages of Artificial Peptide Strategy https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541410/ SHA: f2fcc16391f946c99717b63ec9a24e5384aac381 Authors: Zhu, Xiaojie; Zhu, Yun; Ye, Sheng; Wang, Qian; Xu, Wei; Su, Shan; Sun, Zhiwu; Yu, Fei; Liu, Qi; Wang, Chao; Zhang, Tianhong; Zhang, Zhenqing; Zhang, Xiaoyan; Xu, Jianqing; Du, Lanying; Liu, Keliang; Lu, Lu; Zhang, Rongguang; Jiang, Shibo Date: 2015-08-19 DOI: 10.1038/srep13028 License: cc-by Abstract: Enfuvirtide (T20), is the first HIV fusion inhibitor approved for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, its clinical application is limited because of short half-life, drug resistance and cross-reactivity with the preexisting antibodies in HIV-infected patients. Using an artificial peptide strategy, we designed a peptide with non-native protein sequence, AP3, which exhibited potent antiviral activity against a broad spectrum of HIV-1 strains, including those resistant to T20, and had remarkably longer in vivo half-life than T20. While the preexisting antibodies in HIV-infected patients significantly suppressed T20’s antiviral activity, these antibodies neither recognized AP3, nor attenuated its anti-HIV-1 activity. Structurally different from T20, AP3 could fold into single-helix and interact with gp41 NHR. The two residues, Met and Thr, at the N-terminus of AP3 form a hook-like structure to stabilize interaction between AP3 and NHR helices. Therefore, AP3 has potential for further development as a new HIV fusion inhibitor with improved antiviral efficacy, resistance profile and pharmacological properties over enfuvirtide. Meanwhile, this study highlighted the advantages of artificially designed peptides, and confirmed that this strategy could be used in developing artificial peptide-based viral fusion inhibitors against HIV and other enveloped viruses. Text: The sequences of gp41 NHR-or CHR-derived peptides. The residues corresponding to the NHR pocket region are marked in red. The residues for the PBD are marked in blue, and the MT-hook residues adjacent to the N terminus of PBD are marked in green. 5HRu peptide consists of 5 copies of artificial sequence template (AEELAKK) underlined. The mutant residues in PBD of AP2 and AP3 were highlighted in pink. (b) The inhibitory activity of AP1, AP2, AP3 and T20 on infection by HIV-1 IIIB (subtype B, X4) in MT-2 cells (left panel) by HIV-1 Bal (subtype B, R5) in M7 cells (right panel). Each sample was tested in triplicate and the experiment was repeated twice. The data are presented as means ± SD. Scientific RepoRts | 5:13028 | DOi: 10 .1038/srep13028 To address these obstacles, many efforts have been made to optimize T20 and gp41 CHR-derived peptides. Some of these peptides have better inhibitory activities against T20-resistant strains and/or longer half-life than T20. However, they still have the problem to cross-react with the preexisting antibodies in the sera of HIV-infected patients because they contain some native CHR sequences. Based on the universal artificial peptide template of 5HRu, we previously designed the artificial peptides of AP1 (PBD-m4HR) and AP2 (PBDtrp-m4HR), and have made preliminary research on their inhibitory activity against HIV-1 Env-mediated cell-cell fusion 16 . In the present study, we designed a new artificial peptide, AP3 (Fig. 1a) , aiming to apply the "M-T hook" structure to stabilize the interaction of the artificial peptide with the hydrophobic pocket on the gp41 NHR trimer 17, 18 . After comprehensively studying its antiviral activity, biochemical property, crystal structure, functional mechanism, in vivo half-life and, for the first time, the effect of preexisting antibodies in the sera of HIV-infected patients, we found that the newly designed artificial peptide, AP3, exhibited improved antiviral activity, drug resistance profile and pharmacological properties over T20. Particularly, the preexisting antibodies in the sera of HIV-infected patients did not suppress, but enhanced the anti-HIV-1 activity of AP3. These results suggest that AP3 has potential for development as a new anti-HIV drug and confirm that this strategy can be used for designing artificial antiviral peptides against other enveloped viruses, such as SARS-CoV 19 , MERS-CoV 20 , and paramyxovirus 21 . AP3 inhibited HIV-1 infection with higher potency than T20. Our previously designed artificial peptides AP1 and AP2 could inhibit HIV-1 Env-mediated cell-cell membrane fusion 16 . He and colleagues reported that adding two amino acids of Met and Thr to the N-terminus of a CHR-peptide could enhance their anti-HIV-1 activity 17, 18 . Here we designed a new artificial peptide, AP3, by adding Met and Thr to the N-terminus of AP2 (Fig. 1a) . We then compared AP3 with AP1, AP2 and T20 for their anti-HIV-1 activity against divergent HIV-1 strains, including the laboratory-adapted viruses, IIIB (subtype B, X4) and Bal (subtype B, R5), and a series of primary HIV-1 isolates, as well as the T20-resistant strains. As shown in Fig. 1b , AP3 exhibited higher inhibitory activities on infection by HIV-1 IIIB and HIV-1 Bal strains (IC 50 : 3.06 and 15.09 nM, respectively) than AP1 (IC 50 : 86.25 and 396.14 nM, respectively), AP2 (IC 50 : 23.05 and 49.95 nM, respectively), and T20 (IC 50 : 13.63 and 30.21 nM, respectively). The inhibitory activity of AP3 on infection by divergent primary HIV-1 isolates with distinct genotypes (subtypes A -E and group O) and phenotypes (R5 and X4) was also higher than that of AP2 and T20 (Table 1) . While T20 was not effective against T20-resistant HIV-1 strains at the concentration as high as 2,000 nM, AP3 could effectively inhibit infection of these strains with IC 50 in the range of 13 ~ 90 nM, which was about 2-to 4-fold more effective than AP2 (Table 1 ). These results indicate that the artificial peptide AP3 has remarkably improved anti-HIV-1 activity against a broad spectrum of HIV-1 strains, including T20resistant variants, over T20 and the artificial peptides AP1 and AP2. The preexisting antibodies in HIV-1-infected patients neither recognized AP3, nor attenuated its anti-HIV-1 activity. Previous studies have shown that the preexisting antibodies in HIV-1-infected patients, including those cross-reacting with T20 and those specific for the binding sites of T20 in gp120 (e.g., the C1 and V3 loop regions) and gp41 (e.g., the NHR domain), could significantly block the fusion inhibitory activity of T20 14, 15 . Here we investigated the influence of preexisting antibodies against AP3 peptide. As shown in Fig. 2a , both T20 and C46 reacted with the antibodies in sera from five HIV-1-infected patients; however, none of the three artificial peptides AP1, AP2 and AP3 was recognized by the preexisting antibodies. The inhibitory activity of T20 on HIV-1 IIIB infection was reduced about 1.9-fold to > 3.6-fold in the presence of the sera from HIV-1-infected patients ( Fig. 2b and Supplementary Table S1), confirming that the preexisting antibodies in sera of HIV/AIDS patients can attenuate the anti-HIV-1 activity of T20 14, 15 . However, none of the artificial peptides in the present study showed significant decrease of anti-HIV-1 activity in the presence of patients' sera. Instead, the antiviral activity of AP3 increased in the presence of antisera from HIV-1-infected patients ( Fig. 2b and Supplementary Table S1), suggesting that anti-HIV-1 antibodies actually enhanced the anti-HIV-1 activity of AP3, possibly because the binding of the antibodies to some sites in gp120 or gp41 promote the interaction of AP3 with viral gp41 NHR region. AP3 had longer half-life than T20. Although T20 has shown efficacy in inhibiting HIV-1 infection, its major weakness lies in its short half-life in plasma (about 2 h) [22] [23] [24] . As a result, T20 has to be administered subcutaneously twice daily at 90 mg per dose, often causing serious injection-site reactions 25, 26 . Here, we performed pharmacokinetic studies by intravenous administration of AP3, AP2, and T20, respectively, to SD rat at a dose of 1 mg/kg, in order to compare their in vivo circulation time. As expected, T20 exhibited a shorter half-life and lower AUC (0-t) from systemic circulation, while AP3 and AP2 demonstrated much higher concentration and longer circulation time ( Table 2 ). The pharmacokinetic profiles of AP3 and AP2 fit a non-compartment model. The pharmacokinetic parameters were calculated with PK Solver. The in vivo elimination half-life of AP3 (t 1/2 = 6.02 h) was about 2.8-fold longer than that of T20 (t 1/2 = 1.57 h). This result provided the theoretical basis for reducing the injection frequency and dose of the fusion inhibitor, in conjugation with the improved antiviral potency of AP3. Therefore, replacement of T20 with AP3 may significantly reduce injection-site reactions and the drug cost, which would promote the clinical applications of the HIV fusion inhibitor in resource-poor regions or countries. AP3 was much more resistant than T20 to proteolytic degradation by proteinase K and rat liver homogenate. We compared the stability of T20 and AP3 in the presence of proteinase K (a broad-spectrum serine proteinase) and rat liver homogenate. After treatment with 20 ng/mL of proteinase K for 2 h at 37 °C, only 29% of the parental T20 peptide remained, as detected by LC-MS analysis. Under the same condition, AP3 retained 100% of its prototype (Fig. 3a ). In addition, AP3 showed a significantly enhanced in vitro metabolic stability over T20 in the presence of liver homogenate (Fig. 3b) . These results indicate that the artificial peptide AP3 is much more resistant to proteolytic degradation than the natural peptide T20, which may contribute to its significant longer in vivo half-life than T20 as described above. AP3 formed stable α-helical complex and block gp41 6-HB formation. To investigate the antiviral mechanism of AP3, the thermal stability of AP3/N36 complex was compared with that of AP1/N36, AP2/N36, T20/N36, and C34/N36 complexes by circular-dichroism (CD) spectroscopy 27 . Because T20 lacks the pocket-binding domain (PBD), the T20/N36 complex did not show a typical α -helical conformation, in consistence with our previous studies 8, 9 . Similar to the α -helicity of C34/N36 complex 3 , the AP1/N36, AP2/N36 and AP3/N36 complexes all formed a saddle-shaped negative peak at 208 nm and 222 nm, indicating their α -helical structures (Fig. 4a) Fig. 4b) , indicating that the α -helical complex formed by AP3 and N36 is the most stable among the four complexes. Then we compared the inhibitory activity of AP3 with that of AP1 and AP2 on 6-HB formation between C34 and N36. Since T20 cannot block 6-HB formation 8, 9 , we used a small-molecule HIV-1 fusion inhibitor, ADS-J1 28, 29 , to replace T20 as a control of 6-HB inhibition. As expected, ADS-J1 could effectively inhibit 6-HB formation with IC 50 of 2.75 μ M 8, 9, [27] [28] [29] . AP3 was highly effective against 6-HB formation in a dose-dependent manner with an IC 50 value of 0.24 μ M, about 30-and 15-fold more potent than AP1 and AP2, respectively (Fig. 4c) , confirming that AP3 can potently block gp41 6-HB fusion core formation, thus inhibiting HIV-1 fusion with the target cell membrane. Structural basis for the potent fusion inhibitory activity of the artificial peptide AP3. To elucidate the molecular determinants of these artificial peptides, we successfully solved all three complex structures of AP1/AP2/AP3 peptides binding with gp41 NHR. For AP1 and AP2, an optimized linker Each sample was tested in triplicate and the experiment was repeated twice. The data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. "SGGRGG" was used to assemble the NHR and the artificial peptide into a single recombinant protein (N36-L6-AP1 or N36-L6-AP2). However, a similar strategy failed on the crystallization of AP3; therefore, we decided to cocrystallize the synthetic peptide N45 and AP3 peptide, and eventually the complex crystals were obtained. Interestingly, the crystals of three different inhibitors belong to three distinctive space groups: P2 1 for N36-L6-AP1, R32 for N36-L6-AP2, and P6 3 for N45/AP3. As expected, the NHR portions in three structures all form a trimeric core, while the AP1, AP2 or AP3 portion folds into a single-helix conformation and binds to NHR-trimer to form a typical 6-HB, similar to that of the HIV-1 gp41 core structure formed by the native CHR peptide C34 and N36 (Fig. 5a) . Also, the conserved hydrophobic residues, such as W43, W46 and I50, in the artificial peptides were deeply buried into the hydrophobic Table 2 . Pharmacokinetic parameters of AP2, AP3 and T20 following intravenous administration at 1 mg/kg in male SD rats (n = 2). Figure 3 . Sensitivity of AP3 and T20 to proteolytic degradation by proteinase K and rat liver homogenate. (a) After digestion by proteinase K at pH 7.2 and (b) rat liver homogenate, the residual amount of AP3 and T20 was detected by LC-MS analysis. The experiment was performed in triplicate and the data are presented as means ± SD. The inhibition of AP1, AP2, AP3, T20 and ADS-J1 against 6-HB formation between N36 and C34 was detected by ELISA using the 6-HB-specific mAb NC-1. Each sample was tested in triplicate, and the data are presented as means ± SD. grooves formed between each pair of NHR helices, similar to the corresponding residues of W628, W631 and I635 in the native gp41 CHR (Fig. 5b) . AP peptides exhibited better affinity against gp41 natural CHR. In C34, which contains the natural CHR sequence from W628 to L661, no strong interaction between I642 and Q565 in the viral gp41 NHR-CHR complex was found (Fig. 5c) . However, in the corresponding sequence (from W43 to K76) of AP1 and AP2, a hydrogen bond was established between S57 (corresponding to I642 in CHR) and Q18 (corresponding to Q565 in NHR) in N36-L6-AP1, N36-L6-AP2 and N45/AP3. Thus, S57 in AP1/AP2/AP3 plays a role in stabilizing the interactions between the artificial peptide inhibitor and its NHR target, resulting in their stronger binding affinity. Moreover, in NHR-CHR, L567 and L568 on two adjacent NHRs form a hydrophobic groove, in which T639 is buried (Supplementary Fig. S1a ). However, in N36-L6-AP1, N36-L6-AP2 and N45/AP3, I54 (corresponding to T639 in CHR) can strongly bind to L20 and L21 through fully hydrophobic side chain interactions. Similarly, the interaction of I64 (corresponding to S659 in CHR) with L10 and L11 (corresponding to L567 and L568 in NHR, respectively) in N36-L6-AP1, N36-L6-AP2 and N45/AP3 has been significantly enhanced ( Supplementary Fig. S1b ). Like the gp41 CHR helix, the helices of AP1, AP2 and AP3 also have two different sides, a hydrophobic side facing toward the NHR and a hydrophilic one facing outward. It is expected that the enhancement of the hydrophilicity of the exposed side of the inhibitors can increase their antiviral activity and solubility. To achieve this goal, the amino acid residues with hydrophobicity, or low hydrophilicity, like N637, S640, L641 and S644 in CHR, were changed to the amino acid residues with high hydrophilicity, like E52, K55, K56 and E59 in AP1, AP2 and AP3, respectively. Moreover, the hydrophobic residue M629 in CHR was replaced with a hydrophilic residue E44 in AP2 and AP3 ( Supplementary Fig. S2 ). These hydrophilic residues, such as glutamic acid and lysine, can increase the solubility of whole peptide and, hence, stabilize the complex formed by the inhibitor and its target. It has been proved that the EE-KK double salt bridge can stabilize helix conformation 30 . We have identified this kind of interaction between i and i + 3 or i + 4 positions on the three complex structures. In N36-L6-AP1, R48 interacts with E45 and E52 to form a salt bridge network. In N36-L6-AP2, E45 interacts with K48, and E52 binds to K56, while in N45/AP3, K69 binds to E66 ( Supplementary Fig. S2 ). These strong salt bridges formed by the oppositely charged residues stabilize AP peptide conformation, bringing its inhibitory effect into full play. As previously reported, addition of the "M-T hook" to the CHR peptides C34 and sifuvertide could dramatically improve the anti-HIV-1 activity 17, 18 . As expected, the N-terminal Met and Thr of AP3 forms a hook-like structure (Fig. 5d) . The hydrophobic methionine side chain of M41 accommodates the groove between AP3 and NHR helices, capping the hydrophobic pocket. This interaction leads to a series of conformational changes. The main chain of AP3 at W43 moves 1.91 Å closer to NHR compared to AP2 (Supplementary Fig. S3 ). The side chain of W43 in AP3 flips around 90 degrees and is buried deeper than that of AP2. The side chain of E44 turns back to interact to D47, but the E45 side chain turns back from K48 and interacts with T42. Therefore, this M-T hook structure could further stabilize the binding between AP3 and NHR target. Enfuvirtide, also known as T20, was approved by the U.S. FDA as the first HIV entry inhibitor-based antiviral drug for use with other anti-HIV medicines to treat HIV-1 infected adults and children at ages 6-16 years 23,31,32 (http://www.fuzeon.com). Although T20 is an indispensable anti-HIV drug for HIV/ AIDS patients who have failed to respond to the current antiretroviral therapeutics, its shortcomings have limited its clinical application. T20 has lower anti-HIV activity and shorter half-life than other CHR peptides containing PBD, such as C34 and C38 8, 9, 33 . In addition, T20-resistant HIV-1 variants emerged shortly (e.g., 14 days) after its use in patients 34 . Most of the T20-resistant viruses carried mutations in the GIV motif (residues 36-45: GIVQQQNNLL) in the gp41 NHR domain 10, [34] [35] [36] [37] [38] . The lack of PBD contributes to the major weaknesses of T20 described above. Since the conserved hydrophobic pocket in the gp41 NHR-trimer plays a critical role in stabilizing the interaction between the gp41 NHR and CHR and formation of the fusogenic 6-HB core 1, 39, 40 , the PBD-containing CHR-peptide, like C34, can bind to viral gp41 trimer more strongly and stably, thus possessing more potent anti-HIV activity than T20, a CHR peptide without PBD 8, 9 . In the absence of PBD, T20 mainly interacts with the middle region of the NHR domain containing the GIV motif. Therefore, a virus with mutations in this motif is generally resistant to T20 10, [34] [35] [36] [37] [38] . Compared with other anti-HIV drugs, another weakness of T20 is its cross-reactivity with the preexisting antibodies in HIV-1-infected patients. Besides gp41, T20 could also bind to some regions in gp120. The preexisting antibodies specific for the T20's binding sites in gp120 and gp41 may indirectly suppress the anti-HIV activity of T20 14, 15 . Addition of PBD to the N-terminus of T20, such as T-1249, could significantly improve the anti-HIV-1 potency, half-life and drug-resistance profile 33, [41] [42] [43] . Addition of M-T hook structure to the N-terminus of a PBD-containing CHR-peptides, such as MT-C34 or MT-SFT, could further increase the anti-HIV-1 activity of the corresponding CHR-peptides 17, 18 . Deletion of the GIV-motif-binding domain from a CHR-peptide, such as CP621-652 and CP32M, is another effective approach to increase the genetic barrier to drug resistance 44, 45 . However, none of the above approaches is effective in preventing the cross-reaction of T20 with the preexisting anti-gp41 antibodies in HIV/AIDS patients, since the above-modified peptides mainly contain the native sequences of the HIV-1 gp41 CHR domain. Our previous studies have shown that AP1 and AP2, artificial peptides with non-native protein sequences, could form coiled-coil structure to interact with gp41 NHR and inhibit HIV-1 Env-mediated cell-cell fusion 16 . In the present study, we designed a new artificial peptide, AP3, by adding M-T hook structure to the N-terminus of AP2 (Fig. 1a) , followed by investigating the influence of preexisting anti-gp41 antibodies in HIV-infected patients on AP3, using AP1, AP2 and T20 as controls. We demonstrated that sera of HIV-infected patients could bind to T20 and significantly reduce its potency against HIV-1. However, these same serum samples did not interact with the three artificial peptides and hardly impaired their antiviral activity. Surprisingly, the antibodies in the sera could even enhance AP3's anti-HIV-1 activity (Fig. 2a,b and Supplementary Table S1 ). These results confirmed, for the first time, that replacement of the native viral sequence in T20 with an artificial sequence is an effective approach to overcome a key shortcoming of T20 whereby its anti-HIV activity could be attenuated by preexisting anti-gp41 antibodies in HIV/AIDS patients. It is worthwhile to explore why the antibodies in the sera is able to enhance the anti-HIV-1 activity of AP3. Our recent study has demonstrated that T20's anti-HIV-1 activity is enhanced by a non-neutralizing antibody directed against the NHR domain of the HIV-1 gp41 46 . We thus hypothesize that some of the anti-gp41 antibodies in HIV/AIDS patients may bind to a site in NHR domain adjacent to the AP3's binding region, resulting in increased interaction between AP3 and NHR-trimer and enhanced antiviral activity of AP3. We then compared the inhibitory activity of AP3 with M-T hook and T20/AP2 without M-T hook on infection by divergent HIV-1 strains. AP3 was more effective than either AP2 or T20 in inhibiting infection by the laboratory-adapted strains and the primary isolates of HIV-1, including those resistant to T20 (Fig. 1b, Table 1 ). One may question whether AP3 can also induce drug-resistant viruses in patients if it is used in clinics to treat HIV-infected patients. We believe that AP3 is expected to have much higher genetic barrier to resistance than T20 because AP3 contains PBD, while T20 lacks PBD. Dwyer et al. 33 used T2544, a PBD-containing CHR-peptide, to carry out a passaging experiment, using T20 as a control. They demonstrated that T20 could induce a mutant virus with high resistance (81-fold) to T20 in about 1 month, while T2544 failed to induce a resistant strain in more than 2 months in culture. After extending the passaging experiment for almost 8 months, they identified one strain with a weak resistance (8.3-fold) to T-2544, and the related mutation sites were not in the gp41 pocket region, suggesting that the PBD-containing CHR-peptides, including AP3, may have difficulty to induce drug-resistance. AP3 also had longer half-life than T20 (Table 2) , possibly because the artificial peptide AP3 is less sensitive to the proteolytic enzymes than T20 with native viral protein sequence. Removal of the proteolytic enzymes' cleavage sites in AP3 peptide is expected to further extend its half-life. These results confirmed that replacement of native protein sequence with artificial sequence and addition of the M-T hook to the PBD-containing peptide is a sound strategy for designing HIV fusion inhibitory peptides with improved antiviral activity and pharmacological properties when compared to T20. Since the three-dimensional structures of AP peptides had not been investigated before the present study, the optimization of these artificial peptide inhibitors could not be performed rationally. Our structural studies of the artificial peptides AP1/AP2/AP3 in complex with NHR showed that AP peptides, just like the CHR peptide C34, could bind to gp41 NHR to form a canonical 6-HB structure (Fig. 5a) . It is well known that a deep hydrophobic pocket exists in each groove on the surface of the viral gp41 NHR trimer. The hydrophobic residues I635, W631 and W628 in the gp41 CHR bind with the hydrophobic residues in the wall of this pocket, resulting in the formation of stable 6-HB by the strong interaction between CHR and NHR. This important feature has been well preserved in the AP1/AP2/AP3 6-HB structures (Fig. 5b) , which may account for the potent HIV-1 fusion inhibitory activities of these artificial peptides. A new hydrogen bond, which was established between S57 and Q18 in AP1/AP2/AP3 complexes, does not exist in the viral gp41 CHR-NHR complex, suggesting that S57 may play an important role in stabilizing the interactions between the peptide and NHR, resulting in binding affinities of AP1/AP2/AP3 that are stronger than those of HIV-1 gp41 CHR to NHR. Furthermore, the EE-KK double salt bridge formed between the i and i + 4 positions in the AP1/AP2/AP3 structures could stabilize helix conformation and increase the inhibitory effect of these peptides. Compared with AP1, triple-site mutations were introduced in AP2 and AP3, i.e. M44E, R48K and E49K. Those substitutions not only increase solubility of the peptide, but also trigger a series of rearrangements of certain intrahelical salt bridges to improve the stability of CHR helix structure and HIV-1 fusion inhibitory activity. M-T hook was previously demonstrated to be an effective step toward increasing the stable interaction between a CHR-peptide and the HIV-1 gp41 pocket 17, 18 . Therefore, AP2 was further optimized by incorporating Met and Thr at its N-terminus. CD spectroscopy and thermal denaturation results both indicate that the incorporation of M-T hook contribute to the formation of a more stable 6-HB core structure between AP3 (M-T hook-optimized AP2) and N36. In addition, the EE-KK double salt bridge formed between i and i + 4 positions in the N36-L6-AP3 structure contributed to increased CHR helix and 6-HB stability, resulting in improved potency of AP3, as has been noted in studies of CHR-peptides with EE-KK double mutations 30, 33, 47, 48 . Also, the HIV-1 fusion activity and half-life of AP2 may have been strengthened and extended, respectively, by the addition of M-T hook in the design of AP3. In conclusion, AP3, an artificial peptide with both PBD and M-T hook structures, exhibited improved anti-HIV-1 activity and drug-resistance profile, as well as prolonged half-life. Moreover, it did not react with the preexisting antibodies in the sera of HIV/AIDS patients. Consequently, its antiviral activity Scientific RepoRts | 5:13028 | DOi: 10.1038/srep13028 was not significantly affected by these antibodies. Therefore, AP3 shows promise as a candidate for further development as a new HIV fusion inhibitor for clinical use. This study also provides important structure and activity information for the rational design of novel artificially peptide inhibitors. Besides, our results highlighted the advantages of artificially designed peptides and confirmed that this strategy could be widely used in development of artificial peptide-based virus fusion inhibitors against HIV-1 and other enveloped viruses with class I membrane fusion proteins, such as SARS-CoV 19 , MERS-CoV 20 , and paramyxovirus 49 . Ethics statement. This study did not involve human experimentation; the only human materials used were serum samples obtained from HIV-1-infected individuals with the approval by the Ethics Committee of the Shanghai Public Health Clinical Center, Fudan University (Protocol No. SPHCC-125-2). The methods were carried out in accordance with the approved guidelines. All of these sera samples came from adults; no minor was involved in this study. Written informed consent for the use of the clinical specimens was obtained from all patients involved in this study. Peptide synthesis. A panel of peptides (Fig. 1a) , including T20, C34, C46, AP1, AP2, AP3, as well as NHR-derived N-peptides, N36 and N45, were synthesized with a standard solid-phase FMOC method, as described previously 8, 50 . All peptides were acetylated at the N terminus and amidated at the C terminus. The peptides were found to be about 95% pure by HPLC and were identified by mass spectrometry (Perseptive Biosystems, Framingham, MA, USA). Concentrations of the peptides were determined by UV absorbance and a theoretically calculated molar-extinction coefficient based on tryptophan and tyrosine residues. Qualification assay. Chromatographic analyses were performed using an ODS-C8 column (5 μ m, 100 mm × 2.0 mm ID) kept at ambient temperature. The mobile phase was composed of acetonitrile-water-formic acid in the ratio of 50:50:0.1 (v/v/v) at a flow rate of 0.3 mL/min. The sample injection volume was 10 μ L. Acetonitrile was HPLC grade, and other chemical reagents and solvents were analytical grade. A Thermo TSQ Quantum Discovery MAX triple-quadruple tandem mass spectrometer equipped with ESI source (San Jose, CA) and Surveyor LC pump were used for LC-MS analysis. Data acquisition and data processing were performed by using Xcalibur software and LCQuan 2.0 data analysis program (Thermo Finnigan), respectively. Optimized MS parameters were as below: 4800 V spray voltage, 40.0 psi sheath gas pressure, 1.0 psi auxiliary valve flow, and 300 °C of capillary temperature. When running collision-induced dissociation (CID), the pressure was set to 1.5 mTorr. The selected reaction monitoring (SRM) mode was used for AP3 while the selected ion monitoring (SIM) mode was preformed for T20. The following transitions were recorded: m/z 670.5 for AP3, m/z 1498.6 for T20. The masses of synthetic peptides T20, AP1, AP2 and AP3 were determined by MALDI-TOF-MS (Supplementary Fig. S4 and S5 ). Expression and purification of fusion protein N36-L6-AP1 and N36-L6-AP2. Using overlapping PCR, the DNA fragment encoding AP1 or AP2 peptide was attached to the 3′-end of the cDNA of gp41 NHR ("N36", 546-581), with a short linker ("L6", SGGRGG) between them. Then, the whole sequence was subcloned into the pET-28a vector (Novagen, USA) with an artificial SUMO-tag between the N-terminal His-tag and the target protein. The pET-28a-SUMO-N36-L6-AP1-or pET-28a-SUMO-N36-L6-AP2-transformed E. coli cells were induced by adding 1 mM IPTG and incubating overnight at 16 °C. Fusion protein was purified by Ni-NTA affinity resin (Qiagen, Valencia, CA, USA), and the His-SUMO-tag was cleaved off by Ulp1 enzyme treatment at 4 °C for 2 h. The purified N36-L6-AP1 or N36-L6-AP2 was applied onto a Superdex-75 gel filtration column (GE Healthcare, Piscataway, NJ, USA). Fractions containing N36-L6-AP1 or N36-L6-AP2 trimer were collected and concentrated to different concentrations by ultrafiltration. Crystallization, data collection, and structure determination. The fusion protein N36-L6-AP1 was crystallized at 16 °C using the hanging drop, vapor-diffusion method. The drops were set on a siliconized cover clip by equilibrating a mixture containing 1 μ l protein solution (25 mg/ml N36-L6-AP1 trimer in 20 mM Tris-HCl pH 8.0 and 150 mM NaCl) and 1 μ l reservoir solution (0.1 M Tris-HCl pH 8.5, 32% (w/v) PEG3350, and 0.2 M MgCl 2 ) against a 400 μ l reservoir solution. After one week, single crystals formed and were flash frozen by liquid nitrogen for future data collection. Fusion protein N36-L6-AP2 was crystallized in a similar way with a different reservoir solution (0.1 M Tris-HCl pH 8.0, 34% (w/v) PEG3350, and 0.2 M MgCl 2 ). To obtain the complex crystal of AP3 and NHR, synthesized AP3 was first mixed with peptide N45 at 1:1 molar ratio and then applied onto a Superdex-75 gel filtration column (GE Healthcare, Piscataway, NJ, USA) to isolate the formed 6-HB. Fractions containing N45/AP3 trimer were collected and concentrated to 30 mg/ml, then crystallized at 16 °C using the hanging drop, vapor-diffusion method.The drops were set on a siliconized cover clip by equilibrating a mixture containing 1 μ l protein solution (20 mM Tris-HCl pH 8.0 and 150 mM NaCl) and 1 μ l reservoir solution (0.2 M Ammonium Sulfate, 0.1 M Bis-Tris pH 6.5, and 25% w/v PEG 3350) against a 400 μ l reservoir solution. After 3 days, single crystals formed and were flash frozen by liquid nitrogen for future data collection. The datasets of N36-L6-AP1 were collected at 100 K at beamline 19-ID of the Advanced Photon Source (Argonne National Laboratory, USA). The datasets of N36-L6-AP2 were collected on an in-house x-ray source (MicroMax 007 x-ray generator, Rigaku, Japan) at the Institute of Biophysics, ChineseAcademy of Sciences. The datasets of AP3/N45 complex crystals were collected at beamline BL-19U1 of the Shanghai Synchrotron Radiation Facility, China. X-ray diffraction data were integrated and scaled using the HKL2000 program 51 . The phasing problem of all three structures was solved by the molecular replacement method using PHENIX.phaser 52 with a crystal structure of HIV gp41 NHR-CHR (PDB entry: 1SZT) as a search model. The final models were manually adjusted in COOT 53 and refined with PHENIX.refine 54 . All coordinates were deposited in the Protein Data Bank (N36-L6-AP1: 5CMU; N36-L6-AP2: 5CN0; and N45/AP3: 5CMZ). The statistics of data collection and structure refinement are given in Supplementary Table S2 . Determination of the cross-reactivity of the native and artificial peptides with the preexisting antibodies in HIV-1-infected patients by sandwich ELISA. A sandwich ELISA was conducted to determine the cross-reactivity of the peptides with the preexisting antibodies in HIV-1-infected patients. T20, C46, AP1, AP2 and AP3 were coated onto the wells of 96-well polystyrene plates (Costar, Corning Inc., Corning, NY) at 10 μ g/ml. The wells were then blocked with 1% gelatin, followed by addition of 50 μ l of serially diluted sera from HIV-1-infected patients and incubation at 37 °C for 1 h. Then, HRP-labeled goat-anti-human IgG (Abcam, UK) and TMB were added sequentially. A450 was determined with an ELISA reader (Ultra 384, Tecan). patients. Inhibition of peptides on HIV-1 IIIB (subtype B, X4)infection in the presence of HIV-1-infected patients' sera was determined as previously described 55 . Briefly, each peptide was mixed with serially diluted serum from an HIV-1-infected patient at room temperature for 30 min. Next, the mixture of peptide/serum and HIV-1 (100 TCID 50 ) were added to MT-2 cells (1 × 10 5 /ml) in RPMI 1640 medium containing 10% FBS. After incubation at 37 °C overnight, the culture supernatants were replaced with fresh culture medium. On the fourth day post-infection, culture supernatants were collected for detection of p24 antigen by ELISA. CD Spectroscopy and Thermal Midpoint Analysis. The secondary structure of AP1, AP2 or AP3 peptides mixed with N36 was analyzed by CD spectroscopy as previously described 56 . Briefly, each peptide or peptide mixture was dissolved in phosphate-buffered saline (PBS: 50 mM sodium phosphate and 150 mM NaCl, pH 7.2) at the final concentration of 10 μ M and incubated at 37 °C for 30 min before cooling down to 4 °C. The CD spectra of each sample were acquired on a Jasco spectropolarimeter (Model J-815, Jasco Inc., Japan) at 4 °C using a 5 nm bandwidth, 0.1 nm resolution, 0.1 cm path length, and an average time of 5.0 sec. Spectra were corrected by the subtraction of a blank corresponding to the solvent composition of each sample. Thermal midpoint analysis was used to determine the temperature at which 50% of the 6-HB formed by the CHR and NHR would decompose. It was monitored at 222 nm from 4 °C to 98 °C by applying a thermal gradient of 5 °C/min. The melting curve was smoothed, and the midpoint of the thermal unfolding transition (Tm) values was calculated using Jasco software utilities as described above. Inhibition of gp41 six-helix bundle formation by sandwich ELISA. Inhibition of gp41 six-helix bundle formation by a testing peptide was determined with a sandwich ELISA described previously 57 . Briefly, a testing peptide (ADS-J1 as a control) at graded concentrations was preincubated with peptide N36 (1 μ M) at 37 °C for 30 min, followed by the addition of peptide C34 (1 μ M) and incubation at 37 °C for another 30 min. The mixture was added to a 96-well polystyrene plate (Costar, Corning Inc., Corning, NY) precoated with anti-N36/C34 antibodies (2 μ g/ml) purified from mouse antisera specifically against the gp41 six-helix bundle 58 . Then, mAb NC-1, HRP-labeled rabbit-anti-mouse IgG (Sigma), and TMB were added in order. A450 was determined by an ELISA reader (Ultra 384, Tecan). Inhibition activities of AP1, AP2, and AP3 on HIV-1 infection were determined as previously described 57 . For inhibition of HIV-1 IIIB (subtype B, X4) infection,100 TCID 50 of the virus was added to 1 × 10 5 /ml MT-2 cells in RPMI 1640 medium containing 10% FBS in the presence or absence of the test peptide overnight. Then, the culture supernatants were changed to fresh media. On the fourth day post-infection, culture supernatants were collected for detection of p24 antigen by ELISA. For inhibition of infection by the HIV-1 strain Bal (subtype B, R5), M7 cells (1 × 10 5 /ml) were precultured overnight and infected with Bal at 100 TCID 50 in the presence or absence of the test peptide or protein overnight. Then, the culture supernatants were changed to fresh media. On the fourth day post-infection, the culture supernatants were discarded, and fresh media were complemented again. The supernatants were collected on the seventh day post-infection and tested for p24 antigen by ELISA as previously described 55 . The percent inhibition of p24 production was calculated. Analysis of the half-life of peptide inhibitors. Four male SD rats weighing approximately 200 g each were obtained from the Shanghai Medical School Animal Center and were used for the half-life assay. Animals were treated in accordance with the Animal Welfare Act and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication 86-23, revised 1985). Either AP2 or AP3 was intravenously injected at the concentration of 1 mg/ml. After injection, blood samples were acquired from rat orbit at several time points (8 and 30 min and 1.5, 3, 6, 9, 12, and 24 h after peptide injection) and placed in clean tubes. To study the pharmacokinetics of AP2 and AP3 in rats and provide experimental evidence for the possible pharmacokinetics in human, a double-antibody sandwich ELISA method was established for rapid determination of AP2 and AP3 in rat plasma. Briefly, 96-well polystyrene plates (Costar, Corning Inc., Corning, NY) were precoated with antibody against AP2 or AP3 (5 μ g/ml) purified from rabbit anti-sera 59 . They were then preincubated with serum samples diluted 20 times at 37 °C for 1 h, followed by the addition of anti-AP2 or anti-AP3 antibody (1:1000) purified from mouse antisera specifically against AP2 or AP3 59 at 37 °C for another 1 h. Then, HRP-labeled rabbit-anti-mouse IgG (Sigma, USA) and TMB were added in order. Absorbance at 450 nm was determined by an ELISA reader (Ultra 384, Tecan). The standard peptide parameters were obtained first. Then, the plasma peptide concentrations were determined as a function of time, and the half-life was calculated by using PK Solver for Microsoft Excel to obtain pharmacokinetic parameters. Assessment of sensitivity of peptides to proteolytic digestion by proteinase K and proteolytic enzymes in liver homogenate. The peptides (10 μ g/mL) were prepared in PBS pH 7.2 containing 20 ng/ml proteinase K. The resulting mixture were incubated at 37 °C in a water bath and taken out at different time intervals (0, 5, 15, 30, 60, 120 minutes), followed by quenching the samples with ethyl alcohol and quantitating the peptides by LC-MS analysis as described above. To test the sensitivity of peptides to the proteolytic enzymes in liver homogenate, 3 male SD rats (250 ± 20 g) were sacrificed under anesthesia. The whole liver was quickly removed from each rat, washed in ice-cold PBS (50 mM, pH 7.2), weighed and cut into small pieces, which were resuspended in PBS to 100 mg wet liver tissue/2.5 ml PBS. The samples were pooled and homogenized, followed by centrifugation at 9,000 g for 20 min at 4 °C. The supernatants were collected. The test peptides were added to the liver homogenate at a final concentration of 10 μ g/ml. The resulting mixture was incubated 37 °C in a water bath, and the residue peptides in the mixture were quantitated as described above.
What is marked in green?
false
2,253
{ "text": [ "MT-hook residues adjacent to the N terminus of PBD" ], "answer_start": [ 2185 ] }