Datasets:

longluu

/

covid-qa-split

like 0

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

When was the Email published?

Host resilience to emerging coronaviruses https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079962/ SHA: f7cfc37ea164f16393d7f4f3f2b32214dea1ded4 Authors: Jamieson, Amanda M Date: 2016-07-01 DOI: 10.2217/fvl-2016-0060 License: cc-by Abstract: Recently, two coronaviruses, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, have emerged to cause unusually severe respiratory disease in humans. Currently, there is a lack of effective antiviral treatment options or vaccine available. Given the severity of these outbreaks, and the possibility of additional zoonotic coronaviruses emerging in the near future, the exploration of different treatment strategies is necessary. Disease resilience is the ability of a given host to tolerate an infection, and to return to a state of health. This review focuses on exploring various host resilience mechanisms that could be exploited for treatment of severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus and other respiratory viruses that cause acute lung injury and acute respiratory distress syndrome. Text: The 21st century was heralded with the emergence of two novel coronaviruses (CoV) that have unusually high pathogenicity and mortality [1] [2] [3] [4] [5] . Severe acute respiratory syndrome coronavirus (SARS-Cov) was first identified in 2003 [6] [7] [8] [9] . While there was initially great concern about SARS-CoV, once no new cases emerged, funding and research decreased. However, a decade later Middle East respiratory syndrome coronavirus (MERS-CoV), also known as HCoV-EMC, emerged initially in Saudi Arabia [3, 10] . SARS-CoV infected about 8000 people, and resulted in the deaths of approximately 10% of those infected [11] . While MERS-CoV is not as widespread as SARS-CoV, it appears to have an even higher mortality rate, with 35-50% of diagnosed infections resulting in death [3, [12] [13] . These deadly betacoronavirus viruses existed in animal reservoirs [4] [5] 9, [14] [15] . Recently, other CoVs have been detected in animal populations raising the possibility that we will see a repeat of these types of outbreaks in the near future [11, [16] [17] [18] [19] [20] . Both these zoonotic viruses cause a much more severe disease than what is typically seen for CoVs, making them a global health concern. Both SARS-CoV and MERS-CoV result in severe lung pathology. Many infected patients have acute lung injury (ALI), a condition that is diagnosed based on the presence of pulmonary edema and respiratory failure without a cardiac cause. In some patients there is a progression to the more severe form of ALI, acute respiratory distress syndrome (ARDS) [21] [22] [23] . In order to survive a given infection, a successful host must not only be able to clear the pathogen, but tolerate damage caused by the pathogen itself and also by the host's immune response [24] [25] [26] . We refer to resilience as the ability of a host to tolerate the effects of pathogens and the immune response to pathogens. A resilient host is able to return to a state of health after responding to an infection [24, [27] [28] . Most currently available treatment options for infectious diseases are antimicrobials, For reprint orders, please contact: reprints@futuremedicine.com REviEW Jamieson future science group and thus target the pathogen itself. Given the damage that pathogens can cause this focus on rapid pathogen clearance is understandable. However, an equally important medical intervention is to increase the ability of the host to tolerate the direct and indirect effects of the pathogen, and this is an area that is just beginning to be explored [29] . Damage to the lung epithelium by respiratory pathogens is a common cause of decreased resilience [30] [31] [32] . This review explores some of the probable host resilience pathways to viral infections, with a particular focus on the emerging coronaviruses. We will also examine factors that make some patients disease tolerant and other patients less tolerant to the viral infection. These factors can serve as a guide to new potential therapies for improved patient care. Both SARS-CoV and MERS-CoV are typified by a rapid progression to ARDS, however, there are some distinct differences in the infectivity and pathogenicity. The two viruses have different receptors leading to different cellular tropism, and SARS-CoV is more ubiquitous in the cell type and species it can infect. SARS-CoV uses the ACE2 receptor to gain entry to cells, while MERS-CoV uses the ectopeptidase DPP4 [33] [34] [35] [36] . Unlike SARS-CoV infection, which causes primarily a severe respiratory syndrome, MERS-CoV infection can also lead to kidney failure [37, 38] . SARS-CoV also spreads more rapidly between hosts, while MERS-CoV has been more easily contained, but it is unclear if this is due to the affected patient populations and regions [3] [4] 39 ]. Since MERS-CoV is a very recently discovered virus, [40, 41] more research has been done on SARS-CoV. However, given the similarities it is hoped that some of these findings can also be applied to MERS-CoV, and other potential emerging zoonotic coronaviruses. Both viral infections elicit a very strong inflammatory response, and are also able to circumvent the immune response. There appears to be several ways that these viruses evade and otherwise redirect the immune response [1, [42] [43] [44] [45] . The pathways that lead to the induction of the antiviral type I interferon (IFN) response are common targets of many viruses, and coronaviruses are no exception. SARS-CoV and MERS-CoV are contained in double membrane vesicles (DMVs), that prevents sensing of its genome [1, 46] . As with most coronaviruses several viral proteins suppress the type I IFN response, and other aspects of innate antiviral immunity [47] . These alterations of the type I IFN response appear to play a role in immunopathology in more than one way. In patients with high initial viral titers there is a poor prognosis [39, 48] . This indicates that reduction of the antiviral response may lead to direct viral-induced pathology. There is also evidence that the delayed type I IFN response can lead to misregulation of the immune response that can cause immunopathology. In a mouse model of SARS-CoV infection, the type I IFN response is delayed [49] . The delay of this potent antiviral response leads to decreased viral clearance, at the same time there is an increase in inflammatory cells of the immune system that cause excessive immunopathology [49] . In this case, the delayed antiviral response not only causes immunopathology, it also fails to properly control the viral replication. While more research is needed, it appears that MERS has a similar effect on the innate immune response [5, 50] . The current treatment and prevention options for SARS-CoV and MERS-CoV are limited. So far there are no licensed vaccines for SAR-CoV or MERS-CoV, although several strategies have been tried in animal models [51, 52] . There are also no antiviral strategies that are clearly effective in controlled trials. During outbreaks several antiviral strategies were empirically tried, but these uncontrolled studies gave mixed results [5, 39] . The main antivirals used were ribavirin, lopinavir and ritonavir [38, 53] . These were often used in combination with IFN therapy [54] . However, retrospective analysis of these data has not led to clear conclusions of the efficacy of these treatment options. Research in this area is still ongoing and it is hoped that we will soon have effective strategies to treat novel CoV [3,36,38,40, [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] . The lack of effective antivirals makes it necessary to examine other potential treatments for SARS-CoV and MERS-CoV. Even if there were effective strategies to decrease viral burden, for these viruses, the potential for new emerging zoonotic CoVs presents additional complications. Vaccines cannot be produced in time to stop the spread of an emerging virus. In addition, as was demonstrated during SARS-CoV and MERS-CoV outbreaks, there is always a challenge during a crisis situation to know which Host resilience to emerging coronaviruses REviEW future science group www.futuremedicine.com antiviral will work on a given virus. One method of addressing this is to develop broad-spectrum antivirals that target conserved features of a given class of virus [65] . However, given the fast mutation rates of viruses there are several challenges to this strategy. Another method is to increase the ability of a given patient to tolerate the disease, i.e., target host resilience mechanisms. So far this has largely been in the form of supportive care, which relies on mechanical ventilation and oxygenation [29, 39, 66] . Since SARS-CoV and MERS-CoV were discovered relatively recently there is a lack of both patient and experimental data. However, many other viruses cause ALI and ARDS, including influenza A virus (IAV). By looking at data from other high pathology viruses we can extrapolate various pathways that could be targeted during infection with these emerging CoVs. This can add to our understanding of disease resilience mechanisms that we have learned from direct studies of SARS-CoV and MERS-CoV. Increased understanding of host resilience mechanisms can lead to future host-based therapies that could increase patient survival [29] . One common theme that emerges in many respiratory viruses including SARS-CoV and MERS-CoV is that much of the pathology is due to an excessive inflammatory response. A study from Josset et al. examines the cell host response to both MERS-CoV and SARS-CoV, and discovered that MERS-CoV dysregulates the host transcriptome to a much greater extent than SARS-CoV [67] . It demonstrates that glucocorticoids may be a potential way of altering the changes in the host transcriptome at late time points after infection. If host gene responses are maintained this may increase disease resilience. Given the severe disease that manifested during the SARS-CoV outbreak, many different treatment options were empirically tried on human patients. One immunomodulatory treatment that was tried during the SARS-CoV outbreak was systemic corticosteroids. This was tried with and without the use of type I IFNs and other therapies that could directly target the virus [68] . Retrospective analysis revealed that, when given at the correct time and to the appropriate patients, corticosteroid use could decrease mortality and also length of hospital stays [68] . In addition, there is some evidence that simultaneous treatment with IFNs could increase the potential benefits [69] . Although these treatments are not without complications, and there has been a lack of a randomized controlled trial [5, 39] . Corticosteroids are broadly immunosuppressive and have many physiological effects [5, 39] . Several recent studies have suggested that other compounds could be useful in increasing host resilience to viral lung infections. A recent paper demonstrates that topoisomerase I can protect against inflammation-induced death from a variety of viral infections including IAV [70] . Blockade of C5a complement signaling has also been suggested as a possible option in decreasing inflammation during IAV infection [71] . Other immunomodulators include celecoxib, mesalazine and eritoran [72, 73] . Another class of drugs that have been suggested are statins. They act to stabilize the activation of aspects of the innate immune response and prevent excessive inflammation [74] . However, decreasing immunopathology by immunomodulation is problematic because it can lead to increased pathogen burden, and thus increase virus-induced pathology [75, 76] . Another potential treatment option is increasing tissue repair pathways to increase host resilience to disease. This has been shown by bioinformatics [77] , as well as in several animal models [30-31,78-79]. These therapies have been shown in cell culture model systems or animal models to be effective, but have not been demonstrated in human patients. The correct timing of the treatments is essential. Early intervention has been shown to be the most effective in some cases, but other therapies work better when given slightly later during the course of the infection. As the onset of symptoms varies slightly from patient to patient the need for precise timing will be a challenge. Examination of potential treatment options for SARS-CoV and MERS-CoV should include consideration of host resilience [29] . In addition to the viral effects, and the pathology caused by the immune response, there are various comorbidities associated with SARS-CoV and MERS-CoV that lead to adverse outcomes. Interestingly, these additional risk factors that lead to a more severe disease are different between the two viruses. It is unclear if these differences are due to distinct populations affected by the viruses, because of properties of the virus themselves, or both. Understanding these factors could be a key to increasing host resilience to the infections. MERS-CoV patients had increased morbidity and mortality if they were obese, immunocompromised, diabetic or had cardiac disease [4, 12] . REviEW Jamieson future science group Risk factors for SARS-CoV patients included an older age and male [39] . Immune factors that increased mortality for SARS-CoV were a higher neutrophil count and low T-cell counts [5, 39, 77] . One factor that increased disease for patients infected with SARS-CoV and MERS-CoV was infection with other viruses or bacteria [5, 39] . This is similar to what is seen with many other respiratory infections. A recent study looking at malaria infections in animal models and human patients demonstrated that resilient hosts can be predicted [28] . Clinical studies have started to correlate specific biomarkers with disease outcomes in ARDS patients [80] . By understanding risk factors for disease severity we can perhaps predict if a host may be nonresilient and tailor the treatment options appropriately. A clear advantage of targeting host resilience pathways is that these therapies can be used to treat a variety of different infections. In addition, there is no need to develop a vaccine or understand the antiviral susceptibility of a new virus. Toward this end, understanding why some patients or patient populations have increased susceptibility is of paramount importance. In addition, a need for good model systems to study responses to these new emerging coronaviruses is essential. Research into both these subjects will lead us toward improved treatment of emerging viruses that cause ALI, such as SARS-CoV and MERS-CoV. The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. • Severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus are zoonotic coronaviruses that cause acute lung injury and acute respiratory distress syndrome. • Antivirals have limited effects on the course of the infection with these coronaviruses. • There is currently no vaccine for either severe acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus. • Host resilience is the ability of a host to tolerate the effects of an infection and return to a state of health. • Several pathways, including control of inflammation, metabolism and tissue repair may be targeted to increase host resilience. • The future challenge is to target host resilience pathways in such a way that there are limited effects on pathogen clearance pathways. Future studies should determine the safety of these types of treatments for human patients. Papers of special note have been highlighted as:

Which medical comorbidities most profoundly influenced MERS-CoV outcomes?

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)

What are the key factors preventing the elimination of HCV infection in some patients?

Community-acquired pneumonia in children — a changing spectrum of disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608782/ SHA: eecb946b106a94f26a79a964f0160e8e16f79f42 Authors: le Roux, David M.; Zar, Heather J. Date: 2017-09-21 DOI: 10.1007/s00247-017-3827-8 License: cc-by Abstract: Pneumonia remains the leading cause of death in children outside the neonatal period, despite advances in prevention and management. Over the last 20 years, there has been a substantial decrease in the incidence of childhood pneumonia and pneumonia-associated mortality. New conjugate vaccines against Haemophilus influenzae type b and Streptococcus pneumoniae have contributed to decreases in radiologic, clinical and complicated pneumonia cases and have reduced hospitalization and mortality. The importance of co-infections with multiple pathogens and the predominance of viral-associated disease are emerging. Better access to effective preventative and management strategies is needed in low- and middle-income countries, while new strategies are needed to address the residual burden of disease once these have been implemented. Text: Pneumonia has been the leading cause of death in children younger than 5 years for decades. Although there have been substantial decreases in overall child mortality and in pneumonia-specific mortality, pneumonia remains the major single cause of death in children outside the neonatal period, causing approximately 900,000 of the estimated 6.3 million child deaths in 2013 [1] . Substantial advances have occurred in the understanding of risk factors and etiology of pneumonia, in development of standardized case definitions, and in prevention with the production of improved vaccines and in treatment. Such advances have led to changes in the epidemiology, etiology and mortality from childhood pneumonia. However in many areas access to these interventions remains sub-optimal, with large inequities between and within countries and regions. In this paper we review the impact of recent preventative and management advances in pneumonia epidemiology, etiology, radiologic presentation and outcome in children. The overall burden of childhood pneumonia has been reduced substantially over the last decade, despite an increase in the global childhood population from 605 million in 2000 to 664 million in 2015 [2] . Recent data suggest that there has been a 25% decrease in the incidence of pneumonia, from 0.29 episodes per child year in low-and middle-income countries in 2000, to 0.22 episodes per child year in 2010 [3] . This is substantiated by a 58% decrease in pneumonia-associated disability-adjusted life years between 1990 and 2013, from 186 million to 78 million as estimated in the Global Burden of Disease study [1] . Pneumonia deaths decreased from 1.8 million in 2000 to 900,000 in 2013 [1] . These data do not reflect the full impact of increasingly widespread use of pneumococcal conjugate vaccine in low-and middle-income countries because the incidence of pneumonia and number of deaths are likely to decrease still further as a result of this widespread intervention [4] . Notwithstanding this progress, there remains a disproportionate burden of disease in low-and middle-income countries, where more than 90% of pneumonia cases and deaths occur. The incidence in high-income countries is estimated at 0.015 episodes per child year, compared to 0.22 episodes per child year in low-and middle-income countries [3] . On average, 1 in 66 children in high-income countries is affected by pneumonia per year, compared to 1 in 5 children in low-and middle-income countries. Even within low-and middleincome countries there are regional inequities and challenges with access to health care services: up to 81% of severe pneumonia deaths occur outside a hospital [5] . In addition to a higher incidence of pneumonia, the case fatality rate is estimated to be almost 10-fold higher in low-and middle-income countries as compared to high-income countries [3, 5] . Childhood pneumonia can also lead to significant morbidity and chronic disease. Early life pneumonia can impair longterm lung health by decreasing lung function [6] . Severe or recurrent pneumonia can have a worse effect on lung function; increasing evidence suggests that chronic obstructive pulmonary disease might be related to early childhood pneumonia [7, 8] . A meta-analysis of the risk of long-term outcomes after childhood pneumonia categorized chronic respiratory sequelae into major (restrictive lung disease, obstructive lung disease, bronchiectasis) and minor (chronic bronchitis, asthma, abnormal pulmonary function) groups [9] . The risk of developing at least one of the major sequelae was estimated as 6% after an ambulatory pneumonia event and 14% after an episode of hospitalized pneumonia. Because respiratory diseases affect almost 1 billion people globally and are a major cause of mortality and morbidity [10] , childhood pneumonia might contribute to substantial morbidity across the life course. Chest radiologic changes have been considered the gold standard for defining a pneumonia event [11] because clinical findings can be subjective and clinical definitions of pneumonia can be nonspecific. In 2005, to aid in defining outcomes of pneumococcal vaccine studies, the World Health Organization's (WHO) standardized chest radiograph description defined a group of children who were considered most likely to have pneumococcal pneumonia [12] . The term "end-point consolidation" was described as a dense or fluffy opacity that occupies a portion or whole of a lobe, or the entire lung. "Other infiltrate" included linear and patchy densities, peribronchial thickening, minor patchy infiltrates that are not of sufficient magnitude to constitute primary end-point consolidation, and small areas of atelectasis that in children can be difficult to distinguish from consolidation. "Primary end-point pneumonia" included either end-point consolidation or a pleural effusion associated with a pulmonary parenchymal infiltrate (including "other" infiltrate). Widespread use of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination has decreased the incidence of radiologic pneumonia. In a review of four randomized controlled trials and two case-control studies of Haemophilus influenzae type B conjugate vaccination in high-burden communities, the vaccination was associated with an 18% decrease in radiologic pneumonia [13] . Introduction of pneumococcal conjugate vaccination was associated with a 26% decrease in radiologic pneumonia in California between 1995 and 1998 [14] . In vaccine efficacy trials in low-and middle-income countries, pneumococcal conjugate vaccination reduced radiologic pneumonia by 37% in the Gambia [15] , 25% in South Africa [16] and 26% in the Philippines [17] . The WHO radiologic case definition was not intended to distinguish bacterial from viral etiology but rather to define a sub-set of pneumonia cases in which pneumococcal infection was considered more likely and to provide a set of standardized definitions through which researchers could achieve broad agreement in reporting chest radiographs. However, despite widespread field utilization, there are concerns regarding inter-observer repeatability. There has been good consensus for the description of lobar consolidation but significant disagreement on the description of patchy and perihilar infiltrates [18, 19] . In addition, many children with clinically severe lung disease do not have primary end-point pneumonia: in one pre-pneumococcal conjugate vaccination study, only 34% of children hospitalized with pneumonia had primary end-point pneumonia [20] . A revised case definition of "presumed bacterial pneumonia" has been introduced, and this definition includes pneumonia cases with WHO-defined alveolar consolidation, as well as those with other abnormal chest radiograph infiltrates and a serum C-reactive protein of at least 40 mg/L [21, 22] . This definition has been shown to have greater sensitivity than the original WHO radiologic definition of primary end-point pneumonia for detecting the burden of pneumonia prevented by pneumococcal conjugate vaccination [23] . Using the revised definition, the 10-valent pneumococcal conjugate vaccine (pneumococcal conjugate vaccination-10), had a vaccine efficacy of 22% in preventing presumed bacterial pneumonia in young children in South America [22] , and pneumococcal conjugate vaccination-13 had a vaccine efficacy of 39% in preventing presumed bacterial pneumonia in children older than 16 weeks who were not infected with human immunodeficiency virus (HIV) in South Africa [21] . Thus there is convincing evidence that pneumococcal conjugate vaccination decreases the incidence of radiologic pneumonia; however there is no evidence to suggest that pneumococcal conjugate vaccination modifies the radiologic appearance of pneumococcal pneumonia. Empyema is a rare complication of pneumonia. An increased incidence of empyema in children was noted in some high-income countries following pneumococcal conjugate vaccination-7 introduction, and this was attributed to pneumococcal serotypes not included in pneumococcal conjugate vaccination-7, especially 3 and 19A [24] . In the United States, evidence from a national hospital database suggests that the incidence of empyema increased 1.9-fold between 1996 and 2008 [25] . In Australia, the incidence rate ratio increased by 1.4 times when comparing the pre-pneumococcal conjugate vaccination-7 period (1998 to 2004) to the post-pneumococcal conjugate vaccination-7 period (2005 to 2010) [26] . In Scotland, incidence of empyema in children rose from 6.5 per million between 1981 and 1998, to 66 per million in 2005 [27] . These trends have been reversed since the introduction of pneumococcal conjugate vaccination-13. Data from the United States suggest that empyema decreased by 50% in children younger than 5 years [28] ; similarly, data from the United Kingdom and Scotland showed substantial reduction in pediatric empyema following pneumococcal conjugate vaccination-13 introduction [29, 30] . Several national guidelines from high-income countries, as well as the WHO recommendations for low-and middleincome countries, recommend that chest radiography should not be routinely performed in children with ambulatory pneumonia [31] [32] [33] . Indications for chest radiography include hospitalization, severe hypoxemia or respiratory distress, failed initial antibiotic therapy, or suspicion for other diseases (tuberculosis, inhaled foreign body) or complications. However, point-of-care lung ultrasound is emerging as a promising modality for diagnosing childhood pneumonia [34] . In addition to the effect on radiologic pneumonia, pneumococcal conjugate vaccination reduces the risk of hospitalization from viral-associated pneumonia, probably by reducing bacterial-viral co-infections resulting in severe disease and hospitalization [35] . An analysis of ecological and observational studies of pneumonia incidence in different age groups soon after introduction of pneumococcal conjugate vaccination-7 in Canada, Italy, Australia, Poland and the United States showed decreases in all-cause pneumonia hospitalizations ranging from 15% to 65% [36] . In the United States after pneumococcal conjugate vaccination-13 replaced pneumococcal conjugate vaccination-7, there was a further 17% decrease in hospitalizations for pneumonia among children eligible for the vaccination, and a further 12% decrease among unvaccinated adults [28] . A systematic review of etiology studies prior to availability of new conjugate vaccines confirmed S. pneumoniae and H. influenzae type B as the most important bacterial causes of pneumonia, with Staphylococcus aureus and Klebsiella pneumoniae associated with some severe cases. Respiratory syncytial virus was the leading viral cause, identified in 15-40% of pneumonia cases, followed by influenza A and B, parainfluenza, human metapneumovirus and adenovirus [37] . More recent meta-analyses of etiology data suggest a changing pathogen profile, with increasing recognition that clinical pneumonia is caused by the sequential or concurrent interaction of more than one organism. Severe disease in particular is often caused by multiple pathogens. With high coverage of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination, viral pathogens increasingly predominate [38] . In recent case-control studies, at least one virus was detected in 87% of clinical pneumonia cases in South Africa [39] , while viruses were detected in 81% of radiologic pneumonia cases in Sweden [40] . In a large multi-center study in the United States, viral pathogens were detected in 73% of children hospitalized with radiologic pneumonia, while bacteria were detected in only 15% of cases [41] . A meta-analysis of 23 case-control studies of viral etiology in radiologically confirmed pneumonia in children, completed up to 2014, reported good evidence of causal attribution for respiratory syncytial virus, influenza, metapneumovirus and parainfluenza virus [42] . However there was no consistent evidence that many other commonly described viruses, including rhinovirus, adenovirus, bocavirus and coronavirus, were more commonly isolated from cases than from controls. Further attribution of bacterial etiology is difficult because it is often not possible to distinguish colonizing from pathogenic bacteria when they are isolated from nasal specimens [43] . Another etiology is pertussis. In the last decade there has also been a resurgence in pertussis cases, especially in highincome countries [44] . Because pertussis immunity after acellular pertussis vaccination is less long-lasting than immunity after wild-type infection or whole-cell vaccination, many women of child-bearing age have waning pertussis antibody levels. Their infants might therefore be born with low transplacental anti-pertussis immunoglobulin G levels, making them susceptible to pertussis infection before completion of the primary vaccination series [45] . In 2014, more than 40,000 pertussis cases were reported to the Centers for Disease Control and Prevention in the United States; in some states, population-based incidence rates are higher than at any time in the last 70 years [44] . In contrast, most low-and middleincome countries use whole-cell pertussis vaccines and the numbers of pertussis cases in those countries were stable or decreasing until 2015 [46] . However recent evidence from South Africa (where the acellular vaccine is used) shows an appreciable incidence of pertussis among infants presenting with acute pneumonia: 2% of clinical pneumonia cases among infants enrolled in a birth cohort were caused by pertussis [39] , and 3.7% of infants and young children presenting to a tertiary academic hospital had evidence of pertussis infection [47] . Similarly, childhood tuberculosis is a major cause of morbidity and mortality in many low-and middle-income countries, and Mycobacterium tuberculosis has increasingly been recognized as a pathogen in acute pneumonia in children living in high tuberculosis-prevalence settings. Postmortem studies of children dying from acute respiratory illness have commonly reported M. tuberculosis [48, 49] . A recent systematic review of tuberculosis as a comorbidity of childhood pneumonia reported culture-confirmed disease in about 8% of cases [50] . Because intrathoracic tuberculosis disease is only culture-confirmed in a minority of cases, the true burden could be even higher; tuberculosis could therefore be an important contributor to childhood pneumonia incidence and mortality in high-prevalence areas. Childhood pneumonia and clinically severe disease result from a complex interaction of host and environmental risk factors [37] . Because of the effectiveness of pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination for prevention of radiologic and clinical pneumonia, incomplete or inadequate vaccination must be considered as a major preventable risk factor for childhood pneumonia. Other risk factors include low birth weight, which is associated with 3.2 times increased odds of severe pneumonia in low-and middle-income countries, and 1.8 times increased odds in high-income countries [51] . Similarly, lack of exclusive breastfeeding for the first 4 months of life increases odds of severe pneumonia by 2.7 times in low-and middle-income countries and 1.3 times in highincome countries. Markers of undernutrition are strong risk factors for pneumonia in low-and middle-income countries only, with highly significant odds ratios for underweight for age (4.5), stunting (2.6) and wasting (2.8) . Household crowding has uniform risk, with odds ratios between 1.9 and 2.3 in both low-and middle-income countries and high-income countries. Indoor air pollution from use of solid or biomass fuels increases odds of pneumonia by 1.6 times; lack of measles vaccination by the end of the first year of age increases odds of pneumonia by 1.8 times [51] . It is estimated that the prevalence of these critical risk factors in low-and middle-income countries decreased by 25% between 2000 and 2010, contributing to reductions in pneumonia incidence and mortality in low-and middle-income countries, even in countries where conjugate vaccines have not been available [3] . The single strongest risk factor for pneumonia is HIV infection, which is especially prevalent in children in sub-Saharan Africa. HIV-infected children have 6 times increased odds of developing severe pneumonia or of death compared to HIV-uninfected children [52] . Since the effective prevention of mother-to-child transmission of HIV, there is a growing population of HIV-exposed children who are uninfected; their excess risk of pneumonia, compared to HIV unexposed children, has been described as 1.3-to 3.4-fold higher [53] [54] [55] [56] [57] . The pneumococcal conjugate vaccination and Haemophilus influenzae type B conjugate vaccination have been effective tools to decrease pneumonia incidence, severity and mortality [58, 59] . However, equitable coverage and access to vaccines remains sub-optimal. By the end of 2015, Haemophilus influenzae type B conjugate vaccination had been introduced in 73 countries, with global coverage estimated at 68%. However, inequities are still apparent among regions: in the Americas coverage is estimated at 90%, while in the Western Pacific it is only 25%. By 2015, pneumococcal conjugate vaccination had been introduced into 54 countries, with global coverage of 35% for three doses of pneumococcal conjugate vaccination for infant populations [60] . To address this issue, the WHO's Global Vaccine Access Plan initiative was launched to make life-saving vaccines more equitably available. In addition to securing guarantees for financing of vaccines, the program objectives include building political will in low-and middle-income countries to commit to immunization as a priority, social marketing to individuals and communities, strengthening health systems and promoting relevant local research and development innovations [61] . Maternal vaccination to prevent disease in the youngest infants has been shown to be effective for tetanus, influenza and pertussis [62] . Influenza vaccination during pregnancy is safe, provides reasonable maternal protection against influenza, and also protects infants for a limited period from confirmed influenza infection (vaccine efficacy 63% in Bangladesh [63] and 50.4% in South Africa [64] ). However as antibody levels drop sharply after birth, infant protection does not persist much beyond 8 weeks [65] . Recently respiratory syncytial virus vaccination in pregnancy has been shown to be safe and immunogenic, and a phase-3 clinical trial of efficacy at preventing respiratory syncytial virus disease in infants is under way [66] . Within a decade, respiratory syncytial virus in infancy might be vaccine-preventable, with further decreases in pneumonia incidence, morbidity and mortality [67] . Improved access to health care, better nutrition and improved living conditions might contribute to further decreases in childhood pneumonia burden. The WHO Integrated Global Action Plan for diarrhea and pneumonia highlights many opportunities to protect, prevent and treat children [68] . Breastfeeding rates can be improved by programs that combine education and counseling interventions in homes, communities and health facilities, and by promotion of baby-friendly hospitals [69] . Improved home ventilation, cleaner cooking fuels and reduction in exposure to cigarette smoke are essential interventions to reduce the incidence and severity of pneumonia [70, 71] . Prevention of pediatric HIV is possible by providing interventions to prevent mother-to-child transmission [72] . Early infant HIV testing and early initiation of antiretroviral therapy and cotrimoxazole prophylaxis can substantially reduce the incidence of community-acquired pneumonia among HIV-infected children [73] . Community-based interventions reduce pneumonia mortality and have the indirect effect of improved-careseeking behavior [58] . If these cost-effective interventions were scaled up, it is estimated that 67% of pneumonia deaths in lowand middle-income countries could be prevented by 2025 [58] . Case management of pneumonia is a strategy by which severity of disease is classified as severe or non-severe. All children receive early, appropriate oral antibiotics, and severe cases are referred for parenteral antibiotics. When implemented in highburden areas before the availability of conjugate vaccines, case management as part of Integrated Management of Childhood Illness was associated with a 27% decrease in overall child mortality, and 42% decrease in pneumonia-specific mortality [74] . However the predominance of viral causes of pneumonia and low case fatality have prompted concern about overuse of antibiotics. Several randomized controlled trials comparing oral antibiotics to placebo for non-severe pneumonia have been performed [75] [76] [77] and others are ongoing [78] . In two studies, performed in Denmark and in India, outcomes of antibiotic and placebo treatments were equivalent [76, 77] . In the third study, in Pakistan, there was a non-significant 24% vs. 20% rate of failure in the placebo group, which was deemed to be non-equivalent to the antibiotic group [75] . Furthermore, because WHO-classified non-severe pneumonia and bronchiolitis might be considered within a spectrum of lower respiratory disease, many children with clinical pneumonia could actually have viral bronchiolitis, for which antibiotics are not beneficial [79] . This has been reflected in British [33] and Spanish [31] national pneumonia guidelines, which do not recommend routine antibiotic treatment for children younger than 2 years with evidence of pneumococcal conjugate vaccination who present with non-severe pneumonia. The United States' national guidelines recommend withholding antibiotics in children up to age 5 years presenting with non-severe pneumonia [32] . However, given the high mortality from pneumonia in low-and middle-income countries, the lack of easy access to care, and the high prevalence of risk factors for severe disease, revised World Health Organization pneumonia guidelines still recommend antibiotic treatment for all children who meet the WHO pneumonia case definitions [80] . Use of supplemental oxygen is life-saving, but this is not universally available in low-and middle-income countries; it is estimated that use of supplemental oxygen systems could reduce mortality of children with hypoxic pneumonia by 20% [81] . Identifying systems capacity to increase availability of oxygen in health facilities, and identifying barriers to further implementation are among the top 15 priorities for future childhood pneumonia research [82] . However, up to 81% of pneumonia deaths in 2010 occurred outside health facilities [5] , so there are major challenges with access to health services and health-seeking behavior of vulnerable populations. Identifying and changing the barriers to accessing health care is an important area with the potential to impact the survival and health of the most vulnerable children [82] . Much progress has been made in decreasing deaths caused by childhood pneumonia. Improved socioeconomic status and vaccinations, primarily the conjugate vaccines (against Haemophilus influenzae and pneumococcus), have led to substantial reductions in the incidence and severity of childhood pneumonia. Stronger strategies to prevent and manage HIV have reduced HIV-associated pneumonia deaths. However, despite the substantial changes in incidence, etiology and radiology globally, there remain inequities in access to care and availability of effective interventions, especially in low-and middle-income countries. Effective interventions need to be more widely available and new interventions developed for the residual burden of childhood pneumonia.

What is responsible for the reduction of radiologic pneumonia?

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 Enfuvirtide?

Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054964/ SHA: 77b0c98d1a2ca46b219ad090074814c387c80d8f Authors: Chen, Weilie; Lan, Yun; Yuan, Xiaozhen; Deng, Xilong; Li, Yueping; Cai, Xiaoli; Li, Liya; He, Ruiying; Tan, Yizhou; Deng, Xizi; Gao, Ming; Tang, Guofang; Zhao, Lingzhai; Wang, Jinlin; Fan, Qinghong; Wen, Chunyan; Tong, Yuwei; Tang, Yangbo; Hu, Fengyu; Li, Feng; Tang, Xiaoping Date: 2020-02-26 DOI: 10.1080/22221751.2020.1732837 License: cc-by Abstract: The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites. Text: The 2019 novel coronavirus (2019-nCoV), originally outbreaking from Wuhan China, has transmitted in an extremely short period to 25 countries and infected over 31 000 individuals as of Feb 06, 2020, causing an international alarm. Basic scientific research has achieved significantly in the investigation of viral origination [1, 2] , transmission and evolution [3] , and unprecedented public health control actions in China have been activated and effectively prevented the otherwise dramatic spread. The 2019-nCoV virus seems more infectious in its public transmission capacity compared to the well-known 2003 SARS virus in spite of the unavailability of convincingly scientific evidence. The mechanism of viral transmission is still worthy of further exploration. Currently, one urgent and critical challenge is to treat infected patients and save their lives. Several studies have roughly described the overall clinical features of 2019-nCoV patients [4, 5] . However, the more specific and classified clinical characteristics of the infected patients still require further investigation, particularly for those with severe symptoms, which is roughly estimated to be approximately 15-20 percent of totally confirmed cases based on the local data in our hospital. Clinically, for those severe patients, the main symptoms of 2019-nCoV pneumonia are fever, decreased white blood cell and lymphocyte count, increased C reaction protein and abnormally expressed cytokines [6] . One remaining question to be resolved is whether the 2019-nCoV virus can replicate in extra-pulmonary sites, which might account for the deteriorated clinical manifestation. In this study, we investigated whether the patients with severe clinical symptoms exhibited special profiles of virus replication or/and distribution compared to those only with mild symptoms. Patients, who were confirmed to be infected by the 2019-nCoV virus, were firstly enrolled in or transferred to Guangzhou Eighth People's Hospital for treatment purposes. This study followed the guideline of the Ethics Committee of Guangzhou Eighth People's Hospital. All blood, pharyngeal swab, and anal swab samples were collected for diagnostic purposes in the laboratory and our study added no extra burden to patients. Viral RNA was extracted with Nucleic Acid Isolation Kit (Da'an Gene Corporation, Cat: DA0630) on an automatic workstation Smart 32 (Da'an Gene Corporation) following the guidelines. Real-time reverse transcriptional polymerase chain reaction (RT-PCR) reagent (Da'an Gene cooperation, Cat DA0930) was employed for viral detection per the protocol. In brief, two PCR primer and probe sets, which target orf1ab (FAM reporter) and N (VIC reporter) genes separately, were added in the same reaction tube. Positive and negative controls were included for each batch of detection. Samples were considered to be viral positive when either or both set(s) gave a reliable signal(s). All patients had pneumonia-based diseases but with diversified clinical manifestation. To simplify data analysis, the patients were only classified as either mild or severe clinical symptom groups based on the guideline newly released by Chinese government. Patients who were with at least one of the following symptom should be diagnosed to be severe case, 1) distress of respiratory with respiratory rate > = 30/min; 2) Oxygen saturation < = 93% in the rest state, and 3) arterial oxygen tension (PaO₂) over inspiratory oxygen fraction (FIO₂) of less than 300 mm Hg. In the blood detection cohort (Figure 1 (A)), patients who had at less one serum sample measurement with the PCR method were included. In the 57, 6 cases were detected to be blood positive, all of them (100%) were severe in symptom requiring special care attention, and the blood of the rest 51 cases was without detectable virus in the blood, only 12 of them (23.5%) were severe cases. The ratio of severe symptoms between these two groups was significantly different (p value = 0.0001). In the anal swab cohort (Figure 1 (B)), 11 of 28 cases were detected to be anal swab positive, 8 of them (72.7%) were with severe symptoms, which was significantly higher than that 4 (23.5%) of the rest 17 cases without detectable virus in anal were severe cases. Fortunately, two cases with detectable virus both in blood and anal swab cohort were recorded. Patient 1 (Figure 2 (A)) was admitted to ICU after enrollment evaluation and was highly suspected infection with 2019-nCoV because of his recent travelling from Wuhan and of confirmed pneumonia by radiographic diagnosis with 5-day fever and 1-day continuous dry coughing. He was then confirmed to be infected by the 2019-nCoV virus on illness day 6 by CDC. High concentrations of the viral RNA were detected in the pharyngeal swabs on illness days 5 (Ct = 17 + 25), 7, 8 (Ct = 25 + 26), and 11 (Ct = 15 + 25). In the blood, no viral RNA was detected on day 5 but the sample on day 6 gave a weak positive signal (Ct = Neg+39), and then the signal was gone again on day 8. On day 9, a low level of viral RNA (Ct = 36 + 41) was detected again in the blood. On day 12, the blood lost signal again. A high concentration of virus RNA (Ct = 23 + 27) was detected in the anal sample on day 13, on the day the 2019-nCoV virus was not detected in the pharyngeal swab. Unfortunately, he was transferred out to another hospital after an emergency expert consultation. Patient 2 (Figure 2 (B)), who had a clear infection history and started fever 5-day ago and dry coughing 2-day ago, was admitted with clinically highly suspect of 2019-nCoV infection, considering the radiographical diagnosis which indicated clear pneumonia in the bilateral lung lobes. The virus was detected in his blood on illness day 7 (Ct = 34 + 36) and 8 (Ct = 38 + 38). His infection was also informed by the CDC on day 8. Because his disease advanced very fast, he was transferred to the ICU ward for special medical care requirements on day 9, on which day high titers of virus (Ct = 25 + 36) were detected in the pharyngeal sample. Importantly, virus RNA was detected in all pharyngeal (Ct = 23 + 24), blood (Ct = 34 + 39) and anal (Ct = 24 + 29) samples on day 10. He was transferred out to another hospital after an emergency expert consultation. Finally, we described here the four patients with detectable serum viral RNA. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. Pharyngeal samples were PCR positive on days 14 and 17 and became negative on day 22. Patient 4 (Figure 3(B) ) was transferred to the ICU ward on the illness day 6 with a CDC confirmation. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). On day 9, he was transferred out. Patient 5 (Figure 3(C) ) was admitted on illness day 4 and his blood sample was virus-positive (Ct = 38 + Neg) on day 6. Her disease progressed rapidly to a severe stage within the next 3 days. Patient 6 ( Figure 3 (D)) with a clear history of virus infection was confirmed to be infected on infection day 7. Viral RNA was detected in his blood sample on day 9, one day ahead of his transfer into ICU. As his condition worsens, he was transferred out on day 13. In this retrospective study, we analyzed the PCR data of virus detection in different tissues in our laboratory. Firstly, our observation indicated that the presence of viral RNA outside of the respiratory tract might herald the severity of the disease and alarm the requirement of special care. In the blood test cohort, all the 6 infected patients were in (or later progressed to) severe disease stage when serum viral RNA became detectable, which showed a significant difference compared to the blood negative group (p = 0.0001). Patient 2 (Figure 2(B) ), 5 (Figure 3 (C)) and 6 ( Figure 3(D) ) all had detectable viral RNA in the serum before they progressed to the clinical severe symptom stage. Unfortunately, we missed the earlier time points of patient 1 (Figure 2(A) ) and 3 (Figure 3(A) ) who were directly admitted to ICU on transfer to our hospital because of severe condition, of patient 4 (Figure 3(B) ) who had serum sample collected one day post the diagnosis of severe illness. We, fortunately, observed high serum viral load in serum within their severe illness stage. In the anal swab cohort, we found that the presence of virus RNA in the anal digestive tract was also positively correlated with disease severity (p = 0.0102). The 3 patients detected with anal virus RNA but in mild stage should be monitored whether they will progress to the severe stage. We have summarized the information of approximately 70 percent of the patients in Guangzhou city, and the study represented nearly the whole picture of this region. However, the virus outbroke in such an emergence, allowing no delay in waiting for more patients to further confirm the findings. Secondly, a high concentration of viral RNA in anal swabs suggested the digestive tract might be one extrapulmonary site for virus replication. For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead). For patient 2, higher concentrations of viral RNAs were detected in anal swab (Ct = 24 + 39) and pharyngeal swab (Ct = 23 + 24) than in the blood (Ct = 34 + 39) on the same day. Angiotensin-converting enzyme 2 (ACE2) still is one of the receptors for 2019-nCoV attachment and entry [2] . Intensive structural analysis of the S protein of 2019-nCoV with the SARS-Coronavirus suggested that several critical residues in the viral spike protein might confer favourable interaction with human ACE2 [7] . Of note, ACE2 is also abundantly present in humans in the epithelia of the small intestine besides the respiratory tract and is ubiquitously present in endothelial cells [8] , which might provide possible routes of transmission, and might account for the high transmission capacity of the new virus. We propose that rampant coronavirus replication in pulmonary alveolus results in the breakdown of the alveolar vessel and the subsequent virus leakage into the blood flow, through which the virus is disseminated across the whole body. Then the virus succeeds in establishing reinfection in the digestive tract by using the highly expressed ACE2 receptor, which exacerbated the disease vice versa. Bat originated coronavirus was found to replicate in the swine digestive tract recently, also suggesting the potential replication possibility in the human digestive tract [9] . Nevertheless, confirmation of virus transmission through the digestive tract warrants further virus isolation from the anal swab in high safety level lab. Unfortunately, in our study, we did not collect stool samples from patients and did not pursue viral RNA in the stool. But we believe the existence of virus RNA in the stool samples from these patients because that a large amount of viral RNA was detected in anal swabs and that viral RNA had also been detected in a case reported from the United States [10] . Also, we didn't collect sputum and bronchoalveolar lavage fluid for virus detection because that the dry coughing characteristic of patients infected with 2019-nCoV prevents producing enough amount of sputum and that bronchoalveolar lavage fluid collection requires a sophisticated operation which increases virus exposure possibility of care providers to high concentrations of virus-containing aerosol. In summary, we find that the presence of viral RNA in the blood and anal swab is positively correlated with the severe disease stage and that early monitoring of virus RNA in blood and the digestive tract on top of the respiratory tract might benefit the disease prediction.

Which patients were classified as severe in Chinese guidelines?

"Virus-Vectored Influenza Virus Vaccines\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147686/\n\(...TRUNCATED)

What is a concern with these vaccines?

"Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient wit(...TRUNCATED)

How different is it from SARS-related viruses?

"A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies\n\nhttps://www(...TRUNCATED)

What is the Hepatitis C virus?

"A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies\n\nhttps://www(...TRUNCATED)

What antiviral treatments are used for hepatitis C infection?