Summary Background Human infection with a novel coronavirus named Middle East Respiratory Syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia and the Middle East in September, 2012, ...with 44 laboratory-confirmed cases as of May 23, 2013. We report detailed clinical and virological data for two related cases of MERS-CoV disease, after nosocomial transmission of the virus from one patient to another in a French hospital. Methods Patient 1 visited Dubai in April, 2013; patient 2 lives in France and did not travel abroad. Both patients had underlying immunosuppressive disorders. We tested specimens from the upper (nasopharyngeal swabs) or the lower (bronchoalveolar lavage, sputum) respiratory tract and whole blood, plasma, and serum specimens for MERS-CoV by real-time RT-PCR targeting the upE and Orf1A genes of MERS-CoV. Findings Initial clinical presentation included fever, chills, and myalgia in both patients, and for patient 1, diarrhoea. Respiratory symptoms rapidly became predominant with acute respiratory failure leading to mechanical ventilation and extracorporeal membrane oxygenation (ECMO). Both patients developed acute renal failure. MERS-CoV was detected in lower respiratory tract specimens with high viral load (eg, cycle threshold Ct values of 22·9 for upE and 24 for Orf1a for a bronchoalveolar lavage sample from patient 1; Ct values of 22·5 for upE and 23·9 for Orf1a for an induced sputum sample from patient 2), whereas nasopharyngeal specimens were weakly positive or inconclusive. The two patients shared the same room for 3 days. The incubation period was estimated at 9–12 days for the second case. No secondary transmission was documented in hospital staff despite the absence of specific protective measures before the diagnosis of MERS-CoV was suspected. Patient 1 died on May 28, due to refractory multiple organ failure. Interpretation Patients with respiratory symptoms returning from the Middle East or exposed to a confirmed case should be isolated and investigated for MERS-CoV with lower respiratory tract sample analysis and an assumed incubation period of 12 days. Immunosuppression should also be taken into account as a risk factor. Funding French Institute for Public Health Surveillance, ANR grant Labex Integrative Biology of Emerging Infectious Diseases, and the European Community's Seventh Framework Programme projects EMPERIE and PREDEMICS.
Summary Background The novel Middle East respiratory syndrome coronavirus (MERS-CoV) had, as of Aug 8, 2013, caused 111 virologically confirmed or probable human cases of infection worldwide. We ...analysed epidemiological and genetic data to assess the extent of human infection, the performance of case detection, and the transmission potential of MERS-CoV with and without control measures. Methods We assembled a comprehensive database of all confirmed and probable cases from public sources and estimated the incubation period and generation time from case cluster data. Using data of numbers of visitors to the Middle East and their duration of stay, we estimated the number of symptomatic cases in the Middle East. We did independent analyses, looking at the growth in incident clusters, the growth in viral population, the reproduction number of cluster index cases, and cluster sizes to characterise the dynamical properties of the epidemic and the transmission scenario. Findings The estimated number of symptomatic cases up to Aug 8, 2013, is 940 (95% CI 290–2200), indicating that at least 62% of human symptomatic cases have not been detected. We find that the case-fatality ratio of primary cases detected via routine surveillance (74%; 95% CI 49–91) is biased upwards because of detection bias; the case-fatality ratio of secondary cases was 20% (7–42). Detection of milder cases (or clinical management) seemed to have improved in recent months. Analysis of human clusters indicated that chains of transmission were not self-sustaining when infection control was implemented, but that R in the absence of controls was in the range 0·8–1·3. Three independent data sources provide evidence that R cannot be much above 1, with an upper bound of 1·2–1·5. Interpretation By showing that a slowly growing epidemic is underway either in human beings or in an animal reservoir, quantification of uncertainty in transmissibility estimates, and provision of the first estimates of the scale of the epidemic and extent of case detection biases, we provide valuable information for more informed risk assessment. Funding Medical Research Council, Bill & Melinda Gates Foundation, EU FP7, and National Institute of General Medical Sciences.
The impact of variants of concern (VoC) on SARS-CoV-2 viral dynamics remains poorly understood and essentially relies on observational studies subject to various sorts of biases. In contrast, ...experimental models of infection constitute a powerful model to perform controlled comparisons of the viral dynamics observed with VoC and better quantify how VoC escape from the immune response. Here we used molecular and infectious viral load of 78 cynomolgus macaques to characterize in detail the effects of VoC on viral dynamics. We first developed a mathematical model that recapitulate the observed dynamics, and we found that the best model describing the data assumed a rapid antigen-dependent stimulation of the immune response leading to a rapid reduction of viral infectivity. When compared with the historical variant, all VoC except beta were associated with an escape from this immune response, and this effect was particularly sensitive for delta and omicron variant (p<10-6 for both). Interestingly, delta variant was associated with a 1.8-fold increased viral production rate (p = 0.046), while conversely omicron variant was associated with a 14-fold reduction in viral production rate (p<10-6). During a natural infection, our models predict that delta variant is associated with a higher peak viral RNA than omicron variant (7.6 log10 copies/mL 95% CI 6.8-8 for delta; 5.6 log10 copies/mL 95% CI 4.8-6.3 for omicron) while having similar peak infectious titers (3.7 log10 PFU/mL 95% CI 2.4-4.6 for delta; 2.8 log10 PFU/mL 95% CI 1.9-3.8 for omicron). These results provide a detailed picture of the effects of VoC on total and infectious viral load and may help understand some differences observed in the patterns of viral transmission of these viruses.
Health-care workers are crucial to any health-care system. During the ongoing COVID-19 pandemic, health-care workers are at a substantially increased risk of becoming infected with severe acute ...respiratory syndrome coronavirus 2 (SARS-CoV-2) and could come to considerable harm as a result. Depending on the phase of the pandemic, patients with COVID-19 might not be the main source of SARS-CoV-2 infection and health-care workers could be exposed to atypical patients, infected family members, contacts, and colleagues, or live in communities of active transmission. Clear strategies to support and appropriately manage exposed and infected health-care workers are essential to ensure effective staff management and to engender trust in the workplace. These management strategies should focus on risk stratification, suitable clinical monitoring, low-threshold access to diagnostics, and decision making about removal from and return to work. Policy makers need to support health-care facilities in interpreting guidance during a pandemic that will probably be characterised by fluctuating local incidence of SARS-CoV-2 to mitigate the impact of this pandemic on their workforce.
Abstract
Cellular exonucleases involved in the processes that regulate RNA stability and quality control have been shown to restrict or to promote the multiplication cycle of numerous RNA viruses. ...Influenza A viruses are major human pathogens that are responsible for seasonal epidemics, but the interplay between viral proteins and cellular exonucleases has never been specifically studied. Here, using a stringent interactomics screening strategy and an siRNA-silencing approach, we identified eight cellular factors among a set of 75 cellular proteins carrying exo(ribo)nuclease activities or involved in RNA decay processes that support influenza A virus multiplication. We show that the exoribonuclease ERI1 interacts with the PB2, PB1 and NP components of the viral ribonucleoproteins and is required for viral mRNA transcription. More specifically, we demonstrate that the protein-protein interaction is RNA dependent and that both the RNA binding and exonuclease activities of ERI1 are required to promote influenza A virus transcription. Finally, we provide evidence that during infection, the SLBP protein and histone mRNAs co-purify with vRNPs alongside ERI1, indicating that ERI1 is most probably recruited when it is present in the histone pre-mRNA processing complex in the nucleus.
Soon after the beginning of the COVID-19 pandemic in early 2020, the Betacoronavirus SARS-CoV-2 infection of several mink farms breeding American minks (Neovison vison) for fur was detected in ...various European countries. The risk of a new reservoir being formed and of a reverse zoonosis from minks quickly became a major concern. The aim of this study was to investigate the four French mink farms to see whether SARS-CoV-2 was circulating there in late 2020. The investigations took place during the slaughtering period, thus facilitating different types of sampling (swabs and blood). On one of the four mink farms, 96.6% of serum samples were positive when tested with a SARS-CoV-2 ELISA coated with purified N protein recombinant antigen, and 54 out of 162 (33%) pharyngo-tracheal swabs were positive by RT-qPCR. The genetic variability among 12 SARS-CoV-2 genomes sequenced from this farm indicated the co-circulation of several lineages at the time of sampling. All the SARS-CoV-2 genomes detected were nested within the 20A clade (Nextclade), together with SARS-CoV-2 genomes from humans sampled during the same period. The percentage of SARS-CoV-2 seropositivity by ELISA varied between 0.3 and 1.1% on the other three farms. Interestingly, among these three farms, 11 pharyngo-tracheal swabs and 3 fecal pools from two farms were positive by end-point RT-PCR for an Alphacoronavirus very similar to a mink coronavirus sequence observed on Danish farms in 2015. In addition, a mink Caliciviridae was identified on one of the two farms positive for Alphacoronavirus. The clinical impact of these inapparent viral infections is not known. The co-infection of SARS-CoV-2 with other viruses on mink farms could help explain the diversity of clinical symptoms noted on different infected farms in Europe. In addition, the co-circulation of an Alphacoronavirus and SARS-CoV-2 on a mink farm would potentially increase the risk of viral recombination between alpha and betacoronaviruses as already suggested in wild and domestic animals, as well as in humans.
The present study was set up to evaluate the efficacy of virological surveillance using posterior oropharyngeal saliva samples to monitor the COVID-19 pandemic in general practice.
Posterior ...oropharyngeal saliva samples were collected without restriction on timing or alimentation by general practitioners from patients with acute respiratory infection (ARI) seen in consultation. Saliva samples were tested by real-time reverse transcription polymerase chain reaction for SARS-CoV-2 and 21 other respiratory pathogens. Results for SARS-CoV-2 in saliva samples were compared to results obtained using a nasopharyngeal swab (NPS) collected in a certified medical laboratory before or after the ARI consultation.
Overall, 143 ARI patients were enrolled between 6 June 2020, and 19 January 2021. SARS-CoV-2 RNA was detected in 37.0% (n = 53) of saliva samples and in 39.0% (n = 56) of NPS. Both saliva and NPS were positive in 51 patients. Positive and negative results were concordant between saliva samples and NPS in 51 (96.2%) and in 85 (94.4%) patients, respectively, with a Cohen's Kappa coefficient of 0.89 (95% CI 0.82-0.97,
< 0.001). Other respiratory viruses were detected in 28.0% (n = 40) of the 143 saliva samples.
Findings suggest that saliva samples could represent an attractive alternative to NPS for surveillance of SARS-CoV-2 in patients consulting for an ARI in primary care.
On Dec 31, 2019, China reported a cluster of cases of pneumonia in people at Wuhan, Hubei Province. The responsible pathogen is a novel coronavirus, named severe acute respiratory syndrome ...coronavirus 2 (SARS-CoV-2). We report the relevant features of the first cases in Europe of confirmed infection, named coronavirus disease 2019 (COVID-19), with the first patient diagnosed with the disease on Jan 24, 2020.
In this case series, we followed five patients admitted to Bichat-Claude Bernard University Hospital (Paris, France) and Pellegrin University Hospital (Bordeaux, France) and diagnosed with COVID-19 by semi-quantitative RT-PCR on nasopharyngeal swabs. We assessed patterns of clinical disease and viral load from different samples (nasopharyngeal and blood, urine, and stool samples), which were obtained once daily for 3 days from hospital admission, and once every 2 or 3 days until patient discharge. All samples were refrigerated and shipped to laboratories in the National Reference Center for Respiratory Viruses (The Institut Pasteur, Paris, and Hospices Civils de Lyon, Lyon, France), where RNA extraction, real-time RT-PCR, and virus isolation and titration procedures were done.
The patients were three men (aged 31 years, 48 years, and 80 years) and two women (aged 30 years and 46 years), all of Chinese origin, who had travelled to France from China around mid-January, 2020. Three different clinical evolutions are described: (1) two paucisymptomatic women diagnosed within a day of exhibiting symptoms, with high nasopharyngeal titres of SARS-CoV-2 within the first 24 h of the illness onset (5·2 and 7·4 log10 copies per 1000 cells, respectively) and viral RNA detection in stools; (2) a two-step disease progression in two young men, with a secondary worsening around 10 days after disease onset despite a decreasing viral load in nasopharyngeal samples; and (3) an 80-year-old man with a rapid evolution towards multiple organ failure and a persistent high viral load in lower and upper respiratory tract with systemic virus dissemination and virus detection in plasma. The 80-year-old patient died on day 14 of illness (Feb 14, 2020); all other patients had recovered and been discharged by Feb 19, 2020.
We illustrated three different clinical and biological types of evolution in five patients infected with SARS-CoV-2 with detailed and comprehensive viral sampling strategy. We believe that these findings will contribute to a better understanding of the natural history of the disease and will contribute to advances in the implementation of more efficient infection control strategies.
REACTing (Research & Action Emerging Infectious Diseases).
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.7 and B.1.351 variants were first identified in the United Kingdom and South Africa, respectively, and have since spread to many ...countries. These variants harboring diverse mutations in the gene encoding the spike protein raise important concerns about their immune evasion potential. Here, we isolated infectious B.1.1.7 and B.1.351 strains from acutely infected individuals. We examined sensitivity of the two variants to SARS-CoV-2 antibodies present in sera and nasal swabs from individuals infected with previously circulating strains or who were recently vaccinated, in comparison with a D614G reference virus. We utilized a new rapid neutralization assay, based on reporter cells that become positive for GFP after overnight infection. Sera from 58 convalescent individuals collected up to 9 months after symptoms, similarly neutralized B.1.1.7 and D614G. In contrast, after 9 months, convalescent sera had a mean sixfold reduction in neutralizing titers, and 40% of the samples lacked any activity against B.1.351. Sera from 19 individuals vaccinated twice with Pfizer Cominarty, longitudinally tested up to 6 weeks after vaccination, were similarly potent against B.1.1.7 but less efficacious against B.1.351, when compared to D614G. Neutralizing titers increased after the second vaccine dose, but remained 14-fold lower against B.1.351. In contrast, sera from convalescent or vaccinated individuals similarly bound the three spike proteins in a flow cytometry-based serological assay. Neutralizing antibodies were rarely detected in nasal swabs from vaccinees. Thus, faster-spreading SARS-CoV-2 variants acquired a partial resistance to neutralizing antibodies generated by natural infection or vaccination, which was most frequently detected in individuals with low antibody levels. Our results indicate that B1.351, but not B.1.1.7, may increase the risk of infection in immunized individuals.
The segmented RNA genome of influenza A viruses (IAVs) enables viral evolution through genetic reassortment after multiple IAVs coinfect the same cell, leading to viruses harboring combinations of ...eight genomic segments from distinct parental viruses. Existing data indicate that reassortant genotypes are not equiprobable; however, the low throughput of available virology techniques does not allow quantitative analysis. Here, we have developed a high-throughput single-cell droplet microfluidic system allowing encapsulation of IAV-infected cells, each cell being infected by a single progeny virion resulting from a coinfection process. Customized barcoded primers for targeted viral RNA sequencing enabled the analysis of 18,422 viral genotypes resulting from coinfection with two circulating human H1N1pdm09 and H3N2 IAVs. Results were highly reproducible, confirmed that genetic reassortment is far from random, and allowed accurate quantification of reassortants including rare events. In total, 159 out of the 254 possible reassortant genotypes were observed but with widely varied prevalence (from 0.038 to 8.45%). In cells where eight segments were detected, all 112 possible pairwise combinations of segments were observed. The inclusion of data from single cells where less than eight segments were detected allowed analysis of pairwise cosegregation between segments with very high confidence. Direct coupling analysis accurately predicted the fraction of pairwise segments and full genotypes. Overall, our results indicate that a large proportion of reassortant genotypes can emerge upon coinfection and be detected over a wide range of frequencies, highlighting the power of our tool for systematic and exhaustive monitoring of the reassortment potential of IAVs.
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