Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates.

Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic and no antiviral drug or vaccine is yet available for the treatment of this disease1-3. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes COVID-19-worldwide but there is no definitive evidence that HCQ is effective for treating COVID-194-7. Here we evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in African green monkey kidney cells (Vero E6) but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to a placebo treatment, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor the combination of HCQ and AZTH showed a significant effect on viral load in any of the analysed tissues. When the drug was used as a pre-exposure prophylaxis treatment, HCQ did not confer protection against infection with SARS-CoV-2. Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral drug for the treatment of COVID-19 in humans.

Impact of variants of concern on SARS-CoV-2 viral dynamics in non-human primates.

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.

A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection.

SARS-CoV-2 infection results in impaired interferon response in patients with severe COVID-19. However, how SARS-CoV-2 interferes with host immune responses is incompletely understood. Here, we sequence small RNAs from SARS-CoV-2-infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus-derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3'UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 can potentially employ a virus-derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon-mediated immune response.

Emergence, spread and characterisation of the SARS-CoV-2 variant B.1.640 circulating in France, October 2021 to February 2022.

BackgroundSuccessive epidemic waves of COVID-19 illustrated the potential of SARS-CoV-2 variants to reshape the pandemic. Detecting and characterising emerging variants is essential to evaluate their public health impact and guide implementation of adapted control measures.AimTo describe the detection of emerging variant, B.1.640, in France through genomic surveillance and present investigations performed to inform public health decisions.MethodsIdentification and monitoring of SARS-CoV-2 variant B.1.640 was achieved through the French genomic surveillance system, producing 1,009 sequences. Additional investigation of 272 B.1.640-infected cases was performed between October 2021 and January 2022 using a standardised questionnaire and comparing with Omicron variant-infected cases.ResultsB.1.640 was identified in early October 2021 in a school cluster in Bretagne, later spreading throughout France. B.1.640 was detected at low levels at the end of SARS-CoV-2 Delta variant's dominance and progressively disappeared after the emergence of the Omicron (BA.1) variant. A high proportion of investigated B.1.640 cases were children aged under 14 (14%) and people over 60 (27%) years, because of large clusters in these age groups. B.1.640 cases reported previous SARS-CoV-2 infection (4%), anosmia (32%) and ageusia (34%), consistent with data on pre-Omicron SARS-CoV-2 variants. Eight percent of investigated B.1.640 cases were hospitalised, with an overrepresentation of individuals aged over 60 years and with risk factors.ConclusionEven though B.1.640 did not outcompete the Delta variant, its importation and continuous low-level spread raised concerns regarding its public health impact. The investigations informed public health decisions during the time that B.1.640 was circulating.

Saliva for molecular detection of SARS-CoV-2 in pre-school and school-age children.

SARS-CoV-2 diagnosis is a cornerstone for the management of coronavirus disease 2019 (COVID-19). Numerous studies have assessed saliva performance over nasopharyngeal sampling (NPS), but data in young children are still rare. We explored saliva performance for SARS-CoV-2 detection by RT-PCR according to the time interval from initial symptoms or patient serological status. We collected 509 NPS and saliva paired samples at initial diagnosis from 166 children under 12 years of age (including 57 children under 6), 106 between 12 and 17, and 237 adults. In children under 12, overall detection rate for SARS-CoV-2 was comparable in saliva and NPS, with an overall agreement of 89.8%. Saliva sensitivity was significantly lower than that of NPS (77.1% compared to 95.8%) in pre-school and school-age children but regained 96% when considering seronegative children only. This pattern was also observed to a lesser degree in adolescents but not in adults. Sensitivity of saliva was independent of symptoms, in contrary to NPS, whose sensitivity decreased significantly in asymptomatic subjects. Performance of saliva is excellent in children under 12 at early stages of infection. This reinforces saliva as a collection method for early and unbiased SARS-CoV-2 detection and a less invasive alternative for young children.

RNA-seq accuracy and reproducibility for the mapping and quantification of influenza defective viral genomes.

Like most RNA viruses, influenza viruses generate defective viral genomes (DVGs) with large internal deletions during replication. There is accumulating evidence supporting a biological relevance of such DVGs. However, further understanding of the molecular mechanisms that underlie the production and biological activity of DVGs is conditioned upon the sensitivity and accuracy of detection methods, that is, next-generation sequencing (NGS) technologies and related bioinformatics algorithms. Although many algorithms were developed, their sensitivity and reproducibility were mostly assessed on simulated data. Here, we introduce DG-seq, a time-efficient pipeline for DVG detection and quantification, and a set of biological controls to assess the performance of not only our bioinformatics algorithm but also the upstream NGS steps. Using these tools, we provide the first rigorous comparison of the two commonly used sample processing methods for RNA-seq, with or without a PCR preamplification step. Our data show that preamplification confers a limited advantage in terms of sensitivity and introduces size- but also sequence-dependent biases in DVG quantification, thereby providing a strong rationale to favor preamplification-free methods. We further examine the features of DVGs produced by wild-type and transcription-defective (PA-K635A or PA-R638A) influenza viruses, and show an increased diversity and frequency of DVGs produced by the PA mutants compared to the wild-type virus. Finally, we demonstrate a significant enrichment in DVGs showing direct, A/T-rich sequence repeats at the deletion breakpoint sites. Our findings provide novel insights into the mechanisms of influenza virus DVG production.

SARS-CoV-2 viral dynamics in non-human primates.

Non-human primates infected with SARS-CoV-2 exhibit mild clinical signs. Here we used a mathematical model to characterize in detail the viral dynamics in 31 cynomolgus macaques for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large burst size (>104 virus) and a within-host reproductive basic number of approximately 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly lost with a half-life of 9 hours, with no significant association between cytokine elevation and clearance, leading to a median time to viral clearance of 10 days, consistent with observations in mild human infections. Given these parameter estimates, we predict that a prophylactic treatment blocking 90% of viral production or viral infection could prevent viral growth. In conclusion, our results provide estimates of SARS-CoV-2 viral kinetic parameters in an experimental model of mild infection and they provide means to assess the efficacy of future antiviral treatments.

Epidemiological and clinical insights from SARS-CoV-2 RT-PCR crossing threshold values, France, January to November 2020.

BackgroundThe COVID-19 pandemic has led to an unprecedented daily use of RT-PCR tests. These tests are interpreted qualitatively for diagnosis, and the relevance of the test result intensity, i.e. the number of quantification cycles (Cq), is debated because of strong potential biases.AimWe explored the possibility to use Cq values from SARS-CoV-2 screening tests to better understand the spread of an epidemic and to better understand the biology of the infection.MethodsWe used linear regression models to analyse a large database of 793,479 Cq values from tests performed on more than 2 million samples between 21 January and 30 November 2020, i.e. the first two pandemic waves. We performed time series analysis using autoregressive integrated moving average (ARIMA) models to estimate whether Cq data information improves short-term predictions of epidemiological dynamics.ResultsAlthough we found that the Cq values varied depending on the testing laboratory or the assay used, we detected strong significant trends associated with patient age, number of days after symptoms onset or the state of the epidemic (the temporal reproduction number) at the time of the test. Furthermore, knowing the quartiles of the Cq distribution greatly reduced the error in predicting the temporal reproduction number of the COVID-19 epidemic.ConclusionOur results suggest that Cq values of screening tests performed in the general population generate testable hypotheses and help improve short-term predictions for epidemic surveillance.

Effectiveness of complete primary vaccination against COVID-19 at primary care and community level during predominant Delta circulation in Europe: multicentre analysis, I-MOVE-COVID-19 and ECDC networks, July to August 2021.

IntroductionIn July and August 2021, the SARS-CoV-2 Delta variant dominated in Europe.AimUsing a multicentre test-negative study, we measured COVID-19 vaccine effectiveness (VE) against symptomatic infection.MethodsIndividuals with COVID-19 or acute respiratory symptoms at primary care/community level in 10 European countries were tested for SARS-CoV-2. We measured complete primary course overall VE by vaccine brand and by time since vaccination.ResultsOverall VE was 74% (95% CI: 69-79), 76% (95% CI: 71-80), 63% (95% CI: 48-75) and 63% (95% CI: 16-83) among those aged 30-44, 45-59, 60-74 and ≥ 75 years, respectively. VE among those aged 30-59 years was 78% (95% CI: 75-81), 66% (95% CI: 58-73), 91% (95% CI: 87-94) and 52% (95% CI: 40-61), for Comirnaty, Vaxzevria, Spikevax and COVID-19 Vaccine Janssen, respectively. VE among people 60 years and older was 67% (95% CI: 52-77), 65% (95% CI: 48-76) and 83% (95% CI: 64-92) for Comirnaty, Vaxzevria and Spikevax, respectively. Comirnaty VE among those aged 30-59 years was 87% (95% CI: 83-89) at 14-29 days and 65% (95% CI: 56-71%) at ≥ 90 days between vaccination and onset of symptoms.ConclusionsVE against symptomatic infection with the SARS-CoV-2 Delta variant varied among brands, ranging from 52% to 91%. While some waning of the vaccine effect may be present (sample size limited this analysis to only Comirnaty), protection was 65% at 90 days or more between vaccination and onset.

Estimation of influenza-attributable burden in primary care from season 2014/2015 to 2018/2019, France.

Influenza viruses cause seasonal epidemics whose intensity varies according to the circulating virus type and subtype. We aim to estimate influenza-like illness (ILI) incidence attributable to influenza viruses in France from October 2014 to May 2019. Physicians participating in the French Sentinelles network reported the number of patients with ILI seen in consultation and performed nasopharyngeal swabs in a sample of these patients. The swabs were tested by RT-PCR for the presence of influenza viruses. These clinical and virological data were combined to estimate ILI incidence attributable to influenza viruses by subtypes and age groups. Influenza incidence rates over seasons ranged from 1.9 (95% CI, 1.9; 2.0) to 3.4% (95% CI, 3.2; 3.6) of the population. Each season, more than half of ILI cases were attributable to influenza. Children under 15 years were the most affected, with influenza incidence rates ranging from 3.0 (95% CI, 2.8;3.3) to 5.7% (95% CI, 5.3;6.1). Co-circulation of several (sub)types of influenza viruses was observed each year, except in 2016/2017 where A(H3N2) viruses accounted for 98.0% of the influenza cases. Weekly ILI incidences attributable to each influenza virus (sub)type were mostly synchronized with ILI incidence, except in 2014/2015 and 2017/2018, where incidence attributable to type B viruses peaked few weeks later. The burden of medically attended influenza among patients with ILI is significant in France, varying considerably across years and age groups. These results show the importance of influenza surveillance in primary care combining clinical and virological data.

Early assessment of diffusion and possible expansion of SARS-CoV-2 Lineage 20I/501Y.V1 (B.1.1.7, variant of concern 202012/01) in France, January to March 2021.

The emergence of SARS-CoV-2 variant 20I/501Y.V1 (VOC-202012/1 or GR/501Y.V1) is concerning given its increased transmissibility. We reanalysed 11,916 PCR-positive tests (41% of all positive tests) performed on 7-8 January 2021 in France. The prevalence of 20I/501Y.V1 was 3.3% among positive tests nationwide and 6.9% in the Paris region. Analysing the recent rise in the prevalence of 20I/501Y.V1, we estimate that, in the French context, 20I/501Y.V1 is 52-69% more transmissible than the previously circulating lineages, depending on modelling assumptions.

Prior infection by seasonal coronaviruses, as assessed by serology, does not prevent SARS-CoV-2 infection and disease in children, France, April to June 2020.

BackgroundChildren have a low rate of COVID-19 and secondary severe multisystem inflammatory syndrome (MIS) but present a high prevalence of symptomatic seasonal coronavirus infections.AimWe tested if prior infections by seasonal coronaviruses (HCoV) NL63, HKU1, 229E or OC43 as assessed by serology, provide cross-protective immunity against SARS-CoV-2 infection.MethodsWe set a cross-sectional observational multicentric study in pauci- or asymptomatic children hospitalised in Paris during the first wave for reasons other than COVID (hospitalised children (HOS), n = 739) plus children presenting with MIS (n = 36). SARS-CoV-2 antibodies directed against the nucleoprotein (N) and S1 and S2 domains of the spike (S) proteins were monitored by an in-house luciferase immunoprecipitation system assay. We randomly selected 69 SARS-CoV-2-seropositive patients (including 15 with MIS) and 115 matched SARS-CoV-2-seronegative patients (controls (CTL)). We measured antibodies against SARS-CoV-2 and HCoV as evidence for prior corresponding infections and assessed if SARS-CoV-2 prevalence of infection and levels of antibody responses were shaped by prior seasonal coronavirus infections.ResultsPrevalence of HCoV infections were similar in HOS, MIS and CTL groups. Antibody levels against HCoV were not significantly different in the three groups and were not related to the level of SARS-CoV-2 antibodies in the HOS and MIS groups. SARS-CoV-2 antibody profiles were different between HOS and MIS children.ConclusionPrior infection by seasonal coronaviruses, as assessed by serology, does not interfere with SARS-CoV-2 infection and related MIS in children.

Oseltamivir Resistance in Severe Influenza A(H1N1)pdm09 Pneumonia and Acute Respiratory Distress Syndrome: A French Multicenter Observational Cohort Study.

In a multicenter cohort study including 22 oseltamivir-treated patients with influenza A(H1N1)pdm09 acute respiratory distress syndrome, prevalence of the H275Y substitution in the neuraminidase, responsible for highly reduced sensitivity to oseltamivir, was 23%. Patients infected with the H275Y mutant virus had higher day 28 mortality than others (80% vs 12%; P = .011).

Introductions and early spread of SARS-CoV-2 in France, 24 January to 23 March 2020.

Following SARS-CoV-2 emergence in China, a specific surveillance was implemented in France. Phylogenetic analysis of sequences retrieved through this surveillance suggests that detected initial introductions, involving non-clade G viruses, did not seed local transmission. Nevertheless, identification of clade G variants subsequently circulating in the country, with the earliest from a patient who neither travelled to risk areas nor had contact with travellers, suggests that SARS-CoV-2 might have been present before the first recorded local cases.

Excess cases of influenza-like illnesses synchronous with coronavirus disease (COVID-19) epidemic, France, March 2020.

Several French regions where coronavirus disease (COVID-19) has been reported currently show a renewed increase in ILI cases in the general practice-based Sentinelles network. We computed the number of excess cases by region from 24 February to 8 March 2020 and found a correlation with the number of reported COVID-19 cases so far. The data suggest larger circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the French population than apparent from confirmed cases.

First cases of coronavirus disease 2019 (COVID-19) in France: surveillance, investigations and control measures, January 2020.

A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) causing a cluster of respiratory infections (coronavirus disease 2019, COVID-19) in Wuhan, China, was identified on 7 January 2020. The epidemic quickly disseminated from Wuhan and as at 12 February 2020, 45,179 cases have been confirmed in 25 countries, including 1,116 deaths. Strengthened surveillance was implemented in France on 10 January 2020 in order to identify imported cases early and prevent secondary transmission. Three categories of risk exposure and follow-up procedure were defined for contacts. Three cases of COVID-19 were confirmed on 24 January, the first cases in Europe. Contact tracing was immediately initiated. Five contacts were evaluated as at low risk of exposure and 18 at moderate/high risk. As at 12 February 2020, two cases have been discharged and the third one remains symptomatic with a persistent cough, and no secondary transmission has been identified. Effective collaboration between all parties involved in the surveillance and response to emerging threats is required to detect imported cases early and to implement adequate control measures.

First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region, 24 January to 21 February 2020.

In the WHO European Region, COVID-19 surveillance was implemented 27 January 2020. We detail the first European cases. As at 21 February, nine European countries reported 47 cases. Among 38 cases studied, 21 were linked to two clusters in Germany and France, 14 were infected in China. Median case age was 42 years; 25 were male. Late detection of the clusters' index cases delayed isolation of further local cases. As at 5 March, there were 4,250 cases.

Bidirectional Human-Swine Transmission of Seasonal Influenza A(H1N1)pdm09 Virus in Pig Herd, France, 2018.

In 2018, a veterinarian became sick shortly after swabbing sows exhibiting respiratory syndrome on a farm in France. Epidemiologic data and genetic analyses revealed consecutive human-to-swine and swine-to-human influenza A(H1N1)pdm09 virus transmission, which occurred despite some biosecurity measures. Providing pig industry workers the annual influenza vaccine might reduce transmission risk.

Temporal Association Between Rhinovirus Activity and Kingella kingae Osteoarticular Infections.

OBJECTIVE: To determine whether the seasonal distribution of Kingella kingae osteoarticular infections is similar to that of common respiratory viruses. STUDY DESIGN: Between October 2009 and September 2016, we extracted the results of K kingae-specific real-time polymerase chain reaction analyses performed for bone or joint specimens in patients from 2 pediatric tertiary care centers in Paris. We used data of respiratory virus detection from the Réseau National des Laboratoires network with coordination with the National Influenza Center of France. The Spearman rank correlation was used to assess a correlation between weekly distributions, with P < .05 denoting a significant correlation. RESULTS: During the 7-year study period, 322 children were diagnosed with K kingae osteoarticular infection, and 317 testing episodes were K kingae-negative. We observed high activity for both K kingae osteoarticular infection and human rhinovirus (HRV) during the fall (98 [30.4%] and 2401 [39.1%] cases, respectively) and low activity during summer (59 [18.3%] and 681 [11.1%] cases, respectively). Weekly distributions of K kingae osteoarticular infection and rhinovirus activity were significantly correlated (r = 0.30; P = .03). In contrast, no significant correlation was found between the weekly distribution of K kingae osteoarticular infection and other respiratory viruses (r = -0.17, P = .34 compared with respiratory syncytial virus; r = -0.13, P = .34 compared with influenza virus; and r = -0.22, P = .11 compared with metapneumovirus). CONCLUSION: A significant temporal association was observed between HRV circulation and K kingae osteoarticular infection, strengthening the hypothesis of a role of viral infections in the pathophysiology of K kingae invasive infection.

Performance of influenza case definitions for influenza community surveillance: based on the French influenza surveillance network GROG, 2009-2014.

International case definitions recommended by the Centers for Disease Control and Prevention (CDC), the European Centre for Disease Prevention and Control (ECDC), and the World Health Organization (WHO) are commonly used for influenza surveillance. We evaluated clinical factors associated with the laboratory-confirmed diagnosis of influenza and the performance of these influenza case definitions by using a complete dataset of 14,994 patients with acute respiratory infection (ARI) from whom a specimen was collected between August 2009 and April 2014 by the Groupes Régionaux d'Observation de la Grippe (GROG), a French national influenza surveillance network. Cough and fever ≥ 39 °C most accurately predicted an influenza infection in all age groups. Several other symptoms were associated with an increased risk of influenza (headache, weakness, myalgia, coryza) or decreased risk (adenopathy, pharyngitis, shortness of breath, otitis/otalgia, bronchitis/ bronchiolitis), but not throughout all age groups. The WHO case definition for influenza-like illness (ILI) had the highest specificity with 21.4%, while the ECDC ILI case definition had the highest sensitivity with 96.1%. The diagnosis among children younger than 5 years remains challenging. The study compared the performance of clinical influenza definitions based on outpatient surveillance and will contribute to improving the comparability of data shared at international level.