Positive SARS-CoV-2 RT-qPCR of a nasal swab spot after 30 days of conservation on filter paper at room temperature.

We tested the use of nasal swabs spotted onto filter paper (Whatman 3M) for the molecular diagnosis of SARS-CoV-2 infection. Spots of a positive nasal swab in conservation medium (B.1.177 strain, 21Ct) were still positive (duo E-gene/IP4) after 10, 20, and 30 days of conservation at room temperature, with Ct values of 28, 27, and 26, respectively. Direct spotting of the swab at bedside (omicron strain) still gave a positive result after 10 days in two RT-qPCR systems: 33.7 Ct using duo E-gene/IP4, and 34.8 using a specific Omicron system. Spotting of a dilution range of media spiked with the Delta (strain 2021/FR/0610, lineage B 1.617.2) and Omicron strains (strain UVE/SARS-CoV-2/2021/FR/1514) showed a threshold of 0.04 TCID50 after 10 days of conservation. We show, for the first time, that this simple and low-cost conservation method can be used to store samples for RT-qPCR against SARS-CoV-2 for up to at least 1 month.

Orthopoxvirus Seroprevalence and Infection Susceptibility in France, Bolivia, Laos, and Mali.

To determine a demographic overview of orthopoxvirus seroprevalence, we tested blood samples collected during 2003-2019 from France (n = 4,876), Bolivia (n = 601), Laos (n = 657), and Mali (n = 255) for neutralizing antibodies against vaccinia virus. In addition, we tested 4,448 of the 4,876 samples from France for neutralizing antibodies against cowpox virus. We confirmed extensive cross-immunity between the 2 viruses. Seroprevalence of antibodies was <1% in Bolivia, <5% in Laos, and 17.25% in Mali. In France, we found low prevalence of neutralizing antibodies in persons who were unvaccinated and vaccinated for smallpox, suggesting immunosenescence occurred in vaccinated persons, and smallpox vaccination compliance declined before the end of compulsory vaccination. Our results suggest that populations in Europe, Africa, Asia, and South America are susceptible to orthopoxvirus infections, which might have precipitated the emergence of orthopoxvirus infections such as the 2022 spread of monkeypox in Europe.

Eight Months of Serological Follow-Up of Anti-SARS-CoV-2 Antibodies in France: A Study among an Adult Population.

BACKGROUND: Uncertainties remain regarding the nature and durability of the humoral immune response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). AIM: This study investigated immunoglobulin G response and neutralizing activity to evaluate the mean antibody concentrations and response duration induced by each vaccination regimen in a French adult population. METHODS: A study including blood sampling and questionnaires was carried out from November 2020 to July 2021 with three separate follow-up phases. Spike proteins and neutralizing antibodies were quantified using ELISA and a virus-neutralization test. RESULTS: Overall, 295 participants were included. Seroprevalences were 11.5% (n = 34), 10.5% (n = 31), and 68.1% (n = 201) in phases 1, 2, and 3, respectively. Importantly, 5.8% (n = 17) of participants lost their natural antibodies. Antibody response of participants with only a prior infection was 88.2 BAU/mL, significantly lower than those vaccinated, which was 1909.3 BAU/mL (p = 0.04). Moreover, the antibody response of vaccinated participants with a prior infection was higher (3593.8 BAU/mL) than those vaccinated without prior infection (3402.9 BAU/mL) (p = 0.78). Vaccinated participants with or without prior infection had a higher seroneutralization rate (91.0%) than those unvaccinated with prior infection (65.0%). CONCLUSION: These results demonstrated that single infection does not confer effective protection against SARS-CoV-2.

Reduced neutralizing antibody potency of COVID-19 convalescent vaccinated plasma against SARS-CoV-2 Omicron variant.

BACKGROUND AND OBJECTIVE: The SARS-CoV-2 Omicron variant displays increased infectiveness as well as mutations resulting in reduced neutralizing activity of antibodies acquired after vaccination or infection involving earlier strains. To assess the ability of vaccinated COVID-19 convalescent plasma (CCP-V) collected before November 2021 to seroneutralize Omicron, we compared neutralizing antibody (nAb) titres of 63 samples against Omicron and earlier B.1 (D614G) strains. METHODS AND FINDINGS: Relationship between anti-Omicron titres and IgG anti-S1 levels (binding arbitrary unit: BAU/ml) was studied. Although correlated, anti-Omicron titres were significantly lower than anti-B.1 titres (median = 80 [10-1280] vs. 1280 [160-10,240], p < 0.0001). Omicron nAb titres and IgG anti-S1 levels were correlated (Spearman's rank correlation coefficient = 0.67). Anti-S1 IgG threshold at 7000 BAU/ml may allow to discard CCP-V without anti-Omicron activity (nAb titre <40). Conversely, only those with highest titres (≥160) had systematically anti-S1 IgG levels >7000 BAU/ml. CONCLUSION: A fraction of CCP-V collected before November 2021 retains anti-Omicron seroneutralizing activity that may be selected by quantitative anti-IgG assays, but such assays do not easily allow the identification of 'high-titre' CCP-V. However, collecting plasma from vaccinated donors recently infected with Omicron may be the best option to provide optimal CCP-V for immunocompromised patients infected with this variant.

A Cross-Sectional Study of Exposure Factors Associated with Seropositivity for SARS-CoV-2 Antibodies during the Second Epidemic Wave among a Sample of the University of Corsica (France).

This study aimed to estimate the seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection within the staff and student populations of the University of Corsica (France) during the second wave of the epidemic. METHODS: A cross-sectional survey was conducted from 23 November 2020 to 31 January 2021. The participants underwent blood sampling using a fingerstick procedure and completed an anonymized questionnaire. Sera were tested for the presence of anti-SARS-CoV-2 IgG (ELISA-S) and, if positive, with an in-house virus neutralization test (VNT). RESULTS: A total of 418 persons were included in the study. The overall seroprevalence was 12.8% (95% confidence interval (CI), 9.8-16.6%). A total of 15 (31%) of the 49 individuals who had a positive ELISA-S also had a positive VNT. Seropositivity was associated with living at the city campus during the week and on weekends (OR = 3.74 [1.40-12.00]), using public transportation/carpooling (OR = 2.00 [1.01-4.02]), and being in contact with a person who tested positive for SARS-CoV-2 (OR = 2.32 [1.20-4.40]). The main symptoms associated with seropositivity were "having had an acute respiratory infection" (OR = 3.05 [1.43-6.43]) and "experiencing loss of smell" (OR = 16.4 [5.87-50.7]). CONCLUSION: These results could be useful for SARS-CoV-2 prevention and control on university campuses.

High SARS-CoV-2 Prevalence among Healthcare Workers in Cochabamba, Bolivia.

Healthcare workers (HCWs) are at increased risk of SARS-CoV-2 infection. The aim of the study was to estimate the SARS-CoV-2 seroprevalence among HCWs in Cochabamba, Bolivia and to determine the potential risk factors. In January 2021, a cross-sectional SARS-CoV-2 seroprevalence study was conducted in 783 volunteer clinical and non-clinical HCWs in tertiary care facilities. It was based on IgG detection using ELISA, chemiluminiscence, and seroneutralisation tests from dried blood spots. Analysis revealed a high seroprevalence (43.4%) of SARS-CoV-2 IgG antibodies. The combination of anosmia and ageusia (OR: 68.11; 95%-CI 24.83-186.80) was predictive of seropositivity. Belonging to the cleaning staff (OR: 1.94; 95%-CI 1.09-3.45), having more than two children in the same house (OR: 1.74; 95%-CI 1.12-2.71), and having been in contact with a close relative with COVID-19 (OR: 3.53; 95%-CI 2.24-5.58) were identified as risk factors for seropositivity in a multivariate analysis. A total of 47.5% of participants had received medication for COVID-19 treatment or prevention, and only ~50% of symptomatic subjects accessed PCR or antigenic testing. This study confirms a massive SARS-CoV-2 attack rate among HCWs in Cochabamba by the end of January 2021. The main risk factors identified are having a low-skilled job, living with children, and having been in contact with an infected relative in the household.

COVID-19 convalescent plasma: Evolving strategies for serological screening in France.

Quantitation of anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) neutralizing antibodies (Nabs) is a key parameter in determining the effective dose for treatment with COVID-19 convalescent plasma (CCP). Interpretation of results from clinical trials conducted worldwide requires comparison of Nabs titres obtained from different methods. As virus neutralization tests (VNTs) are not standardized scalable or commercially available, strategies based on intensity of ELISA (Enzyme Linked Immunosorbent Assay) or chemiluminescent binding serological tests were implemented to allow comparisons and establish criteria for determining 'high-titres' of anti-SARS-CoV-2 antibodies (Abs). To this end, the FDA (Food and Drug Administration) has proposed criteria to define high-titre plasmas using different serological assays, including the one used in France for the CCP SARS-CoV-2 Abs screening (Euroimmun anti-S1 IgG). A retrospective study revealed that when using the FDA criteria (ELISA signal-to-cut-off [S/C ratio] ≥3.5), 91% of CCP had Nabs titres ≥40 as assessed with an in-house VNT. French strategy to ensure sufficient stocks of CCP of increasing titre has evolved over time. Recently, we improved our strategy by collecting only plasma from vaccinated convalescent donors as we confirmed that the mean IgG antibody level (ELISA S/C ratio) was significantly higher in plasma from vaccinated convalescent donors compared to donations from unvaccinated convalescent donors: 9.31 (CI 95%: 8.46-10.16) versus 3.22 (CI 95%: 3.05-3.39) (p < 0.001).

Determining the best window for BNT162b2 mRNA vaccination for SARS-CoV-2 in patients with multiple sclerosis receiving anti-CD20 therapy.

We studied the serologic response to the BNT162b2 mRNA vaccine at four weeks after the second dose in patients with RRMS treated with rituximab with extended-interval dosing (n = 26). At four weeks, 73% of patients were seropositive. No patient without B cells at the first dose (n = 4) was seropositive. Four of seven (57%) patients with B-cell proportion >0% and ≤5% were seropositive. All patients with B-cell proportion >5% (n = 15) were seropositive. In all patients, quantitative ELISA measures after vaccination were correlated with B-cell counts measured before vaccination. In patients receiving rituximab, seropositivity after BNT162b2 mRNA vaccination emerged only after B-cell repopulation.

Impact of the Second Epidemic Wave of SARS-CoV-2: Increased Exposure of Young People.

We aimed to use serological surveillance based on serial cross-sectional sampling of residual sera obtained from clinical laboratories to compare the differences in age and sex profiles of infected persons in the first and second waves of SARS-CoV-2 in Corsica, France. Residual sera were obtained, including samples from individuals of all ages collected for routine screening or clinical management by clinical laboratories. All the sera collected were tested for the presence of anti-SARS-CoV-2 IgG using a kit for semi-quantitative detection of IgG antibodies against the S1 domain of the viral spike protein (ELISA-S). Samples that were borderline and positive in ELISA-S were tested with an in-house virus neutralization test. During the second-wave period, we collected between 6 November, 2020 and 12 February, 2021, 4,505 sera from patients aged 0-101 years (60.4% women). The overall weighted seroprevalence of residual sera collected during the second-wave period [8.04% (7.87-9.61)] was significantly higher than the overall weighted seroprevalence estimated at the end of the first wave between 16 April and 15 June, 2020 [5.46% (4.37-7.00)] (p-value = 0.00025). Ninety-eight (30.1%) of the 326 samples tested in the VNT assay had a positive neutralization antibody titer. Estimated seroprevalence increased significantly for men [odds ratio (OR) OR = 1.80 (1.30-2.54); p-value = 0.00026] and for people under 30 years of age [OR = 2.17 (1.46-3.28); p-value = 0.000032]. This increase was observed in young adults aged 20-29 years among whom antibody frequencies were around four-fold higher than those observed at the end of the first wave. In conclusion, our seroprevalence estimates, including the proportion of the participants who had produced neutralizing antibodies, indicate that in February, 2021 the population of Corsica was still far from being protected against SARS-Cov-2 by "herd immunity."

SARS-CoV-2 neutralising antibody testing in Europe: towards harmonisation of neutralising antibody titres for better use of convalescent plasma and comparability of trial data.

We compared the performance of SARS-CoV-2 neutralising antibody testing between 12 European laboratories involved in convalescent plasma trials. Raw titres differed almost 100-fold differences between laboratories when blind-testing 15 plasma samples. Calibration of titres in relation to the reference reagent and standard curve obtained by testing a dilution series reduced the inter-laboratory variability ca 10-fold. The harmonisation of neutralising antibody quantification is a vital step towards determining the protective and therapeutic levels of neutralising antibodies.

Evolution of Chikungunya virus in mosquito cells.

It has been observed that replication of Chikungunya virus (CHIKV) in C6/36 Aedes albopictus cells has little effect on virus evolution. To characterize evolutionary patterns associated with CHIKV replication in mosquito cells, we performed serial passages of the LR2006 strain in Ae. albopictus cells (75 and 30 passages in C6/36 and U4.4 respectively) and Ae. aegypti cells (100 passages in AA-A20 and in AE) and studied genotypic changes accompanying adaptation during this evolutionary process. Quantitative analysis revealed cell specific patterns: low mutation rates in C6/36 cells except when a CHIKV strain pre-adapted to mammalian was used and typical features of adaptation to cell culture conditions with a high number of fixed mutations in AE and AA-A20 cells probably due to the weak permissiveness of these latter cell lines. Altogether, these results suggested that both cell line and viral strain influence rates of viral evolution. In contrast, characteristics and distribution of mutations were qualitatively very similar in all mosquito cells with a high level of parallel evolution including 4 deletion mutations. Serial passage in mammalian cells of viruses pre-adapted to mosquito cells revealed disappearance of almost all shared mutations suggesting that many of these mutational patterns are vector-specific.

SuPReMe: a rapid reverse genetics method to generate clonal populations of recombinant RNA viruses.

Reverse genetics systems enable the manipulation of viral genomes and are proving to be essential for studying RNA viruses. Methods for generating clonal virus populations are particularly useful for studying the impact of genomic modifications on viral properties. Here, by exploiting a chikungunya virus model, we compare viral populations and their replicative fitness when generated using either the rapid and user-friendly PCR-based ISA (Infectious Subgenomic Amplicons) method or classical infectious clone technology. As anticipated, the ISA method resulted in greater genetic diversity of the viral populations, but no significant difference in viral fitness in vitro was observed. On the basis of these results, a new ISA-derived reverse genetics procedure was developed. This method, designated 'SuPReMe' (Subgenomic Plasmids Recombination Method), in which digested plasmids containing subgenomic DNA fragments were directly transfected into permissive cells, retains the following major advantages of the ISA method: it is rapid, flexible and does not require the cloning of complete genomes. Moreover, SuPReMe has been shown to produce virus populations with genetic diversity and replicative fitness similar to those obtained using conventional infectious clone technology. SuPReMe, therefore, represents an effective and promising option for the rapid generation of clonal recombinant populations of single-stranded positive-sense RNA viruses.

Toscana virus cap-snatching and initiation of transcription.

Toscana virus (TOSV) is an arthropod-borne phlebovirus within the family Phenuiviridae in the order Bunyavirales. It seems to be an important agent of human meningoencephalitis in the warm season in the Mediterranean area. Because the polymerase of Bunyavirales lacks a capping activity, it cleaves short-capped RNA leaders derived from the host cell, and uses them to initiate viral mRNA synthesis. To determine the size and nucleotide composition of the host-derived RNA leaders, and to elucidate the first steps of TOSV transcription initiation, we performed a high-throughput sequencing of the 5' end of TOSV mRNAs in infected cells at different times post-infection. Our results indicated that the viral polymerase cleaved the host-capped RNA leaders within a window of 11-16 nucleotides. A single population of cellular mRNAs could be cleaved at different sites to prime the synthesis of several viral mRNA species. The majority of the mRNA resulted from direct priming, but we observed mRNAs resulting from several rounds of prime-and-realign events. Our data suggest that the different rounds of the prime-and-realign mechanism result from the blocking of the template strand in a static position in the active site, leading to the slippage of the nascent strand by two nucleotides when the growing duplex is sorted out from the active site. To minimize this rate-limiting step, TOSV polymerase cleaves preferentially capped RNA leaders after GC, so as to greatly reduce the number of cycles of priming and realignment, and facilitate the separation of the growing duplex.

Bunyaviridae RdRps: structure, motifs, and RNA synthesis machinery.

Bunyaviridae family is the largest and most diverse family of RNA viruses. It has more than 350 members divided into five genera: Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus, and Tospovirus. They are present in the five continents, causing recurrent epidemics, epizootics, and considerable agricultural loss. The genome of bunyaviruses is divided into three segments of negative single-stranded RNA according to their relative size: L (Large), M (Medium) and S (Small) segment. Bunyaviridae RNA-dependent RNA polymerase (RdRp) is encoded by the L segment, and is in charge of the replication and transcription of the viral RNA in the cytoplasm of the infected cell. Viral RdRps share a characteristic right hand-like structure with three subdomains: finger, palm, and thumb subdomains that define the formation of the catalytic cavity. In addition to the N-terminal endonuclease domain, eight conserved motifs (A-H) have been identified in the RdRp of Bunyaviridae. In this review, we have summarized the recent insights from the structural and functional studies of RdRp to understand the roles of different motifs shared by RdRps, the mechanism of viral RNA replication, genome segment packaging by the nucleoprotein, cap-snatching, mRNA transcription, and other RNA mechanisms of bunyaviruses.