The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance. (preprint)

Investment in Africa over the past year with regards to SARS-CoV-2 genotyping has led to a massive increase in the number of sequences, exceeding 100,000 genomes generated to track the pandemic on the continent. Our results show an increase in the number of African countries able to sequence within their own borders, coupled with a decrease in sequencing turnaround time. Findings from this genomic surveillance underscores the heterogeneous nature of the pandemic but we observe repeated dissemination of SARS-CoV-2 variants within the continent. Sustained investment for genomic surveillance in Africa is needed as the virus continues to evolve, particularly in the low vaccination landscape. These investments are very crucial for preparedness and response for future pathogen outbreaks.

Capturing SARS-CoV-2 from patient samples with low viral abundance: a comparative analysis (preprint)

Since the beginning of the SARS-CoV-2 coronavirus pandemic, genome sequencing is essential to monitor viral mutations over time and by territory. This need for complete genetic information is further reinforced by the rapid spread of variants of concern. In this paper, we assess the ability of the hybridization technique, Capture-Seq, to detect the SARS-CoV-2 genome, either partially or in its integrity on patients samples. We studied 20 patient nasal swab samples broken down into five series of four samples of equivalent viral load from CT25 to CT36+. For this, we tested 3 multi-virus panel as well as 2 SARS-CoV-2 only panels. The panels were chosen based on their specificity, global or specific, as well as their technological difference in the composition of the probes: ssRNA, ssDNA and dsDNA. The multi-virus panels are able to capture high-abundance targets but fail to capture the lowest-abundance targets, with a high percentage of off-target reads corresponding to the abundance of the host sequences. Both SARS-CoV-2-only panels were very effective, with high percentage of reads corresponding to the target. Overall, capture followed by sequencing is very effective for the study of SARS-CoV-2 in low-abundance patient samples and is suitable for samples with CT values up to 35.

Rapid characterization of a Delta-Omicron SARS-CoV-2 recombinant detected in Europe (preprint)

Recombination is a crucial process in the evolution of many organisms. Although the evolutionary reasons behind its occurrence in RNA viruses are debated, this phenomenon has been associated with major epidemiological events such as virus host range expansion, antigenic shift or variation in virulence 1,2, and this process occurs frequently in positive strand RNA viruses such as coronaviruses. The SARS-CoV-2 pandemic has been associated with the repeated emergence of variants of concern presenting increased transmissibility, severity or immune escape 3. The recent extensive circulation of Delta worldwide and its subsequent replacement by viruses of the Omicron lineage 4 (BA.1 then BA.2), have created conditions for genetic exchanges between viruses with both genetic diversity and phenotypic specificities 5-7. Here we report the identification and in vitro and in vivo characterization of a Delta-Omicron recombinant in Europe. This recombinant exhibits immune escape properties similar to Omicron, while its behavior in mice expressing the human ACE2 receptor is more similar to Delta. This recombinant provides a unique and natural opportunity to better understand the genotype to phenotype links in SARS-CoV-2.

Antibody escape and global spread of SARS-CoV-2 lineage A.27 (preprint)

In spring 2021, an increasing number of infections was observed caused by the hitherto rarely described SARS-CoV-2 variant A.27 in south-west Germany. From December 2020 to June 2021 this lineage has been detected in 31 countries. Phylogeographic analyses of A.27 sequences obtained from national and international databases reveal a global spread of this lineage through multiple introductions from its inferred origin in Western Africa. Variant A.27 is characterized by a mutational pattern in the spike gene that includes the L18F, L452R and N501Y spike amino acid substitutions found in various variants of concern but lacks the globally dominant D614G. Neutralization assays demonstrated an escape of A.27 from convalescent and vaccine-elicited antibody-mediated immunity. Moreover, the therapeutic monoclonal antibody Bamlanivimab and partially the REGN-COV2 cocktail failed to block infection by A.27. Our data emphasize the need for continued global monitoring of novel lineages because of the independent evolution of new escape mutations.

A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa (preprint)

The progression of the SARS-CoV-2 pandemic in Africa has so far been heterogeneous and the full impact is not yet well understood. Here, we describe the genomic epidemiology using a dataset of 8746 genomes from 33 African countries and two overseas territories. We show that the epidemics in most countries were initiated by importations, predominantly from Europe, which diminished following the early introduction of international travel restrictions. As the pandemic progressed, ongoing transmission in many countries and increasing mobility led to the emergence and spread within the continent of many variants of concern and interest, such as B.1.351, B.1.525, A.23.1 and C.1.1. Although distorted by low sampling numbers and blind-spots, the findings highlight that Africa must not be left behind in the global pandemic response, otherwise it could become a breeding ground for new variants.

A new SARS-CoV-2 variant poorly detected by RT-PCR on nasopharyngeal samples, with high lethality (preprint)

Background: In early January 2021, an outbreak of nosocomial cases of COVID 19 emerged in Western France, with RT PCR tests repeatedly negative on nasopharyngeal samples but positive on lower respiratory tract samples. Whole genome sequencing (WGS) revealed a new variant, currently defining a novel SARS CoV 2 lineage: B.1.616. In March, WHO classified this variant as "under investigation" (VUI). We analyzed the characteristics and outcomes of COVID 19 cases related to this new variant. Methods: Clinical, virological, and radiological data were retrospectively collected from medical charts in the two hospitals involved. We enrolled patients with at least one of the following: i) positive SARS CoV 2 RT PCR on a respiratory sample; ii) seroconversion with anti SARS CoV 2 IgG/IgM; iii) suggestive symptoms and typical features of COVID 19 on chest CT scan. Cases were categorized as either: i) B.1.616; ii) variant of concern (VOC); iii) unknown. Findings: From January 1st to March 24th, 2021, 114 patients fulfilled the inclusion criteria: B.1.616 (n=34), VOC (n=32), and unknown (n=48). B.1.616 related cases were older than VOC related cases (81 years [73-88], vs 73 years [67-82], P<0.05) and their first RT PCR tests were less often positive (5/34, 15% vs 31/32, 97%, P<0.05). The B.1.616 variant was independently associated with severe disease (multivariable Cox model HR 4.2 [1.3 , 13.5], P=0.018), and increased lethality (logrank test P=0.01): 28day mortality 15/34 (44%) with B.1.616, vs. 5/32 (16%) for VOC, P=0.036. Interpretation: We report a nosocomial outbreak of COVID-19 cases related to a new variant, B.1.616, poorly detected by RT PCR on nasopharyngeal samples, with high lethality.

Prospective evaluation of ID NOW COVID-19 assay used as point-of-care test in an Emergency Department (preprint)

Background : Rapid testing for COVID-19 has been clearly identified as an essential component of the strategy to control the SARS-CoV-2 epidemic, worldwide. The ID NOW COVID-19 assay is a simple, user-friendly, rapid molecular biology test based on nicking and extension amplification reaction (NEAR). Objectives : The aim of this study was to evaluate the ID NOW COVID-19 assay when used as a point-of-care test (POCT) in our Emergency Department (ED). Type of study : This prospective study enrolled 395 consecutive patients; paired nasopharyngeal swabs were collected from each study participant. The first swab was tested with the ID NOW COVID-19 assay at the point-of-care by ED nurses. The second swab was diluted in viral transport medium (VTM) and sent to the clinical microbiology department for analysis by both the RT-PCR Simplexa test COVID-19 Direct assay as the study reference method, and the ID NOW COVID-19 assay performed in the laboratory. Results : Nasopharyngeal swabs directly tested with the ID NOW COVID-19 assay yielded a sensitivity, specificity, PPV and NPV of 98.0%, 97.5%, 96.2% and 98.7%, respectively, in comparison with the RT-PCR study reference assay. When the ID NOW COVID-19 assay was performed in the laboratory using the VTM samples, the sensitivity decreased to 62.5% and the NPV to 79.7%. Three false negative test results were reported with the ID NOW COVID-19 assay when performed using undiluted swabs directly in the ED; these results were obtained from patients with elevated CT values (>30). Conclusion : We demonstrated that the ID NOW COVID-19 assay, performed as a point of care test in the ED using dry swabs, provides a rapid and reliable alternative to laboratory-based RT-PCR methods

The B1.351 and P.1 variants extend SARS-CoV-2 host range to mice (preprint)

Receptor recognition is a major determinant of viral host range, as well as infectivity and pathogenesis. Emergences have been associated with serendipitous events of adaptation upon encounters with a novel host, and the high mutation rate of RNA viruses has been proposed to explain their frequent host shifts. SARS-CoV-2 extensive circulation in humans has been associated with the emergence of variants, including variants of concern (VOCs) with diverse mutations in the spike and increased transmissibility or immune escape. Here we show that unlike the initial virus, VOCs are able to infect common laboratory mice, replicating to high titers in the lungs. This host range expansion is explained in part by the acquisition of changes at key positions of the receptor binding domain that enable binding to the mouse angiotensin-converting enzyme 2 (ACE2) cellular receptor, although differences between viral lineages suggest that other factors are involved in the capacity of SARS-CoV-2 VOCs to infect mice. This abrogation of the species barrier raises the possibility of wild rodent secondary reservoirs and provides new experimental models to study disease pathophysiology and countermeasures.

Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies (preprint)

SARS-CoV-2 B.1.1.7 and B.1.351 variants emerged respectively in United Kingdom and South Africa and spread in many countries. Here, we isolated infectious B.1.1.7 and B.1.351 strains and examined their sensitivity to anti-SARS-CoV-2 antibodies present in sera and nasal swabs, in comparison with a D614G reference virus. We established a novel rapid neutralization assay, based on reporter cells that become GFP+ after overnight infection. B.1.1.7 was neutralized by 79/83 sera from convalescent patients collected up to 9 months post symptoms, almost similar to D614G. There was a mean 6-fold reduction in titers and even loss of activity against B.1.351 in 40% of convalescent sera after 9 months. Early sera from 19 vaccinated individuals were almost as potent against B.1.1.7 but less efficacious against B.1.351, when compared to D614G. Nasal swabs from vaccine recipients were not neutralizing, except in individuals who were diagnosed COVID-19+ before vaccination. Thus, faster-spreading variants acquired a partial resistance to humoral immunity generated by natural infection or vaccination, mostly visible in individuals with low antibody levels.

Nasopharyngeal and serological anti SARS-CoV-2 IgG/IgA responses in COVID-19 patients. (preprint)

BackgroundThe systemic antibody responses to SARS-CoV-2 in COVID-19 patients has been extensively studied. However, much less is known about the mucosal responses in the upper airways at the site of initial SARS-CoV-2 replication. Local antibody responses in the nasopharyngeal epithelium, that are likely to determine the course of infection, have not been analysed so far nor their correlation with antibody responses in serum. MethodsThe IgG and IgA antibody responses were analysed in the plasma as well as in nasopharyngeal swabs (NPS) from the first four COVID-19 patients confirmed by RT-qPCR in France. Two were pauci-symptomatic while two developed severe disease. Taking advantage of a comprehensive series of plasma and nasopharyngeal samples, we characterized their antibody profiles from the second week post symptoms onset, by using an in-house ELISA to detect anti-SARS-CoV-2 Nucleoprotein (N) IgG and IgA. ResultsAnti-N IgG and IgA antibodies were detected in the NPS of severe patients. Overall, the levels of IgA and IgG antibodies in plasma and NPS appeared specific to each patient. ConclusionsAnti-N IgG and IgA antibodies are detected in NPS, and their levels are related to antibody levels in plasma. The two patients with severe disease exhibited different antibody profiles that may reflect different disease outcome. For the pauci-symptomatic patients, one showed a low anti-N IgG and IgA response in the plasma only, while the other one did not exhibit overt serological response.

Absence of association between 2019–20 influenza vaccination and COVID-19: results of the European I-MOVE-COVID-19 primary care project, March–August 2020 (preprint)

Background: Claims of influenza vaccination increasing COVID-19 risk are circulating. Within the I-MOVE-COVID-19 primary care multicentre study, we measured the association between 2019–20 influenza vaccination and COVID-19. Methods We conducted a multicentre test-negative case-control study at primary care level, in study sites in five European countries, from March–August 2020. Patients presenting with acute respiratory infection were swabbed, with demographic, 2019–20 influenza vaccination and clinical information documented. Using logistic regression we measured the adjusted odds ratio (aOR), adjusting for study site and age, sex, calendar time, presence of chronic conditions. The main analysis included patients swabbed ≤7 days after onset from the three countries with <15% of missing influenza vaccination. In secondary analyses, we included five countries, using multiple imputation with chained equations to account for missing data. Results We included 257 COVID-19 cases and 1631 controls in the main analysis (three countries). The overall aOR between influenza vaccination and COVID-19 was 0.93 (95% CI: 0.66–1.32). The aOR was 0.92 (95% CI: 0.58–1.46) and 0.92 (95%CI: 0.51–1.67) among those aged 20–59 and ≥60 years, respectively. In secondary analyses, we included 6457 cases and 69272 controls. The imputed aOR was 0.87 (95% CI: 0.79–0.95) among all ages and any delay between swab and symptom onset. Conclusions There was no evidence that COVID-19 cases were more likely to be vaccinated against influenza than controls. Influenza vaccination should be encouraged among target groups for vaccination. I-MOVE-COVID-19 will continue documenting influenza vaccination status in 2020-21, in order to learn about effects of recent influenza vaccination.

Highly performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification. (preprint)

In order to respond to the urgent request of massive testing, developed countries perform nucleic acid amplification tests (NAAT) of SARS-CoV-2 in centralized laboratories. Real-time RT - PCR (Reverse transcription - Polymerase Chain Reaction) is used to amplify the viral RNA and enable its detection. Although PCR is 37 years old, it is still considered, without dispute, as the gold standard. PCR is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings. In the present work, by harnessing progress made in the past two decades in DNA amplification, microfluidics and membrane technologies, we succeeded to create a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT - LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or highly fluorescent probes. Depending on the viral load, the detection takes between twenty minutes and one hour. Using pools of naso-pharyngal clinical samples, we estimated a sensitivity comparable to RT-qPCR (up to a Cycle threshold of 39, equivalent to <0.1 TCID50 per mL) and a 100% specificity, for other human coronaviruses and eight respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called "COVIDISC" to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment paves the way towards a large dissemination of this device. The perspective of a reliable SARS-CoV-2 point of care detection, highly performing, that would deliver on-site results in less than one hour, with a self-testing potential, opens up a new efficient approach to manage the pandemics.

Viral dynamic modeling of SARS-CoV-2 in non-human primates (preprint)

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 infected with 106 pfu of SARS-CoV-2 for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large daily viral production (>104 virus) and a within-host reproductive basic number of 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly cleared with a half-life of 9 hours, with no significant association between cytokine elevation and clearance. Translating our model to the context of human-to-human infection, human mild infection may be characterized by a peak occurring 4 days after infection, a viral shedding of ~11 days and a generation time of 4 days. These results improve the understanding of SARS-CoV-2 viral replication and better understand the infection to SARS-CoV-2 in humans.

Early phylodynamics analysis of the COVID-19 epidemics in France (preprint)

France was one of the first countries to be reached by the COVID-19 pandemic. Here, we analyse 196 SARS-Cov-2 genomes collected between Jan 24 and Mar 24 2020, and perform a phylodynamics analysis. In particular, we analyse the doubling time, reproduction number (Rt) and infection duration associated with the epidemic wave that was detected in incidence data starting from Feb 27. Different models suggest a slowing down of the epidemic in Mar, which would be consistent with the implementation of the national lock-down on Mar 17. The inferred distributions for the effective infection duration and Rt are in line with those estimated from contact tracing data. Finally, based on the available sequence data, we estimate that the French epidemic wave originated between mid-Jan and early Feb. Overall, this analysis shows the potential to use sequence genomic data to inform public health decisions in an epidemic crisis context and calls for further analyses with denser sampling.

Delayed Laboratory Response to COVID-19 Caused by Molecular Diagnostic Contamination

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created an exceptional situation in which numerous laboratories in Europe simultaneously implemented SARS-CoV-2 diagnostics. These laboratories reported in February 2020 that commercial primer and probe batches for SARS-CoV-2 detection were contaminated with synthetic control material, causing delays of regional testing roll-out in various countries.

Hydroxychloroquine in the treatment and prophylaxis of SARS-CoV-2 infection in non-human primates (preprint)

COVID-19 has become a pandemic that has caused over 200,000 deaths worldwide, with no antiviral drug or vaccine yet available. 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 worldwide but definitive evidence for HCQ efficacy in treatment of COVID-19 is still missing.We evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in monkey African green monkey kidney (VeroE6) cells but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to placebo, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor HCQ+AZTH showed a significant effect on the viral load levels in any of the tested compartments. When the drug was used as a pre-exposure prophylaxis (PrEP), HCQ did not confer protection against acquisition of infection.Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral treatment for COVID-19 in humans.

Introductions and early spread of SARS-CoV-2 in France (preprint)

Following the emergence of coronavirus disease (COVID-19) in Wuhan, China in December 2019, specific COVID-19 surveillance was launched in France on January 10, 2020. Two weeks later, the first three imported cases of COVID-19 into Europe were diagnosed in France. We sequenced 97 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from samples collected between January 24 and March 24, 2020 from infected patients in France. Phylogenetic analysis identified several early independent SARS-CoV-2 introductions without local transmission, highlighting the efficacy of the measures taken to prevent virus spread from symptomatic cases. In parallel, our genomic data reveals the later predominant circulation of a major clade in many French regions, and implies local circulation of the virus in undocumented infections prior to the wave of COVID-19 cases. This study emphasizes the importance of continuous and geographically broad genomic sequencing and calls for further efforts with inclusion of asymptomatic infections.

SARS-CoV-2 serological analysis of COVID-19 hospitalized patients, pauci-symptomatic individuals and blood donors. (preprint)

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their antibody response profile. Here, we performed a pilot study to assess the levels of anti-SARS-CoV-2 antibodies in samples taken from 491 pre- epidemic individuals, 51 patients from Hopital Bichat (Paris), 209 pauci-symptomatic individuals in the French Oise region and 200 contemporary Oise blood donors. Two in-house ELISA assays, that recognize the full-length nucleoprotein (N) or trimeric Spike (S) ectodomain were implemented. We also developed two novel assays: the S-Flow assay, which is based on the recognition of S at the cell surface by flow-cytometry, and the LIPS assay that recognizes diverse antigens (including S1 or N C- terminal domain) by immunoprecipitation. Overall, the results obtained with the four assays were similar, with differences in sensitivity that can be attributed to the technique and the antigen in use. High antibody titers were associated with neutralisation activity, assessed using infectious SARS-CoV- 2 or lentiviral-S pseudotypes. In hospitalized patients, seroconversion and neutralisation occurred on 5-14 days post symptom onset, confirming previous studies. Seropositivity was detected in 29% of pauci-symptomatic individuals within 15 days post-symptoms and 3 % of blood of healthy donors collected in the area of a cluster of COVID cases. Altogether, our assays allow for a broad evaluation of SARS-CoV2 seroprevalence and antibody profiling in different population subsets.

Absence of SARS-CoV-2 infection in cats and dogs in close contact with a cluster of COVID-19 patients in a veterinary campus (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which originated in Wuhan, China, in 2019, is responsible for the COVID-19 pandemic. It is now accepted that the wild fauna, probably bats, constitute the initial reservoir of the virus, but little is known about the role pets can play in the spread of the disease in human communities, knowing the ability of SARS-CoV-2 to infect some domestic animals. We tested 21 domestic pets (9 cats and 12 dogs) living in close contact with their owners (belonging to a veterinary community of 20 students) in which two students tested positive for COVID-19 and several others (n = 11/18) consecutively showed clinical signs (fever, cough, anosmia, etc.) compatible with COVID-19 infection. Although a few pets presented many clinical signs indicative for a coronavirus infection, no animal tested positive for SARS-CoV-2 by RT-PCR and no antibodies against SARS-CoV-2 were detectable in their blood using an immunoprecipitation assay. These original data can serve a better evaluation of the host range of SARS-CoV-2 in natural environment exposure conditions.