Protection from successive Omicron variants with SARS-CoV-2 vaccine and monoclonal antibodies in kidney transplant recipients.

Introduction: Kidney transplant recipients (KTRs) are at high risk of severe COVID-19, even when they are fully vaccinated. Additional booster vaccinations or passive immunization with prophylactic monoclonal antibodies are recommended to increase their protection against severe COVID-19. Methods: Here, we describe the neutralization of SARS-CoV-2 Delta, Omicron BA.1, BA.2, BA.4, and BA.5 variants, firstly by 39 serum samples from vaccinated KTRs exhibiting anti-spike antibody concentrations ≥264 binding antibody units (BAU)/mL and, secondly, by tixagevimab/cilgavimab. Results: No neutralization was observed for 18% of the KTRs, while serum from only 46% of patients could neutralize the five variants. Cross-neutralization of the Delta and Omicron variants occurred for 65-87% of sera samples. The anti-spike antibody concentration correlated with neutralization activity for all the variants. The neutralization titers against the Delta variant were higher in vaccinated KTRs who had previously presented with COVID-19, compared to those KTRs who had only been vaccinated. Breakthrough infections occurred in 39% of the KTRs after the study. Tixagevimab/cilgavimab poorly neutralizes Omicron variants, particularly BA.5, and does not neutralize BQ.1, which is currently the most prevalent strain. Discussion: As a result, sera from seropositive vaccinated KTRs had poor neutralization of the successive Omicron variants. Several Omicron variants are able to escape tixagevimab/cilgavimab.

Correlation of Redondovirus and Entamoeba gingivalis Detections in the Human Oral Cavity Suggests That This Amoeba Is Possibly the Redondovirus Host.

The virome of the human oral cavity and the relationships between viruses and diseases such as periodontitis are scarcely deciphered. Redondoviruses were reported in the human oral cavity in 2019, including in periodontitis patients. Here, we aimed at detecting redondoviruses and at searching for a potential viral host in human saliva. Non-stimulated saliva was collected between December 2020 and June 2021. These samples were tested using real-time PCR regarding the presence of redondovirus and Entamoeba gingivalis DNA. Similarity searches were performed using BLAST against eukaryotic and prokaryotic sequences from GenBank. The redondovirus DNA was detected in 46% of the 28 human saliva samples. In addition, short fragments of redondovirus genomes were detected in silico within Entamoeba sequences. Finally, Entamoeba gingivalis DNA was detected in 46% of the 28 saliva samples, with a strong correlation between redondovirus DNA and E. gingivalis DNA detections, in 93% of the cases. Regarded together, these findings and previous ones strongly support the presence of redondoviruses in the human oral cavity and their association to E. gingivalis as their likely host.

Presence of SARS-CoV-2 in a Cornea Transplant.

BACKGROUND: The SARS-CoV-2 pandemic has impacted tissue transplantation procedures since conjunctivas were found to be associated with coronavirus infection. Here, we investigated infection of a cornea graft from a COVID-19-positive donor. METHODS: In order to evaluate the presence of SARS-CoV-2 in the cornea graft we first carried out a qRT-PCR and then we investigated the presence of SARS-CoV-2 by fluorescence and electron microscopy. CONCLUSIONS: Although the cornea graft was found to be negative by qRT-PCR, we were able to show the presence of SARS-CoV-2 in corneal cells expressing the SARS-CoV-2 receptor, ACE2. Taken together, our findings may have important implications for the use of corneal tissue in graft indications and open the debate on SARS-CoV-2 transmissibility.

Antimalarial drugs inhibit the replication of SARS-CoV-2: An in vitro evaluation.

In December 2019, a new severe acute respiratory syndrome coronavirus (SARS-CoV-2) causing coronavirus diseases 2019 (COVID-19) emerged in Wuhan, China. African countries see slower dynamic of COVID-19 cases and deaths. One of the assumptions that may explain this later emergence in Africa, and more particularly in malaria endemic areas, would be the use of antimalarial drugs. We investigated the in vitro antiviral activity against SARS-CoV-2 of several antimalarial drugs. Chloroquine (EC50 = 2.1 µM and EC90 = 3.8 µM), hydroxychloroquine (EC50 = 1.5 µM and EC90 = 3.0 µM), ferroquine (EC50 = 1.5 µM and EC90 = 2.4 µM), desethylamodiaquine (EC50 = 0.52 µM and EC90 = 1.9 µM), mefloquine (EC50 = 1.8 µM and EC90 = 8.1 µM), pyronaridine (EC50 = 0.72 µM and EC90 = 0.75 µM) and quinine (EC50 = 10.7 µM and EC90 = 38.8 µM) showed in vitro antiviral effective activity with IC50 and IC90 compatible with drug oral uptake at doses commonly administered in malaria treatment. The ratio Clung/EC90 ranged from 5 to 59. Lumefantrine, piperaquine and dihydroartemisinin had IC50 and IC90 too high to be compatible with expected plasma concentrations (ratio Cmax/EC90 < 0.05). Based on our results, we would expect that countries which commonly use artesunate-amodiaquine or artesunate-mefloquine report fewer cases and deaths than those using artemether-lumefantrine or dihydroartemisinin-piperaquine. It could be necessary now to compare the antimalarial use and the dynamics of COVID-19 country by country to confirm this hypothesis.

The emergence, spread and vanishing of a French SARS-CoV-2 variant exemplifies the fate of RNA virus epidemics and obeys the Mistigri rule.

The nature and dynamics of mutations associated with the emergence, spread, and vanishing of SARS-CoV-2 variants causing successive waves are complex. We determined the kinetics of the most common French variant ("Marseille-4") for 10 months since its onset in July 2020. Here, we analyzed and classified into subvariants and lineages 7453 genomes obtained by next-generation sequencing. We identified two subvariants, Marseille-4A, which contains 22 different lineages of at least 50 genomes, and Marseille-4B. Their average lifetime was 4.1 ± 1.4 months, during which 4.1 ± 2.6 mutations accumulated. Growth rate was 0.079 ± 0.045, varying from 0.010 to 0.173. Most of the lineages exhibited a bell-shaped distribution. Several beneficial mutations at unpredicted sites initiated a new outbreak, while the accumulation of other mutations resulted in more viral heterogenicity, increased diversity and vanishing of the lineages. Marseille-4B emerged when the other Marseille-4 lineages vanished. Its ORF8 gene was knocked out by a stop codon, as reported in SARS-CoV-2 of mink and in the Alpha variant. This subvariant was associated with increased hospitalization and death rates, suggesting that ORF8 is a nonvirulence gene. We speculate that the observed heterogenicity of a lineage may predict the end of the outbreak.

Simple Wastewater Preparation Protocol Applied to Monitor the Emergence of the Omicron 21L/BA.2 Variant by Genome Sequencing.

Detecting and monitoring viruses in wastewater samples have been reported as useful ways of tracking SARS-CoV-2 epidemic trends. However, there is currently no unanimously recognised method of processing samples to identify and quantify SARS-CoV-2 variants in wastewater. We aimed to implement a method that was as simple as possible in order to be used universally. In a study performed between January 2022 and June 2022 in the city of Marseille, France, we first evaluated the impact of the sample preservation strategy. We then compared ultracentrifugation to ultrafiltration and several steps of filtration to determine the optimal approach for virus concentration. As a proof-of-concept, the definitive protocol was applied to next-generation sequencing of SARS-CoV-2 in wastewater to monitor the emergence of the Omicron variant in the city. For sewage water to be processed in the week following the sampling, storage at +4 °C is sufficient, with less than 1 Ct loss. Filtration with a 5 µm syringe filter, then with a 0.8 µm filtration unit, followed by ultrafiltration was the optimal protocol, leading to an average increase of 3.24 Ct when the starting Ct was on average 38 in the wastewater. This made it possible to observe the emergence of the Omicron 21L/BA.2 variant after Omicron 21K/BA.1 by genome sequencing over a period ranging from 20 February to 10 April 2022 in agreement with observations based on patient data. To conclude, by using a simple method requiring only basic filters and a centrifuge as equipment, it is possible to accurately track the relative incidence rates and the emergence of SARS-CoV-2 variants based on sewage samples.

Macrophages and γδ T cells interplay during SARS-CoV-2 variants infection.

Introduction: The emergence of several SARS-CoV-2 variants during the COVID pandemic has revealed the impact of variant diversity on viral infectivity and host immune responses. While antibodies and CD8 T cells are essential to clear viral infection, the protective role of innate immunity including macrophages has been recognized. The aims of our study were to compare the infectivity of different SARS-CoV-2 variants in monocyte-derived macrophages (MDM) and to assess their activation profiles and the role of ACE2 (Angiotensin-converting enzyme 2), the main SARS-CoV-2 receptor. We also studied the ability of macrophages infected to affect other immune cells such as γδ2 T cells, another partner of innate immune response to viral infections. Results: We showed that the SARS-CoV-2 variants α-B.1.1.7 (United Kingdom), ß-B.1.351 (South Africa), γ-P.1 (Brazil), δ-B.1.617 (India) and B.1.1.529 (Omicron), infected MDM without replication, the γ-Brazil variant exhibiting increased infectivity for MDM. No clear polarization profile of SARS-CoV-2 variants-infected MDM was observed. The ß-B.1.351 (South Africa) variant induced macrophage activation while B.1.1.529 (Omicron) was rather inhibitory. We observed that SARS-CoV-2 variants modulated ACE2 expression in MDM. In particular, the ß-B.1.351 (South Africa) variant induced a higher expression of ACE2, related to MDM activation. Finally, all variants were able to activate γδ2 cells among which γ-P.1 (Brazil) and ß-B.1.351 (South Africa) variants were the most efficient. Conclusion: Our data show that SARS-CoV-2 variants can infect MDM and modulate their activation, which was correlated with the ACE2 expression. They also affect γδ2 T cell activation. The macrophage response to SARS-CoV-2 variants was stereotypical.

Quasispecies Analysis of SARS-CoV-2 of 15 Different Lineages during the First Year of the Pandemic Prompts Scratching under the Surface of Consensus Genome Sequences.

The tremendous majority of SARS-CoV-2 genomic data so far neglected intra-host genetic diversity. Here, we studied SARS-CoV-2 quasispecies based on data generated by next-generation sequencing (NGS) of complete genomes. SARS-CoV-2 raw NGS data had been generated for nasopharyngeal samples collected between March 2020 and February 2021 by the Illumina technology on a MiSeq instrument, without prior PCR amplification. To analyze viral quasispecies, we designed and implemented an in-house Excel file ("QuasiS") that can characterize intra-sample nucleotide diversity along the genomes using data of the mapping of NGS reads. We compared intra-sample genetic diversity and global genetic diversity available from Nextstrain. Hierarchical clustering of all samples based on the intra-sample genetic diversity was performed and visualized with the Morpheus web application. NGS mapping data from 110 SARS-CoV-2-positive respiratory samples characterized by a mean depth of 169 NGS reads/nucleotide position and for which consensus genomes that had been obtained were classified into 15 viral lineages were analyzed. Mean intra-sample nucleotide diversity was 0.21 ± 0.65%, and 5357 positions (17.9%) exhibited significant (>4%) diversity, in ≥2 genomes for 1730 (5.8%) of them. ORF10, spike, and N genes had the highest number of positions exhibiting diversity (0.56%, 0.34%, and 0.24%, respectively). Nine hot spots of intra-sample diversity were identified in the SARS-CoV-2 NSP6, NSP12, ORF8, and N genes. Hierarchical clustering delineated a set of six genomes of different lineages characterized by 920 positions exhibiting intra-sample diversity. In addition, 118 nucleotide positions (0.4%) exhibited diversity at both intra- and inter-patient levels. Overall, the present study illustrates that the SARS-CoV-2 consensus genome sequences are only an incomplete and imperfect representation of the entire viral population infecting a patient, and that quasispecies analysis may allow deciphering more accurately the viral evolutionary pathways.

Microscopic observations of SARS-CoV-2 like particles in different oral samples.

The emerging coronavirus pneumonia epidemic caused by the SARS-CoV-2 infection has spread rapidly around the world. The main routes of transmission of SARS-CoV-2 are currently recognised as aerosol/droplet inhalation. However, the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly known. The current data indicates the presence of viral RNA in oral samples, suggesting the implication of saliva in SARS-CoV-2 transmission, however, no direct observation of SARS-CoV-2 particles in different oral samples has been reported. In this study, we investigated whether particles of SARS-CoV-2 were present in oral samples collected from three symptomatic COVID-19 patients. Using scanning electron microscopy (SEM), the correlative strategy of light microscopy and electron microscopy and immunofluorescence staining, we showed the presence of SARS-like particles in RT-qPCR SARS-CoV-2-positive saliva, dental plaque and gingival crevicular fluid (GCF) samples. In the saliva samples, we demonstrated the presence of epithelial oral cells with morphogenetic features of SARS-CoV-2 infected cells. Inside those cells, vacuoles filled with nascent particles were observed, suggesting the potential infection and replication of SARS-CoV-2 in oral tissues. Our results corroborate previous studies and confirm that the oral cavity may be a potential niche for SARS-CoV-2 infection and a potential source of transmission.

Matrix-Encoding Gene Diversity of 624 Influenza A/H3N2 Genomes Does Not Show Association with Impaired Viral Detection by Commercialized qPCR Assays.

As for the case of SARS-CoV-2, genome sequencing of influenza viruses is of potential interest to raise and address virological issues. Recently, false-negativity of real-time reverse transcription-PCR (qPCR) assays that detect influenza A/H3N2 virus RNA were reported and associated with two mutations (A37T and C161T) in the Matrix-encoding (M1) gene located on viral segment 7. This triggered a national alert in France. The present study sought to assess the association between the presence of these mutations and potential false negative results of influenza A/H3N2 virus RNA detection by commercialized qPCR assays at the clinical virology laboratory of our university hospitals in southern France. This study focused on the genetic diversity in the M1 gene and segment 7 of 624 influenza A/H3N2 virus genomes obtained from respiratory samples having tested qPCR-positive with M1 gene-targeting assays in our clinical virology laboratory. A total of 585 among the 624 influenza A/H3N2 virus genomes (93.7%) were of clade 3C.2a1b.2a.2, and 39 (6.3%) were of clade 3C.2a1b.1a. M1 gene substitutions A37T and C161T were both present in 582 (93.3%) genomes, only of clade 3C.2a1b.2a.2. Substitution A37T was present in 621 (99.5%) genomes. Substitution C161T was present in 585 genomes (93.8%), all of clade 3C.2a1b.2a.2. Moreover, 21 other nucleotide positions were mutated in ≥90% of the genomes. The present study shows that A37T/C and C161T mutations, and other mutations in the M1 gene and segment 7, were widely present in influenza A/H3N2 virus genomes recovered from respiratory samples diagnosed qPCR-positive with commercialized assays.

First cases of infection with the 21L/BA.2 Omicron variant in Marseille, France.

The SARS-CoV-2 21K/BA.1, 21L/BA.2, and BA.3 Omicron variants have recently emerged worldwide. To date, the 21L/BA.2 Omicron variant has remained very minority globally but became predominant in Denmark instead of the 21K/BA.1 variant. Here, we describe the first cases diagnosed with this variant in south-eastern France. We identified 13 cases using variant-specific qPCR and next-generation sequencing between 28/11/2021 and 31/01/2022, the first two cases being diagnosed in travelers returning from Tanzania. Overall, viral genomes displayed a mean (±standard deviation) number of 65.9 ± 2.5 (range, 61-69) nucleotide substitutions and 31.0 ± 8.3 (27-50) nucleotide deletions, resulting in 49.6 ± 2.2 (45-52) amino acid substitutions (including 28 in the spike protein) and 12.4 ± 1.1 (12-15) amino acid deletions. Phylogeny showed the distribution in three different clusters of these genomes, which were most closely related to genomes from England and South Africa, from Singapore and Nepal, or from France and Denmark. Structural predictions highlighted a significant enlargement and flattening of the surface of the 21L/BA.2 N-terminal domain of the spike protein compared to that of the 21K/BA.1 Omicron variant, which may facilitate initial viral interactions with lipid rafts. Close surveillance is needed at global, country, and center scales to monitor the incidence and clinical outcome of the 21L/BA.2 Omicron variant.

Choosing a cellular model to study SARS-CoV-2.

As new pathogens emerge, new challenges must be faced. This is no different in infectious disease research, where identifying the best tools available in laboratories to conduct an investigation can, at least initially, be particularly complicated. However, in the context of an emerging virus, such as SARS-CoV-2, which was recently detected in China and has become a global threat to healthcare systems, developing models of infection and pathogenesis is urgently required. Cell-based approaches are crucial to understanding coronavirus infection biology, growth kinetics, and tropism. Usually, laboratory cell lines are the first line in experimental models to study viral pathogenicity and perform assays aimed at screening antiviral compounds which are efficient at blocking the replication of emerging viruses, saving time and resources, reducing the use of experimental animals. However, determining the ideal cell type can be challenging, especially when several researchers have to adapt their studies to specific requirements. This review strives to guide scientists who are venturing into studying SARS-CoV-2 and help them choose the right cellular models. It revisits basic concepts of virology and presents the currently available in vitro models, their advantages and disadvantages, and the known consequences of each choice.

SARS-CoV-2 quasi-species analysis from patients with persistent nasopharyngeal shedding.

At the time of a new and unprecedented viral pandemic, many questions are being asked about the genomic evolution of SARS-CoV-2 and the emergence of different variants, leading to therapeutic and immune evasion and survival of this genetically highly labile RNA virus. The nasopharyngeal persistence of infectious virus beyond 17 days proves its constant interaction with the human immune system and increases the intra-individual mutational possibilities. We performed a prospective high-throughput sequencing study (ARTIC Nanopore) of SARS-CoV-2 from so-called "persistent" patients, comparing them with a non-persistent population, and analyzing the quasi-species present in a single sample at time t. Global intra-individual variability in persistent patients was found to be higher than in controls (mean 5.3%, Standard deviation 0.9 versus 4.6% SD 0.3, respectively, p < 0.001). In the detailed analysis, we found a greater difference between persistent and non-persistent patients with non-severe COVID 19, and between the two groups infected with clade 20A. Furthermore, we found minority N501Y and P681H mutation clouds in all patients, with no significant differences found both groups. The question of the SARS-CoV-2 viral variants' genesis remains to be further investigated, with the need to prevent new viral propagations and their consequences, and quasi-species analysis could be an important key to watch out.

Limited permissibility of ENL-R and Mv-1-Lu mink cell lines to SARS-CoV-2.

The SARS-CoV-2 pandemic started in the end of 2019 in Wuhan, China, which highlighted the scenario of frequent cross-species transmission events. From the outbreak possibly initiated by viral spill-over into humans from an animal reservoir, now we face the human host moving globally while interacting with domesticated and peridomestic animals. The emergence of a new virus into the ecosystem leads to selecting forces and species-specific adaptations. The adaptation of SARS-CoV-2 to other animals represents a risk to controlling the dissemination of this coronavirus and the emergence of new variants. Since 2020, several mink farms in Europe and the United States have had SARS-CoV-2 outbreaks with human-mink and mink-human transmission, where the mink-selected variants possibly hold evolutionary concerning advantages. Here we investigated the permissibility of mink lung-derived cells using two cell lines, Mv-1-Lu and ENL-R, against several lineages of SARS-CoV-2, including some classified as variants of concern. The viral release rate and the infectious titers indicate that these cells support infections by different SARS-CoV-2 lineages. The viral production occurs in the first few days after infection with the low viral release by these mink cells, which is often absent for the omicron variant for lung cells. The electron microscopy reveals that during the viral replication cycle, the endomembrane system of the mink-host cell undergoes typical changes while the viral particles are produced, especially in the first days of infection. Therefore, even if limited, mink lung cells may represent a selecting source for SARS-CoV-2 variants, impacting their transmissibility and pathogenicity and making it difficult to control this new coronavirus.

Concentration of SARS-CoV-2-Infected Cell Culture Supernatants for Detection of Virus-like Particles by Scanning Electron Microscopy.

There is currently a need for new rapid viral diagnostic electron microscopy methods. Although the gold standard remains the transmission electron microscopy (TEM) negative staining method for electron microscopic examination of samples containing a virus, difficulties can arise when the virus particle content of the sample that has to be examined is poor. Such samples include supernatants of virus-infected cells that can be difficult to examine, as sometimes only a few virus particles are released in the culture medium upon infection. In addition to TEM, scanning electron microscopy (SEM) can also be used for visualizing virus particles. One advantage of SEM over TEM is its ability to rapidly screen several large specimens, such as microscopy slides. In this study, we investigated this possibility and tested different coating molecules as well as the effect of centrifugation for analyzing SARS-CoV-2-virus-infected cell culture supernatants deposited on microscopy glass slides by SEM. We found that centrifugation of 25XConcanavalinA-coated microscopy glass slides in shell vials provided an improved method for concentrating SARS-CoV-2-virus-infected cell supernatants for virus-like particle detection by SEM.

First Detection of the SARS-CoV-2 Omicron BA.5/22B in Monaco.

The Omicron BA.5/22B variant has been designated as a "variant of concern" by the World Health Organization. We describe, here, the first evidence in Monaco of infection with an Omicron BA.5/22B variant, probably imported from the Republic of Seychelles, harboring a rare combination of non-BA.5/22B signature amino acid changes. SARS-CoV-2 neutralizing antibodies were measured with a surrogate virus neutralization test. SARS-CoV-2 genotype screening was performed on nasopharyngeal samples with a multiplex qPCR assay. The SARS-CoV-2 genome was obtained by next-generation sequencing with the Illumina COVID-seq protocol, then assembly using bioinformatics pipelines and software was performed. The BA.5/22B spike protein structure was obtained by molecular modeling. Two spouses were SARS-CoV-2-diagnosed the day they returned from a one-week trip in the Republic of Seychelles. SARS-CoV-2 qPCR screening for variant-specific mutations identified an Omicron variant BA.1/21K, BA.4/22A, or BA.5/22B. A SARS-Co-2 BA.5/22B variant genome was recovered from one of the spouses. Aside from BA.5/22B-defining amino acid substitutions, four other amino acid changes were encoded including Q556K in ORF1a, K2557R in ORF1b, and A67V and A829T in spike; only 13 genomes in sequence databases harbored these four mutations concurrently. Structural analysis of this BA.5/22B variant predicted that A829T in spike may result in a compaction that may affect conformational plasticity. Overall, our findings warrant performing genome-based genotypic surveillance to survey accurately the emergence and circulation of SARS-CoV-2 variants worldwide and point out that their first occurrence in a country is often through international travel despite implemented countermeasures.

A 21L/BA.2-21K/BA.1 "MixOmicron" SARS-CoV-2 hybrid undetected by qPCR that screen for variant in routine diagnosis.

Among the multiple SARS-CoV-2 variants identified since summer 2020, several have co-circulated, creating opportunities for coinfections and potentially genetic recombinations that are common in coronaviruses. Viral recombinants are indeed beginning to be reported more frequently. Here, we describe a new SARS-CoV-2 recombinant genome that is mostly that of a Omicron 21L/BA.2 variant but with a 3' tip originating from a Omicron 21K/BA.1 variant. Two such genomes were obtained in our institute from adults sampled in February 2022 in university hospitals of Marseille, southern France, by next-generation sequencing carried out with the Illumina or Nanopore technologies. The recombination site was located between nucleotides 26,858-27,382. In the two genomic assemblies, mean sequencing depth at mutation-harboring positions was 271 and 1362 reads and mean prevalence of the majoritary nucleotide was 99.3 ± 2.2% and 98.8 ± 1.6%, respectively. Phylogeny generated trees with slightly different topologies according to whether genomes analyzed were depleted or not of the 3' tip. This 3' terminal end brought in the Omicron 21L/BA.2 genome a short transposable element of 41 nucleotides named S2m that is present in most SARS-CoV-2 except a few variants among which the Omicron 21L/BA.2 variant and may be involved in virulence. Importantly, this recombinant is not detected by currently used qPCR that screen for variants in routine diagnosis. The present observation emphasizes the need to survey closely the genetic pathways of SARS-CoV-2 variability by whole genome sequencing, and it could contribute to gain a better understanding of factors that lead to observed differences between epidemic potentials of the different variants.

SARS-CoV-2 Testing of Aircraft Wastewater Shows That Mandatory Tests and Vaccination Pass before Boarding Did Not Prevent Massive Importation of Omicron Variant into Europe.

BACKGROUND: Most new SARS-CoV-2 epidemics in France occurred following the importation from abroad of emerging viral variants. Currently, the risk of new variants being imported is controlled based on a negative screening test (PCR or antigenic) and proof of up-to-date vaccine status, such as the International Air Transport Association travel pass. METHODS: The wastewater from two planes arriving in Marseille (France) from Addis Ababa (Ethiopia) in December 2021 was tested by RT-PCR to detect SARS-CoV2 and screen for variants. These tests were carried out between landing and customs clearance and were then sequenced by MiSeq Illumina. Antigenic tests and sequencing by NovaSeq were carried out on respiratory samples collected from the 56 passengers on the second flight. RESULTS: SARS-CoV-2 RNA suspected of being from the Omicron BA.1 variant was detected in the aircraft's wastewater. SARS-CoV2 RNA was detected in 11 [20%) passengers and the Omicron BA.1 variant was identified. CONCLUSION: Our work shows the efficiency of aircraft wastewater testing to detect SARS-CoV-2 cases among travellers and to identify the viral genotype. It also highlights the low efficacy of the current control strategy for flights entering France from outside Europe, which combines a requirement to produce a vaccine pass and proof of a negative test before boarding.

Sequential Appearance and Isolation of a SARS-CoV-2 Recombinant between Two Major SARS-CoV-2 Variants in a Chronically Infected Immunocompromised Patient.

Genetic recombination is a major evolutionary mechanism among RNA viruses, and it is common in coronaviruses, including those infecting humans. A few SARS-CoV-2 recombinants have been reported to date whose genome harbored combinations of mutations from different mutants or variants, but only a single patient's sample was analyzed, and the virus was not isolated. Here, we report the gradual emergence of a hybrid genome of B.1.160 and Alpha variants in a lymphoma patient chronically infected for 14 months, and we isolated the recombinant virus. The hybrid genome was obtained by next-generation sequencing, and the recombination sites were confirmed by PCR. This consisted of a parental B.1.160 backbone interspersed with two fragments, including the spike gene, from an Alpha variant. An analysis of seven sequential samples from the patient decoded the recombination steps, including the initial infection with a B.1.160 variant, then a concurrent infection with this variant and an Alpha variant, the generation of hybrid genomes, and eventually the emergence of a predominant recombinant virus isolated at the end of the patient's follow-up. This case exemplifies the recombination process of SARS-CoV-2 in real life, and it calls for intensifying the genomic surveillance in patients coinfected with different SARS-CoV-2 variants, and more generally with several RNA viruses, as this may lead to the appearance of new viruses.

Analysis of SARS-CoV-2 Variants From 24,181 Patients Exemplifies the Role of Globalization and Zoonosis in Pandemics.

After the end of the first epidemic episode of SARS-CoV-2 infections, as cases began to rise again during the summer of 2020, we at IHU Méditerranée Infection in Marseille, France, intensified the genomic surveillance of SARS-CoV-2, and described the first viral variants. In this study, we compared the incidence curves of SARS-CoV-2-associated deaths in different countries and reported the classification of SARS-CoV-2 variants detected in our institute, as well as the kinetics and sources of the infections. We used mortality collected from a COVID-19 data repository for 221 countries. Viral variants were defined based on ≥5 hallmark mutations along the whole genome shared by ≥30 genomes. SARS-CoV-2 genotype was determined for 24,181 patients using next-generation genome and gene sequencing (in 47 and 11% of cases, respectively) or variant-specific qPCR (in 42% of cases). Sixteen variants were identified by analyzing viral genomes from 9,788 SARS-CoV-2-diagnosed patients. Our data show that since the first SARS-CoV-2 epidemic episode in Marseille, importation through travel from abroad was documented for seven of the new variants. In addition, for the B.1.160 variant of Pangolin classification (a.k.a. Marseille-4), we suspect transmission from farm minks. In conclusion, we observed that the successive epidemic peaks of SARS-CoV-2 infections are not linked to rebounds of viral genotypes that are already present but to newly introduced variants. We thus suggest that border control is the best mean of combating this type of introduction, and that intensive control of mink farms is also necessary to prevent the emergence of new variants generated in this animal reservoir.