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 1,362 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 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.

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 a single patients sample was analyzed, and the virus was not isolated. Here, we re-port the gradual creation 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 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. 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 infec-tion 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 patients follow-up. This case exemplifies the recombination process of SARS-CoV-2 in real life, and it calls for intensifying genomic surveillance in patients coinfected with different SARS-CoV-2 variants, and more gener-ally with several RNA viruses, as this may lead to the creation of new viruses.

Culture and identification of a Deltamicron SARS-CoV-2 in a three cases cluster in southern France

Multiple SARS-CoV-2 variants have successively, or concommitantly spread worldwide since summer 2020. A few co-infections with different variants were reported and genetic recombinations, common among coronaviruses, were reported or suspected based on co-detection of signature mutations of different variants in a given genome. Here we report three infections in southern France with a Delta 21J/AY.4-Omicron 21K/BA.1 "Deltamicron" recombinant. The hybrid genome harbors signature mutations of the two lineages, supported by a mean sequencing depth of 1,163-1,421 reads and mean nucleotide diversity of 0.1-0.6%. It is composed of the near full-length spike gene (from codons 156-179) of an Omicron 21K/BA.1 variant in a Delta 21J/AY.4 lineage backbone. Importantly, we cultured an isolate of this recombinant and sequenced its genome. It was observed by scanning electron microscopy. As it is misidentified with current variant screening qPCR, we designed and implemented for routine diagnosis a specific duplex qPCR. Finally, structural analysis of the recombinant spike suggested its hybrid content could optimize viral binding to the host cell membrane. These findings prompt further studies of the virological, epidemiological, and clinical features of this recombinant.

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

The nature and dynamics of mutations associated with the emergence, spread and vanishing of SARS-CoV-2 variants causing successive waves are complex1-5. We determined the kinetics of the most common French variant ("Marseille-4") for 10 months since its onset in July 20205. Here, we analysed and classified into subvariants and lineages 7,453 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. All the lineages exhibited a "gamma" 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 several mink lineages 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.

Analysis of SARS-CoV-2 variants from 24,181 patients exemplifies the role of globalisation 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 Mediterranee 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 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 analysing 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 mink farms. 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.

Implementation of an in-house real-time reverse transcription-PCR assay to detect the emerging SARS-CoV-2 N501Y variants

The real-time detection of emerging SARS-CoV-2 variants is critical to manage patients appropriately, and monitor and assess their epidemiological and clinical features. Sequencing is not a feasible comprehensive detection strategy considering the very large number of SARS-CoV-2 cases in our current setting. SARS-CoV-2 variants currently of greatest concern carry the N501Y substitution within the spike receptor binding domain. They have become predominant in England (20I/501Y.V1) and were detected in South Africa (20H/501Y.V2), Brazil and dozens of other countries worldwide. The 20I/501Y.V1 variant has started to spread worldwide including in France. It has been reported as 50-74% more transmissible than preexisting strains, suspected to evade anti-spike antibodies, and it caused a reinfection. We have implemented an in-house one-step real-time reverse transcription-PCR (qPCR) assay that specifically detects SARS-CoV-2 N501Y. It was found reliable to detect specifically the N501Y substitution and preliminarily allowed estimating 20I/501Y.V1 variant prevalence to 4% among our current SARS-CoV-2 diagnoses since January.

Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant

IntroductionThe SARS-CoV-2 pandemic has been associated with the occurrence since summer 2020 of several viral variants that overlapped or succeeded each other in time. Those of current concern harbor mutations within the spike receptor binding domain (RBD) that may be associated with viral escape to immune responses. In our geographical area a viral variant we named Marseille-4 harbors a S477N substitution in this RBD. Materials and methodsWe aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay with a hydrolysis probe that specifically detects the SARS-CoV-2 Marseille-4 variant. ResultsAll 6 cDNA samples from Marseille-4 variant strains identified in our institute by genome next-generation sequencing (NGS) tested positive using our Marseille-4 specific qPCR, whereas all 32 cDNA samples from other variants tested negative. In addition, 39/42 (93%) respiratory samples identified by NGS as containing a Marseille-4 variant strain and 0/26 samples identified as containing non-Marseille-4 variant strains were positive. Finally, 1,585/2,889 patients SARS-CoV-2-diagnosed in our institute, 10/277 (3.6%) respiratory samples collected in Algeria, and none of 207 respiratory samples collected in Senegal, Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR. DiscussionOur in-house qPCR system was found reliable to detect specifically the Marseille-4 variant and allowed estimating it is involved in more than half of our SARS-CoV-2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical characterics based on comprehensive sets of data.

Introduction into the Marseille geographical area of a mild SARS-CoV-2 variant originating from sub-Saharan Africa

BACKGROUNDIn Marseille, France, the COVID-19 incidence evolved unusually with several successive epidemic episodes. The second outbreak started in July, was associated with North Africa, and involved travelers and an outbreak on passenger ships. This suggested the involvement of a new viral variant. METHODSWe sequenced the genomes from 916 SARS-CoV-2 strains from COVID-19 patients in our institute. The patients demographic and clinical features were compared according to the infecting viral variant. RESULTSFrom June 26th to August 14th, we identified a new viral variant (Marseille-1). Based on genome sequences (n=89) or specific qPCR (n=53), 142 patients infected with this variant were detected. It is characterized by a combination of 10 mutations located in the nsp2, nsp3, nsp12, S, ORF3a, ORF8 and N/ORF14 genes. We identified Senegal and Gambia, where the virus had been transferred from China and Europe in February-April as the sources of the Marseille-1 variant, which then most likely reached Marseille through Maghreb when French borders reopened. In France, this variant apparently remained almost limited to Marseille. In addition, it was significantly associated with a milder disease compared to clade 20A ancestor strains. CONCLUSIONOur results demonstrate that SARS-CoV-2 can genetically diversify rapidly, its variants can diffuse internationally and cause successive outbreaks.