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

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 were 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. It is similar to those reported for 15 other patients sampled since January 2022 in Europe. 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.

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

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 thirteen cases using variant-specific qPCR and next-generation sequencing between 28/11/2021 and 31/01/2022, the first two cases being diagnosed in travellers 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 pointed out a significant enlargement and flattening of the 21L/BA.2 N-terminal domain surface compared with that of the 21K/BA.2 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.

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 (preprint)

Summary The nature and dynamics of mutations associated with the emergence, spread and vanishing of SARS-CoV-2 variants causing successive waves are complex 1-5 . We determined the kinetics of the most common French variant (“Marseille-4”) for 10 months since its onset in July 2020 5 . 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.

Emergence in Southern France of a new SARS-CoV-2 variant of probably Cameroonian origin harbouring both substitutions N501Y and E484K in the spike protein (preprint)

SARS-CoV-2 variants have become a major virological, epidemiological and clinical concern, particularly with regard to the risk of escape from vaccine-induced immunity. Here we describe the emergence of a new variant. For twelve SARS-CoV-positive patients living in the same geographical area of southeastern France, qPCR testing that screen for variant-associated mutations showed an atypical combination. The index case returned from a travel in Cameroon. The genomes were obtained by next-generation sequencing with Oxford Nanopore Technologies on GridION instruments within approximately 8 h. Their analysis revealed 46 mutations and 37 deletions resulting in 30 amino acid substitutions and 12 deletions. Fourteen amino acid substitutions, including N501Y and E484K, and 9 deletions are located in the spike protein. This genotype pattern led to create a new Pangolin lineage named B.1.640.2, which is a phylogenetic sister group to the old B.1.640 lineage renamed B.1.640.1. Both lineages differ by 25 nucleotide substitutions and 33 deletions. The mutation set and phylogenetic position of the genomes obtained here indicate based on our previous definition a new variant we named 'IHU'. These data are another example of the unpredictability of the emergence of SARS-CoV-2 variants, and of their introduction in a given geographical area from abroad.

Distinct evolution of infection-enhancing and neutralizing epitopes in the spike protein of SARS-CoV-2 variants (from alpha to omicron) : a structural and molecular epidemiology study (preprint)

Infection-enhancing antibodies may limit the efficiency of Covid-19 vaccines. We analyzed the evolution ofneutralizing and facilitating epitopes in 1,860,489 SARS-CoV-2 genomes stored in the Los Alamos databasefrom June to November 2021. The structural dynamics of these epitopes was determined by molecular modelingof the spike protein on a representative panel of SARS-CoV-2 variants. D614, which belongs to an antibody-dependent-enhancement (ADE) epitope common to SARS-CoV-1 and SARS-CoV-2, has mutated to D614G in2020, which could explain why ADE has not been detected following mass vaccination. A second epitopelocated in the N-terminal domain (NTD), specific of SARS-CoV-2, is highly conserved among most variants. Incontrast, the neutralizing epitope of the NTD showed extensive variations in SARS-CoV-2 variants. The balancebetween facilitating and neutralizing antibodies is in favor of neutralization for the Wuhan strain, alpha and betavariants, but not for gamma, delta, lambda, and mu. The recently emerging omicron variant is atypic as itsmutational profiles affects both neutralization and ADE epitopes. Overall, our data reveal that the evolution ofSARS-CoV-2 has dramatically affected the ADE/neutralization balance. Future vaccines should consider thesefindings to design new formulations adapted to SARS-CoV-2 variants and lacking ADE epitopes in the spikeprotein.

Distinct evolution of infection-enhancing and neutralizing epitopes in the spike protein of SARS-CoV-2 variants (alpha, beta, gamma, delta, lambda and µ) : a structural and molecular epidemiology study (preprint)

Objectives: The efficiency of Covid-19 vaccination is determined by cellular and humoral immune responses, and for the latter, by the balance between neutralizing and infection-enhancing antibodies. Here we analyzed the evolution of neutralizing and facilitating epitopes in the spike protein among SARS-CoV-2 variants. Methods. Amino acid alignments were performed on 929,203 spike sequences over the 4 last months. Molecular modeling studies of the N-terminal domain (NTD) and rod-like regions of the spike protein were performed on a representative panel of SARS-CoV-2 variants that were structurally compared with the original Wuhan strain. Results. D614, which belongs to an antibody-dependent-enhancement (ADE) epitope common to SARS-CoV-1 and SARS-CoV-2, has rapidly mutated to D614G in the first months of 2020, explaining why ADE has not been detected following mass vaccination. We show that this epitope is conformationally linked to the main ADE epitope of the SARS-CoV-2 NTD which is highly conserved among most variants. In contrast, the neutralizing epitope of the NTD showed extensive variations in SARS-CoV-2 variants. Conclusions. This molecular epidemiology study coupled with structural analysis of the spike protein indicates that the balance between facilitating and neutralizing antibodies in vaccinated people is in favor of neutralization for the Wuhan strain, alpha and beta variants, but not for gamma, delta, lambda and mu. The evolution of SARS-CoV-2 has dramatically affected the ADE/neutralization balance which is nowadays in favor of ADE. Future vaccines should consider these data to design new formulations adapted to SARS-CoV-2 variants and lacking ADE epitopes in the spike protein.

High individual heterogeneity of neutralizing activities against the original 4 strain and 9 different variants of SARS-CoV-2 (preprint)

Background: Since the beginning of the COVID-19 pandemic, several SARS-CoV-2 variants have sequentially emerged. In France, most cases were due to spike D641G-harbouring viruses that descended initially from the Wuhan strain, then by variant of B.1.160 lineage we called Marseille-4 since the summer of 2020, which was followed by the alpha (UK) and beta (South African) variants in early 2021, then delta (Indian) now.Methods and Findings. We determined the neutralizing antibody (nAb) titres in sera from convalescent individuals previously infected by these 4 major local variants and from vaccine recipients to the original Wuhan strain and 9 variants, including two recent circulating delta (Indian) isolates. The results show high inter-individual heterogeneity in nAbs, especially according to the variant tested. Unexpectedly, the major variations among nAbs are based on the genotype responsible for the infection. Patients previously infected with the beta and B.1.160 variants had the lowest nAb titres. We show that this heterogeneity is well explained by spike protein mutants modelling using in silico approaches. The highest titres were observed in patients vaccinated with the Pfizer/BioNTech COVID-19 vaccine, even against the delta variant.Conclusions. Immunity acquired naturally after infection is highly dependent on the infecting variant and unexpectedly mRNA-based vaccine efficacy is shown to be often better than natural immunity in eliciting neutralizing antibodies.