key: cord-0820518-h2sryzwn authors: Colson, P.; Fournier, P.-E.; Delerce, J.; Million, M.; Bedotto, M.; Houhamdi, L.; Yahi, N.; Bayette, J.; LEVASSEUR, A.; Fantini, J.; Raoult, D.; LA SCOLA, B. title: Culture and identification of a Deltamicron SARS-CoV-2 in a three cases cluster in southern France date: 2022-03-08 journal: nan DOI: 10.1101/2022.03.03.22271812 sha: b373095fdcd95378d9c36684f9eeb0c0cf48c342 doc_id: 820518 cord_uid: h2sryzwn 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. this recombinant and sequenced its genome. It was observed by scanning electron 48 microscopy. As it is misidentified with current variant screening qPCR, we designed and 49 implemented for routine diagnosis a specific duplex qPCR. Finally, structural analysis of the 50 recombinant spike suggested its hybrid content could optimize viral binding to the host cell 51 membrane. These findings prompt further studies of the virological, epidemiological, and 52 clinical features of this recombinant. 53 54 55 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 8, 2022. ; https://doi.org/10.1101/2022.03.03.22271812 doi: medRxiv preprint processed with the ARTIC field bioinformatics pipeline (https://github.com/artic-131 network/fieldbioinformatics). Nucleotide and amino acid changes relatively to the Wuhan-132 Hu-1 isolate genome were obtained using the Nextclade tool (https://clades.nextstrain.org/) 133 (Aksamentov et al., 2021; Rambaut et al., 2020) . Nextstrain clades and Pangolin lineages 134 were determined using the Nextclade web application (https://clades.nextstrain.org/) 135 (Aksamentov et al., 2021) and the Pangolin tool (https://cov-lineages.org/pangolin.html) 136 (Rambaut et al., 2020) , respectively. Genome sequences described here were deposited in the 137 GISAID sequence database (https://www.gisaid.org/) (EPI_ISL_10528736, 138 EPI_ISL_10531214, EPI_ISL_10529499, EPI_ISL_10640045) (Alm et al., 2020) . The 139 Simplot software (https://sray.med.som.jhmi.edu/SCRoftware/SimPlot/) was used for 140 recombination analysis. Phylogeny was reconstructed by the nextstrain/ncov tool 141 (https://github.com/nextstrain/ncov) and visualized with Auspice 142 (https://docs.nextstrain.org/projects/auspice/en/stable/). Genomes the closest genetically to 143 those obtained here were selected among Delta 21J/AY.4 genomes recovered from GISAID 144 based on their mutation pattern, then incorporated in the phylogeny together with the genome 145 of the Wuhan-Hu-1 isolate. 146 SARS-CoV-2 culture isolation was performed by inoculating 200 µL of respiratory 147 sample on Vero E6 cells as previously described (La Scola et al., 2020). Cytopathic effect was 148 observed by inverted microscopy. Viral particles were visualized in the culture supernatant by 149 scanning electron microscopy with a SU5000 microscope (Hitachi High-Technologies 150 Corporation, Tokyo, Japan), as previously described (Colson et al., 2020) . 151 Structural predictions of the spike protein were performed as previously described 152 CoV-2 20B spike and missing amino acids were incorporated with the Robetta protein 155 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 8, 2022. ; https://doi.org/10.1101/2022.03.03.22271812 doi: medRxiv preprint ORF3 protein whose gene is contiguous to the spike gene. The Simplot recombination 206 analysis tool provided congruent results. 207 The respiratory sample from which the first recombinant genome was obtained was 208 inoculated on Vero E6 cells the day following recombinant identification, and cytopathic 209 effect was observed after 4 days (Figures 3a, b) . The same day, supernatant was collected 210 and next-generation genome sequencing was performed using Nanopore technology on a 211 GridION instrument after PCR amplification with Artic primers, which allowed obtaining the 212 genome sequence of the viral isolate 8 h later (GISAID accession no. EPI_ISL_10640045). At 213 mutated positions compared to the Wuhan-Hu-1 isolate genome, mean sequencing depth was 214 2,771 reads and mean prevalence of the majoritary nucleotide was 99.1% (minimum, 95.1%), 215 and the consensus genome was identical to that obtained from the respiratory sample, 216 showing unambiguously that the virus isolated was the Delta 21J/AY.4-Omicron 21K/BA.1 217 recombinant. Finally, viral particles were observed in the culture supernatant by scanning 218 electron microscopy with a SU5000 microscope within minutes after supernatant collection 219 (Figure 3c) . 220 The overall structure of the recombinant spike protein was predicted (Figures 4a-c) . It is increasingly demonstrated that the genomes of most biological entities, whatever 255 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 8, 2022. ; https://doi.org/10.1101/2022.03.03.22271812 doi: medRxiv preprint GENomique microbienne EMERGEN; 305 https://www.santepubliquefrance.fr/dossiers/coronavirus-covid-19/consortium-emergen). 306 The dataset generated and analyzed during the current study is available in the GISAID 308 database (https://www.gisaid.org/). 309 c. Nextclade: clade assignment, mutation 327 calling and quality control for viral genomes WHO European Region sequencing laboratories and 331 GISAID EpiCoV group; WHO European Region sequencing laboratories and GISAID 332 EpiCoV group. 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