key: cord-0977235-c89oddsg authors: Quéromès, Grégory; Destras, Grégory; Bal, Antonin; Regue, Hadrien; Burfin, Gwendolyne; Brun, Solenne; Fanget, Rémi; Morfin, Florence; Valette, Martine; Lina, Bruno; Frobert, Emilie; Josset, Laurence title: Characterization of SARS-CoV-2 ORF6 deletion variants detected in a nosocomial cluster during routine genomic surveillance, Lyon, France date: 2020-08-10 journal: bioRxiv DOI: 10.1101/2020.08.07.241653 sha: 64808ddfbdd65f0e23947f60afe5f419ee1f2955 doc_id: 977235 cord_uid: c89oddsg Through routine genomic surveillance of the novel SARS-CoV-2 virus (n=229 whole genome sequences), 2 different frameshifting deletions were newly detected in the open reading frame (ORF) 6, starting at the same position (27267). While the 26-nucleotide deletion variant was only found in one sample in March 2020, the 34-nucleotide deletion variant was found within a single geriatric hospital unit in 5/9 patients sequenced and one health care worker with samples collected between April 2nd and 9th, 2020. Both the presence of the 34-nucleotide deletion variant limited to this unit and the clustering of the corresponding whole genome sequences by phylogeny analysis strongly suggested a nosocomial transmission between patients. Interestingly, prolonged viral excretion of the 34-nucleotide deletion variant was identified in a stool sample 14 days after initial diagnosis for one patient. Clinical data revealed no significant difference in disease severity between patients harboring the wild-type or the 34-nucleotide deletion variants. The in vitro infection of the two deletion variants on primate endothelial kidney cells (BGM) and human lung adenocarcinoma cells (Calu-3) yielded comparable replication kinetics with the wild-type strain. Furthermore, high viral loads were found in vivo regardless of the presence or absence of the ORF6 deletion. Our study highlights the transmission and replication capacity of two newly described deletion variants in the same ORF6 region. Importance While the SARS-CoV-2 genome has remained relatively stable since its emergence in the human population, genomic deletions are an evolutionary pattern previously described for the related SARS-CoV. Real-time genomic monitoring of the circulating variants is paramount to detect strain prevalence and transmission dynamics. Given the role of ORF6 in interferon modulation, further characterization, such as mechanistic interactions and interferon monitoring in patients, is crucial in understanding the viral-host factors driving disease evolution. Samples were collected for regular clinical management during hospital stay, with no additional 2 5 3 samples for the purpose of this study. Patients were informed of the research and their non-objection 2 5 4 approval was confirmed. This study was presented by the ethics committee of the Hospices Civils de 2 5 5 Lyon (HCL), Lyon, France and registered on the HCL database of RIPHN studies (AGORA N°41). Data availability 2 5 7 The SARS-CoV-2 genomes sequenced in this study were deposited on the GISAID database 2 5 8 (https://www.gisaid.org/) on a regular basis, accession numbers can be found in Supplementary Table 2 5 9 1. We would like to thank all the patients, laboratory technicians, and clinicians who contributed to this 2 6 3 investigation. We gratefully acknowledge all the members of CoV-GLUE, Nextstrain.org, and 2 6 4 virological.org for sharing their analysis in real time. The authors declare no competing interests. BL is a member of the French Scientific Committee for SARS-CoV-2. (n=229). The phylogenetic tree was constructed using R software using ape package and the 2 7 5 neighbor joining evolutionary method (hCoV19/Wuhan/IPBCAMSWH01/2019 as the root). The 2 7 6 colored branches denote the hospital unit origin of the sequence and ORF6 status. On the right, 2 7 7 multiple sequence alignment from nucleotide position 27267-27303 (Wuhan-Hu-1 numbering) is 2 7 8 illustrated, with the 26-nt and 34-nt deletions depicted in black. The deletion sites of interest were not 2 7 9 included for genetic distance calculation. lines. SARS-CoV-2 strains were inoculated on confluent BGM and Calu-3 cells at an MOI of 10 -3 and 2 9 0 then incubated at 36°C with 5% CO 2 for 7 days. Supernatant samples were collected at regular 2 9 1 intervals, for which viral particle quantification was performed by qRT-PCR. Each data point is the 2 9 2 average of three replicates, with standard deviation as error bars. Statistical analysis was performed Brisebarre A, Barbet M, Derrar F, Werf S van der, Enouf V, Simon-Loriere E. 2020. Introductions and early spread of SARS-CoV-2 in France, 24 January to 23 March 2020. Eurosurveillance 25:2001200. Origin and ORF6 mutation I I M R T L H H S 17 18 19 20 21 22 23 24 25 26 27 28 29 I K P H N S stop 27250 27260 27293 27266 27300 27310 I I M R T F K V S I W N L D stop 17 18 19 20 21 22 23 24 25 26 27 28 29 30 62 27250 27260 27270 27280 * h C o V − 1 9 / F r a n c e / A R A 2 4 0 2 3 / 2 0 2 0 * h C o V − 1 9 / F r a n c e / A R A 2 0 8 2 9 / 2 0 2 0 D 3 4 * h C o V − 1 9 / F r a n c e / A R A 1 8 6 2 5 / 2 0 2 0 D 2 6 * h C o V − 1 9 / F r a n c e / A R A 0 7 3 1 / 2 0 2 0 D 3 4 * h C o V − 1 9 / F r a n c e / A R A 2 2 6 4 7 / 2 0 2 0 D 3 4 * h C o V − 1 9 / F r a n c e / A R A 2 2 6 5 0 / 2 0 2 0 D 3 4 * h C o V − 1 9 / F r a n c e / A R A 2 1 1 6 3 / 2 0 2 0 D 3 4 * h C o V − 1 9 / F r a n c e / A R A 2 0 6 8 / 2 0 2 0 D 3 4 * h C o V − 1 9 / F r a n c e / A R A 2 1 3 6 0 / 2 0 2 0 WT and deletion strains. Mismatch count between consensus sequences generated by each method compared 2 by 2 for each sample. Blue tiles correspond to perfect identity and orange tiles correspond to mismatches (number of mismatches is indicated inside the tile). Matrices were generated with an R script using Decipher (alignment), ape (distance matrices) and ggplot2 (charts) libraries. Of note, undetermined bases and deletions were notconsidered in the calculation of mismatches. Human Female 44 Original Nasopharyngeal swab B.1 G hCoV-19/France Human Male 47 Original Nasopharyngeal swab B.1 G hCoV-19/France/Clermont Human Male 16 Original Nasopharyngeal swab B.1 G hCoV-19/France/Clermont Human Female 49 Original Nasopharyngeal swab B.1 G hCoV-19/France Human Male 50 Original Nasopharyngeal swab B.1 GH hCoV-19/France Human Male 82 Original Nasopharyngeal swab B.1 G hCoV-19/France Human Female 88 Original Nasopharyngeal swab B.1 G hCoV-19/France/Bourg Human Male 79 Original Nasopharyngeal swab B.1 GH hCoV-19/France Human Male 48 Original Nasopharyngeal swab B.1 G hCoV-19/France Human Female 50 Original Nasopharyngeal swab B.1 G hCoV-19/France Human Male 74 Original Nasopharyngeal swab B L hCoV-19/France Human Male 79 Original Nasopharyngeal swab B.1 GH hCoV-19/France/Lyon_06625/2020 Human Male 78 Original Nasopharyngeal swab B.1 G hCoV-19/France/Macon_06756/2020 Human Female 63 Original Nasopharyngeal swab B.1 GH hCoV-19/France Human Female 86 Original 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hCoV-19/France Human Female 89 Original Nasopharyngeal swab B.1 GH hCoV-19/France Human Female 82 Original Nasopharyngeal swab B.1 GH hCoV-19/France Human Female 94 Original Nasopharyngeal swab B.1 GH hCoV-19/France Human Female 56 Original Nasopharyngeal swab B.1 GH hCoV-19/France Human Female 57 Original Nasopharyngeal swab B.1 G hCoV-19/France ARA / Lyon Human Female 30 Original Nasopharyngeal swab B.1 GH hCoV-19/France Human Male 41 Original Nasopharyngeal swab B.1 G hCoV-19/France Human Female 32 Original Nasopharyngeal swab B.1 G hCoV-19/France Human Male 48 Original Nasopharyngeal swab B.1 G hCoV-19/France Human Female 91 Original Nasopharyngeal swab B.1 GH hCoV-19/France Human Male 29 Original Nasopharyngeal swab B.1 G hCoV-19/France Human Female 71 Original Nasopharyngeal swab B.1 GH hCoV-19/France Human Male 72 Original Nasopharyngeal swab B.1 G hCoV-19/France ARA / Lyon Human Male 30 Original Nasopharyngeal swab B.1 G hCoV-19/France Human Female 86 Original Nasopharyngeal 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hCoV-19/France ARA / Oyonnax Human Male 58 Original Nasopharyngeal swab B.1 GH