key: cord-0289295-v29jmh6s
authors: Colson, P.; Delerce, J.; Burel, E.; Dahan, J.; Jouffret, A.; Fenollar, F.; Yahi, N.; Fantini, J.; LA SCOLA, B.; Raoult, D.
title: 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
date: 2021-12-29
journal: nan
DOI: 10.1101/2021.12.24.21268174
sha: 4f4456c548b8ee904044be39ad979917b94a32d9
doc_id: 289295
cord_uid: v29jmh6s

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.

geographical area exhibited the same combination of mutations screened by qPCR. They were 80 two adults and five children (<15 years of age) ( Table 1 ). The respiratory samples from these 81 eight patients were sent to university hospital institute Méditerranée Infection for SARS-CoV-82 2 genome sequencing as recommended by French public health authorities. A rapid NGS 83 procedure was launched overnight. It allowed obtaining SARS-CoV-2 genotype identification 84 in 8 hours. Briefly, viral RNA was extracted from 200 µL of nasopharyngeal swab fluid 85 using the KingFisher Flex system (Thermo Fisher Scientific, Waltham, MA, USA) following 86 the manufacturer's instructions. Extracted RNA was reverse-transcribed using SuperScript IV 87 (Thermo Fisher Scientific) and cDNA second strand was synthesized with LunaScript RT 88 SuperMix kit (New England Biolabs) then amplified using a multiplex PCR protocol 89 according to the ARTIC procedure (https://artic.network/) with ARTIC nCoV-2019 V3 panel 90 of primers (IDT, Coralville, IA, USA). Finally, NGS was performed with the ligation 91 sequencing kit and a GridION instrument of Oxford Nanopore Technologies (Oxford, UK) 92 following manufacturer's instructions. Subsequently, fastq files were processed using the 93 ARTIC field bioinformatics pipeline (https://github.com/artic-network/fieldbioinformatics). 94 NGS reads were basecalled using Guppy (4.0.14) and aligned to the Wuhan-Hu-1 reference 95 genome GenBank accession no. MN908947.3 using minimap2 (v2.17-r941) 96 (https://github.com/lh3/minimap2) [8] . The ARTIC tool align_trim was used to softmask 97 primers from read alignment and to cap sequencing depth at a maximum of 400. The 98 identification of consensus-level variant candidates was performed using the Medaka (0.11.5) 99 workflow developed by ARTIC (https://github.com/artic-network/artic-ncov2019). This 100 strategy allowed assemblying the complete genome from NGS reads obtained within 30 min 101 of run for cycle threshold values (Ct) of qPCR comprised between 15 and 27. SARS-CoV-2 102 . CC-BY 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted December 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 deposited in the GISAID sequence database (https://www.gisaid.org/) [14] (Table 1) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted December 29, 2021. other. This interaction is conserved between substituted amino acids 96Q and 190S, which 165 suggests the co-evolution of these changes. In the receptor binding domain (RBD), aside the 166 well-known substitutions N501Y and E484K, several changes were predicted to significantly 167 affect the neutralizing epitopes. Particularly, P681H is located in the cleavage site of S1-S2 168 subunits of the spike and is observed in other variants including the recently emerging 169

Omicron [15] . Besides, D1139H substitution implies an amino acid involved in the fusion 170 between the virus and the infected cell.Also, D614G is combined with T859N in the IHU 171 variant. Interestingly, in the Wuhan-Hu-1 isolate, amino acids D614 and T859 from two 172 subunits of the trimeric spike are face to face and lock the trimer in a closed conformation. 173 Substitution D614G allows unlocking the trimer conformation, but this is predicted to be still 174 easier in case of additional presence of substitution T859N. 175

Respiratory samples collected until end of November 2021 from four other SARS-176

CoV-2 positive patients living in the same city or borough than the index case could be 177 . CC-BY 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Waltham, USA) that provided positive signals for all three genes targeted (ORF1, S, and N). 182

Thus, the IHU variant can be distinguished by screening with qPCR assays from the Delta 183 (L452R-positive) and Omicron (L452R-negative and negative for S gene detection by the 184

TaqPath COVID-19 assay) variants that currently co-circulate in our geographical area. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 29, 2021. ; https://doi.org/10. 1101 /2021 Funding sources had no role in the design and conduct of the study; collection, management, 228 analysis, and interpretation of the data; and preparation, review, or approval of the 229 manuscript. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174 doi: medRxiv preprint sequencing. arXiv.org. https://arxiv.org/abs/1207.3907 (accessed 10 December 2021). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted December 29, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 variants: A health monitoring strategy for anticipating Covid-19 outbreaks. J Infect 83: 313 197-206. 314 20. Bedotto M, Fournier PE, Houhamdi L, Colson P, Raoult D (2021) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Deletion 11288-11296  T11418C  --C11514T  C11956T  C11956T  C14408T  C14408T  C16575T  -C16869T  C16869T  -C17004T  -T17348C  -A17916G  -C18175T  -C18804T  -G19348T  -T19680C  C20283T  --A21258G  C21588T  C21588T  G21848C  G21848C  Deletion 21968-21994  Deletion 21968-21994  G22132T  G22132T  T22191C  --G22205C  A22600C  A22600C  A22743G  A22743G  T22907A  T22907A  -G23012A  T23030C  -T23031G  T23031C  A23063T  A23063T  A23403G  A23403G  C23604A  C23604A  C24138A  C24138A  G24368C  --G24977C  C25487T  C25487T  G25563T  G25563T  A26492T  -T26767C  T26767G  C27513T  C27513T  C27807T  C27807T  -T27833C  C27972T  C27972T  -T28002C  Deletion 28271  Deletion 28271  T28297C  C28312T  G28337T  G28337T  C28887T  C28887T  T29377C  T29377C  G29405C  -Deletion 29738-29758  -G29779T  G29779T 14 . CC-BY 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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for all three genes EPI_ISL_7552483 12 Adult 15 L452R-neg