Ιαν 07

Ολόκληρη η εργασία για την παραλλαγή Καμερούν με πρωτεργάτες τους Philippe COLSON και τον  Didier Raoult

TITLE PAGE
1 2
Full-length title:
3
Emergence in Southern France of a new SARS-CoV-2 variant of probably Cameroonian
4
origin harbouring both substitutions N501Y and E484K in the spike protein
 5 6
Short title (for the running head):
 7
A new SARS-CoV-2 variant with spike substitutions N501Y and E484K
 8 9
Author list: Philippe COLSON
1,2,3
, Jérémy DELERCE
1
, Emilie BUREL
1
, Jordan
10
DAHAN
4
, Agnès JOUFFRET
5
, Florence FENOLLAR
1,2,6
, Nouara YAHI
7
, Jacques
11
FANTINI
7
, Bernard LA SCOLA
1,2,3
, Didier RAOULT
1,2
*
12
Affiliations:
1
 IHU Méditerranée Infection, 19-21 boulevard Jean Moulin, 13005 Marseille, 13 France;
2
Aix-Marseille Univ., Institut de Recherche pour le Développement (IRD), Microbes 14 Evolution Phylogeny and Infections (MEPHI), 27 boulevard Jean Moulin, 13005 Marseille, 15 France;
3
Assistance Publique-Hôpitaux de Marseille (AP-HM), 264 rue Saint-Pierre, 13005 16 Marseille, France;
4
 Laboratoire de Biologie Médicale SYNLAB Provence Marseille, 25 rue 17 Rabattu, 13015 Marseille, France;
5
 Laboratoire de Biologie Médicale SYNLAB Provence 18 Forcalquier, rue du Souvenir Français, 04300 Forcalquier, France;
6
 Aix-Marseille Univ., 19 Institut de Recherche pour le Développement (IRD), Vecteurs
 – 
 Infections Tropicales et 20 Méditerranéennes (VITROME), 27 boulevard Jean Moulin, 13005 Marseille, France;
 7
 Aix-21 Marseille Université, INSERM UMR S 1072, 51 boulevard Pierre Dramard, 13015 Marseille, 22 France. 23
* Corresponding author:
 Didier Raoult, IHU Méditerranée Infection, 19-21 boulevard Jean 24 Moulin, 13005 Marseille, France. Tel.: +33 413 732 401, Fax: +33 413 732 402; email: 25
. CC-BY 4.0 International licenseIt 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(which was not certified by peer review)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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

2 didier.raoult@gmail.com 26

Keywords:
 SARS-CoV-2, variant, emergence, travel, southern France 27
Word counts:
 abstract, 198; text, 1,778 28
Figure:
1;
 Table:
 1
References:
23
 
29
Supplementary Material: Figure:
1;
 Tables:
 2;
References:
2 30 31 32
. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint
3
ABSTRACT
33 34 SARS-CoV-2 variants have become a major virological, epidemiological and clinical 35 concern, particularly with regard to the risk of escape from vaccine-induced immunity. Here 36 we describe the emergence of a new variant. For twelve SARS-CoV-positive patients living in 37 the same geographical area of southeastern France, qPCR testing that screen for variant-38 associated mutations showed an atypical combination. The index case returned from a travel 39 in Cameroon. The genomes were obtained by next-generation sequencing with Oxford 40  Nanopore Technologies on GridION instruments within
8 h. Their analysis revealed 46 41 mutations and 37 deletions resulting in 30 amino acid substitutions and 12 deletions. Fourteen 42 amino acid substitutions, including N501Y and E484K, and 9 deletions are located in the 43 spike protein. This genotype pattern led to create a new Pangolin lineage named B.1.640.2, 44 which is a phylogenetic sister group to the old B.1.640 lineage renamed B.1.640.1. Both 45 lineages differ by 25 nucleotide substitutions and 33 deletions. The mutation set and 46  phylogenetic position of the genomes obtained here indicate based on our previous definition 47 a new
 
variant we named “IHU”.
 These data are another example of the unpredictability of the 48 emergence of SARS-CoV-2 variants, and of their introduction in a given geographical area 49 from abroad. 50 51 52
. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint
4
TEXT
53 54 SARS-CoV-2 has emerged in China in December 2019 and has been declared a 55  pandemic 21 months ago [1]. We have shown since the summer of 2020 that several SARS-56 CoV-2 variants have emerged in our geographical area and caused distinct epidemics, either 57 successive or superimposed [2,3]. In addition, we described that the origin of these variants 58 was often their introduction from abroad but could also be mink. This was observed by 59 genotyping, as of 09/12/2021, SARS-CoV-2 from almost 40,000 patients using next-60 generation sequencing (NGS) of complete genomes for more than 22,000 patients and 61 implementing multiple qPCR specific of each variant for a more exhaustive assessment of 62 their spread. Since then and with the emergence of the Alpha variant at the end of 2020, 63 SARS-CoV-2 variants have become a major virological, epidemiological, and clinical 64 concern, particularly with regard to the risk of escape from vaccine-induced immunity [4-7]. 65 Here we describe the emergence in south-eastern France of a new variant of probably 66 Cameroonian origin. 67 68 The index case was an adult first diagnosed as infected with SARS-CoV-2 by real-69 time reverse transcription PCR (qPCR) performed in a private medical biology laboratory on 70 a nasopharyngeal sample collected mid-November 2021 (Table 1). He was vaccinated against 71 SARS-CoV-2 and returned from a travel to Cameroon three days before. He developed mild 72 respiratory symptoms the day before diagnosis. He lives in a small town of southeastern 73 France. Subsequent detection by qPCR of three mutations in the spike gene to screen for 74 variants, as systematically performed in France in case of SARS-CoV-2 positivity, revealed 75 an atypical combination with L452R-negativity, E484K-positivity, and E484Q-negativity 76 (Pentaplex assay, ID Solution, France) that did not correspond to the pattern of the Delta 77
. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint

5 variant involved in almost all SARS-CoV-2 infections at that time (Table 1). Respiratory 78 samples collected from seven other SARS-CoV-2-positive patients living in the same 79 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 licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint

6 genomes were classified into Nextclade and Pangolin lineages using web applications 103 (https://clades.nextstrain.org/;https://cov-lineages.org/pangolin.html) [10,11,13]. They were 104 deposited in the GISAID sequence database (https://www.gisaid.org/) [14] (Table 1). 105 Phylogenies were reconstructed with the nextstrain/ncov tool 106 (https://github.com/nextstrain/ncov) then visualized with Auspice 107 (https://docs.nextstrain.org/projects/auspice/en/stable/). 108 The analysis of viral genomes revealed the presence of 46 nucleotide substitutions and 109 37 deletions, resulting in 30 amino acid substitutions and 12 deletions (Figure 1a; 110 Supplementary Tables S1 and S2). Fourteen amino acid substitutions and 9 amino acid 111 deletions are located in the spike protein. Substitutions N501Y and E484K are combined as in 112 the Beta, Gamma, Theta and Omicron variants [5,15]. Substitution F490S is present as in the 113 Lambda variant, and substitution P681H is present as in the Lambda and Omicron variants. In 114 other structural proteins than the spike, amino acid changes include two substitutions in the 115 nucleocapsid protein and one in the membrane protein. In non-structural proteins, amino acid 116 changes include one substitution in proteins Nsp2, Nsp3, Nsp4, Nsp6, Nsp12 (RNA-117 dependent RNA polymerase), and Nsp13 (helicase); two substitutions in Nsp14 (

3’
–118
5’exo
nuclease); four substitutions in Nsp8 (which is part of the replication complex with 119  Nsp7 and Nsp12); and three deletions in Nsp6. Finally, in regulatory proteins, amino acid 120 changes include four substitutions in ORF3a, one in ORF9b and one in ORF8. In addition, 121 codon 27 of ORF8 gene is changed into a stop codon, as in the Alpha variant [16]; some 122 members of the Marseille-4 variant (B.1.160) that predominated in our geographical area 123  between August 2020 and February 2021 also exhibit a stop codon in ORF8 gene but at 124 another position [3]. 125  Nextclade identified a 20A lineage. Pangolin identified a B.1.640 lineage in primary 126 analysis but a B.1 lineage with the -usher (Ultrafast Sample placement on Existing tRee; 127
. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint

7 https://genome.ucsc.edu/cgi-bin/hgPhyloPlace) option, which showed the phylogenetic 128  placement of the genomes we obtained as an outgroup of the B.1.640 lineage and their 129 clustering with a genome obtained late October in France (Ile-de-France) 130 (EPI_ISL_5926666). The B.1.640 lineage corresponds to a variant first identified in France in 131 April 2021, in Indonesia in August 2021, and in Republic of the Congo (Brazzaville) in 132 September 2021, and it was involved in a cluster of cases in Brittany, France around mid-133 October 2021 [17]. As of 09/12/2021, 157 genomes were available from the GISAID database 134 including 92 from France and 36 from the Republic of the Congo. The sets of spike mutations 135 of the B.1.640 lineage and of genomes obtained here are similar, with 11 common nucleotide 136 substitutions and 1 common deletion of 9 codons (Supplementary Figure S1, Tables S1-2). 137 However, spike genes of both lineages differ by 7 mutations. In addition, 25 nucleotide 138 substitutions and 33 nucleotide deletions located elsewhere in the genomes differ between the 139 two genotypes. The pattern of mutations of present genomes hence indicates a new

 
variant, 140
which we named “IHU” (in reference to our institute)
, based on our previous definition [3]. 141 Phylogeny performed with nextstrain/ncov tool (https://github.com/nextstrain/ncov) also 142 showed that B.1.640 and IHU variants were most closely related between each other but 143 comprised two divergent branches (Figure 1b). Their last common ancestor is estimated to 144 date from January 2021 but no genome is currently available from GISAID that corresponds 145 to it. Accordingly, a new Pangolin clade corresponding to the IHU variant was created on 146 07/12/2021 that was named B.1.640.2, the old B.1.640 clade being renamed B.1.640.1 147 (https://github.com/cov-lineages/pango-designation/issues/362). It encompasses present 148 genomes and three other genomes comprising a sister group including the one recovered late 149 October 2021 in France (Ile-de-France) (EPI_ISL_5926666) and two additional genomes 150 obtained from samples collected late November in England (EPI_ISL_7181977) and Wales 151 (EPI_ISL_7402094). As the index case was probably infected with the IHU variant during his 152
. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint

8 stay in Cameroon, we sought for this variant in GISAID among genomes from this country 153  but as of 09/12/2021 none of the 556 available genomes belong to the B.1.640.1 or B.1.640.2 154 lineages. 155 We analyzed a complete structure of the spike protein of the IHU variant generated by 156 incorporating its specific mutational profile to the original 20B SARS-CoV-2 (Wuhan-Hu-1 157 isolate with D614G substitution) [18] and fixing all gaps in the pdb file by incorporating the 158 missing amino acids with the Robetta protein structure prediction tool 159 [https://robetta.bakerlab.org/], followed by energy minimization with the Polak-Ribière 160 algorithm as previously reported (Figure 1c) [19]. In the N-terminal domain (NTD), the 134-161 145 amino acid deletion is predicted to significantly affect the neutralizing epitope. Other 162 changes involve amino acids at positions 96 and 190: in Wuhan-Hu-1 isolate, E96 and R190 163 induce a turn in NTD secondary structure through electrostatic interactions between each 164 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 licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint

9 identified as containing the IHU variant by NGS within 24 hours after their reception (Table 178 1). All 12 IHU variant-positive samples showed the same combination of spike mutations as 179 screened by real-time qPCR techniques: negativity for 452R and 484Q; positivity for 484K, 180 501Y [20], and 681H [3]. We also used the TaqPath COVID-19 kit (Thermo Fisher Scientific, 181 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. 185 Finally, scanning electron microscopy using a SUV 5000 microscope (Hitachi High-186 Technologies Corporation, Tokyo, Japan) [21] allowed a quick visualization of the virus from 187 a respiratory sample (Figure 1d). 188 189 Overall, these observations show once again the unpredictability of the emergence of 190 new SARS-CoV-2 variants and their introduction from abroad, and they exemplify the 191 difficulty to control such introduction and subsequent spread. They also warrant the 192 implementation of genomic surveillance of SARS-CoV-2 that we started from the very 193  beginning of the pandemic in our geographical area as soon as we diagnosed the first SARS-194 CoV-2 infection [21] and that we expanded during summer 2020 [2,3]. This surveillance has 195  been implemented at the country scale in 2021 through the French Emergen consortium 196 (https://www.santepubliquefrance.fr/dossiers/coronavirus-covid-19/consortium-emergen). It is 197 too early to speculate on virological, epidemiological or clinical features of this IHU variant 198  based on these 12 cases. For this purpose, respiratory samples from infected patients were 199 inoculated on Vero E6 cells as previously described [22] to be able assessing the sensibility to 200 neutralization by anti-spike antibodies elicited by vaccine immunization, or by prior infection 201 [23]. 202

. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint

 13 resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study. 265 Science 374: 472-478. 266 5. Harvey WT, Carabelli AM, Jackson B, Gupta RK, Thomson EC, Harrison EM, Ludden 267 C, Reeve R, Rambaut A (2021) COVID-19 Genomics UK (COG-UK) Consortium, 268 Peacock SJ, Robertson DL. SARS-CoV-2 variants, spike mutations and immune escape. 269  Nat Rev Microbiol 19: 409-424. 270 6. Tao K, Tzou PL, Nouhin J, Gupta RK, de Oliveira T, Kosakovsky Pond SL, Fera D, 271 Shafer RW (2021) The biological and clinical significance of emerging SARS-CoV-2 272 variants. Nat Rev Genet 22: 757-773. 273 7. Wilder-Smith A (2021) What is the vaccine effect on reducing transmission in the 274 context of the SARS-CoV-2 delta variant? Lancet Infect Dis S1473-3099(21)00690-3. 275 doi: 10.1016/S1473-3099(21)00690-3. Epub ahead of print. PMID: 34756187; PMCID: 276 PMC8554481. 277 8. Li H (2018). Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 278 34: 3094-3100. 279 9. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, 280 Durbin R, 1000 Genome Project Data Processing Subgroup (2009). The Sequence 281 Alignment/Map format and SAMtools. Bioinformatics 25: 2078-2079. 282 10. Hadfield J, Megill C, Bell SM, Huddleston J, Potter B, Callender C, Sagulenko P, 283 Bedford T, Neher RA (2018). Nextstrain: real-time tracking of pathogen evolution. 284 Bioinformatics 34: 4121-4123. 285 11. Aksamentov I, Roemer C, Hodcroft EB, Neher RA (2021). Nextclade: clade 286 assignment, mutation calling and quality control for viral genomes. Zenodo 287 https://doi.org/10.5281/zenodo.5607694. 288

. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint

14 12. Garrison E, Marth G (2012). Haplotype-based variant detection from short-read 289 sequencing. arXiv.org. https://arxiv.org/abs/1207.3907 (accessed 10 December 2021). 290 13. Rambaut A, Holmes EC, O’Toole Ã, Hill V, McCrone JT, Ruis C, du Plessis L, Pybus 291 OG (2020). A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist 292 genomic epidemiology. Nat. Microbiol 5: 1403-1407. 293 14. Alm E, Broberg EK, Connor T, Hodcroft EB, Komissarov AB, Maurer-Stroh S, 294 Melidou A, Neher RA, O’Toole A, Pereyaslov D, WHO European Region sequencing 295 laboratories and GISAID EpiCoV group; WHO European Region sequencing 296 laboratories and GISAID EpiCoV group (2020) Geographical and temporal distribution 297 of SARS-CoV-2 clades in the WHO European Region, January to June 2020. Euro. 298 Surveill 25: 2001410. 299 15. Karim SSA, Karim QA (2021) Omicron SARS-CoV-2 variant: a new chapter in the 300 COVID-19 pandemic. Lancet 398: 2126-2128. 301 16. Rambaut A, Loman N, Pybus O, Barclay W, Barrett J, Carabelli A, Connor T, Peacock 302 T, Robertson DL, Volz E, on behalf of COVID-19 Genomics Consortium UK (CoG-303 UK) (2020) Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage 304 in the UK defined by a novel set of spike mutations. Virological Pre-print. 305 https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-306 2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563 307 17. Le Page M (2021) New variant gains ground. New Sci 252: 8. 308 18. Benton DJ, Wrobel AG, Roustan C, Borg A, Xu P, Martin SR, Rosenthal PB, Skehel JJ, 309 Gamblin SJ (2021). The effect of the D614G substitution on the structure of the spike 310 glycoprotein of SARS-CoV-2. Proc. Natl. Acad. Sci. U S A. 118:e2022586118. 311

. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint
15 19. Fantini J, Yahi N, Azzaz F, Chahinian H (2021) Structural dynamics of SARS-CoV-2 312 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) Implementation of an 315 in-house real-time reverse transcription-PCR assay to detect the emerging SARS-CoV-2 316  N501Y variants. J Clin Virol 140: 104868. 317 21. Colson P, Lagier JC, Baudoin JP, Bou Khalil J, La Scola B, Raoult D (2020) Ultrarapid 318 diagnosis, microscope imaging, genome sequencing, and culture isolation of SARS-319 CoV-2. Eur J Clin Microbiol Infect Dis 39: 1601-1603. 320 22. Wurtz N, Penant G, Jardot P, Duclos N, La Scola B (2021) Culture of SARS-CoV-2 in 321 a panel of laboratory cell lines, permissivity, and differences in growth profile. Eur J 322 Clin Microbiol Infect Dis 40: 477-484. 323 23. Jaafar R, Boschi C, Aherfi S, Bancod A, Le Bideau M, Edouard S, Colson P, Chahinian 324 H, Raoult D, Yahi N, Fantini J, La Scola B (2021) High individual heterogeneity of 325 neutralizing activities against the original strain and nine different variants of SARS-326 CoV-2. Viruses 13: 2177. 327 328
. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint

10 203 204

Acknowledgments
205 We are thankful to the Emergen French consortium 206 (https://www.santepubliquefrance.fr/dossiers/coronavirus-covid-19/consortium-emergen). We 207 are also grateful to Laurence Thomas, Claudia Andrieu, Ludivine Brechard, Mamadou Beye, 208 Marielle Bedotto, Elsa Prudent, Sofiane Bakour, Jacques Bou Khalil, and Clio Grimaldier for 209 their technical help. 210 211
Author contributions
212 Conceived and designed the experiments: PC, DR, JF, BLS. Contributed materials/analysis 213 tools: PC, JDe, EB, JDa, AJ, FF, NY, JF. Analyzed the data: PC, DR, BLS, JD, EB, JF, NY. 214 Wrote the paper: PC, JF, DR. All authors approved the last version of the manuscript. 215 216
Funding
217
This work was supported by the French Government under the “Investments for the Future”
218  program managed by the National Agency for Research (ANR), Méditerranée-Infection 10-219 IAHU-03 and was also supported by Région Provence Alpes Côte
d’Azur and European
220 funding FEDER PRIMMI (Fonds Européen de Développement Régional-Plateformes de 221 Recherche et d’Innovation Mutualisées Méditerranée Infection), FEDER PA 0000320 222 PRIMMI, and by Hitachi High-Technologies Corporation, Tokyo, Japan. 223 224
Conflicts of interest
225 DR has a conflict of interest being a consultant for Hitachi High-Technologies Corporation, 226 Tokyo, Japan from 2018 to 2020. All other authors have no conflicts of interest to declare. 227
. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint
11 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. 230 231
Ethics
232 This study has been approved by the ethics committee of University Hospital Institute (IHU) 233 Méditerranée Infection (N°2021-
029). Access to the patients’ biological and registry data
234 issued from the hospital information system was approved by the data protection committee 235 of Assistance Publique-Hôpitaux de Marseille (APHM) and was recorded in the European 236 General Data Protection Regulation registry under number RGPD/APHM 2019-73. 237 238 239
. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint

12

REFERENCES
240 241 1. Cucinotta D, Vanelli M (2020) WHO Declares COVID-19 a Pandemic. Acta Biomed 242 91: 157-160. 243 2. Colson P, Levasseur A, Delerce J, Chaudet H, Bossi V, Ben Khedher M, Fournier PE, 244 Lagier JC, Raoult D (2020) Dramatic increase in the SARS-CoV-2 mutation rate and 245 low mortality rate during the second epidemic in summer in Marseille. IHU pre-prints 246 https://doi.org/10.35088/68c3-ew82 (accessed 10 December 2021). 247 3. Colson P, Fournier PE, Chaudet H, Delerce J, Giraud-Gatineau A, Houhamdi L, 248 Andrieu C, Brechard L, Bedotto M, Prudent E, Gazin C, Beye M, Burel E, Dudouet P, 249 Tissot-Dupont H, Gautret P, Lagier JC, Million M, Brouqui P, Parola P, Drancourt M, 250 La Scola B, Levasseur A, Raoult D (2021) Analysis of SARS-CoV-2 variants from 251 24,181 patients exemplifies the role of globalisation and zoonosis in pandemics. 252 medRxiv doi: https://doi.org/10.1101/2021.09.10.21262922 (accessed 10 December 253 2021). 254 4. Hastie KM, Li H, Bedinger D, Schendel SL, Dennison SM, Li K, Rayaprolu V, Yu X, 255 Mann C, Zandonatti M, Diaz Avalos R, Zyla D, Buck T, Hui S, Shaffer K, Hariharan C, 256 Yin J, Olmedillas E, Enriquez A, Parekh D, Abraha M, Feeney E, Horn GQ; CoVIC-257 DB team1, Aldon Y, Ali H, Aracic S, Cobb RR, Federman RS, Fernandez JM, Glanville 258 J, Green R, Grigoryan G, Lujan Hernandez AG, Ho DD, Huang KA, Ingraham J, Jiang 259 W, Kellam P, Kim C, Kim M, Kim HM, Kong C, Krebs SJ, Lan F, Lang G, Lee S, 260 Leung CL, Liu J, Lu Y, MacCamy A, McGuire AT, Palser AL, Rabbitts TH, 261 Rikhtegaran Tehrani Z, Sajadi MM, Sanders RW, Sato AK, Schweizer L, Seo J, Shen 262 B, Snitselaar JL, Stamatatos L, Tan Y, Tomic MT, van Gils MJ, Youssef S, Yu J, Yuan 263 TZ, Zhang Q, Peters B, Tomaras GD, Germann T, Saphire EO (2021) Defining variant-264
. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint

13 resistant epitopes targeted by SARS-CoV-2 antibodies: A global consortium study. 265 Science 374: 472-478. 266 5. Harvey WT, Carabelli AM, Jackson B, Gupta RK, Thomson EC, Harrison EM, Ludden 267 C, Reeve R, Rambaut A (2021) COVID-19 Genomics UK (COG-UK) Consortium, 268 Peacock SJ, Robertson DL. SARS-CoV-2 variants, spike mutations and immune escape. 269  Nat Rev Microbiol 19: 409-424. 270 6. Tao K, Tzou PL, Nouhin J, Gupta RK, de Oliveira T, Kosakovsky Pond SL, Fera D, 271 Shafer RW (2021) The biological and clinical significance of emerging SARS-CoV-2 272 variants. Nat Rev Genet 22: 757-773. 273 7. Wilder-Smith A (2021) What is the vaccine effect on reducing transmission in the 274 context of the SARS-CoV-2 delta variant? Lancet Infect Dis S1473-3099(21)00690-3. 275 doi: 10.1016/S1473-3099(21)00690-3. Epub ahead of print. PMID: 34756187; PMCID: 276 PMC8554481. 277 8. Li H (2018). Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 278 34: 3094-3100. 279 9. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, 280 Durbin R, 1000 Genome Project Data Processing Subgroup (2009). The Sequence 281 Alignment/Map format and SAMtools. Bioinformatics 25: 2078-2079. 282 10. Hadfield J, Megill C, Bell SM, Huddleston J, Potter B, Callender C, Sagulenko P, 283 Bedford T, Neher RA (2018). Nextstrain: real-time tracking of pathogen evolution. 284 Bioinformatics 34: 4121-4123. 285 11. Aksamentov I, Roemer C, Hodcroft EB, Neher RA (2021). Nextclade: clade 286 assignment, mutation calling and quality control for viral genomes. Zenodo 287 https://doi.org/10.5281/zenodo.5607694. 288

. CC-BY 4.0 International licenseIt 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 The copyright holder for thisthis version posted December 29, 2021. ; https://doi.org/10.1101/2021.12.24.21268174doi: medRxiv preprint
Source:https://www.zerohedge.com/medical/new-variant-46-mutations-discovered-southern-france

ΙΩΑΝΝΗΣ ΠΑΤΜΙΟΣ

Αφήστε μια απάντηση

Η διεύθυνση του email σας δεν θα δημοσιευθεί.