Emergence and outcomes of the SARS-CoV-2 ‘Marseille-4’ variant

(1)

HAL Id: hal-03245782

https://hal-amu.archives-ouvertes.fr/hal-03245782

Submitted on 2 Jun 2021

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

Distributed under a Creative Commons Attribution - NonCommercial - NoDerivatives| 4.0

International License

Emergence and outcomes of the SARS-CoV-2

‘Marseille-4’ variant

Pierre-Edouard Fournier, Philippe Colson, Anthony Levasseur, Christian

Devaux, Philippe Gautret, Marielle Bedotto, Jeremy Delerce, Ludivine

Brechard, Lucile Pinault, Jean-Christophe Lagier, et al.

To cite this version:

Pierre-Edouard Fournier, Philippe Colson, Anthony Levasseur, Christian Devaux, Philippe Gautret,

et al.. Emergence and outcomes of the SARS-CoV-2 ‘Marseille-4’ variant. International Journal of

Infectious Diseases, Elsevier, 2021, 106, pp.228 - 236. �10.1016/j.ijid.2021.03.068�. �hal-03245782�

(2)

Emergence

and

outcomes

of

the

SARS-CoV-2

‘Marseille-4’

variant

Pierre-Edouard

Fournier

a,b,

*

,

Philippe

Colson

a,c

,

Anthony

Levasseur

a,c

,

Christian

A. Devaux

a,c

,

Philippe

Gautret

a,b

,

Marielle

Bedotto

a

,

Jeremy

Delerce

a

,

Ludivine

Brechard

a

,

Lucile

Pinault

a

,

Jean-Christophe

Lagier

a,c

,

Florence

Fenollar

a,b

,

Didier

Raoult

a,c,

*

a_IHU_{Méditerranée}_Infection,_Marseille,_France b

Vecteurs–InfectionsTropicalesetMéditerranéennes(VITROME),Marseille,France

c

MicrobesEvolutionPhylogenyandInfections(MEPHI),Aix–MarseilleUniversité,Marseille,France

ARTICLE INFO Articlehistory:

Received1February2021

Receivedinrevisedform20March2021 Accepted23March2021 Keywords: SARS-CoV-2 COVID-19 Variant Marseille-4 Mutations Spike Molecularepidemiology ABSTRACT

Background:InMarseille, France,followingaﬁrstsevereacuterespiratorysyndromecoronavirus2

(SARS-CoV-2)outbreakinMarch–May2020,asecondepidemicphaseoccurredfromJune,involving10

newvariants.TheMarseille-4variantcausedanepidemicthatstartedinAugustandisstillongoing.

Methods:The1038SARS-CoV-2wholegenomesequencesobtainedinourlaboratorybynext-generation

sequencingwithIlluminatechnologywereanalysedusingNextcladeandnextstrain/ncovpipelinesand

IQ-TREE.AMarseille-4-speciﬁcqPCRassaywasimplemented.Demographicandclinicalfeatureswere

comparedbetweenpatientswiththeMarseille-4variantandthosewithearlierstrains.

Results:Marseille-4harbours13hallmarkmutations.OneleadstoanS477Nsubstitutioninthereceptor

bindingdomainofthespikeproteintargetedbycurrentvaccines.UsingaspeciﬁcqPCR,itwasobserved

thatMarseille-4caused12–100%ofSARS-CoV-2infectionsinMarseillefromSeptember2020,being

involvedin2106diagnoses.Thisvariantwasmorefrequentlyassociatedwithhypoxemiathanwereclade

20AstrainsbeforeMay2020.Itcausedare-infectionin11patientsdiagnosedwithdifferentSARS-CoV-2

strainsbeforeJune2020,suggestingeithershort-termprotectiveimmunityoralackofcross-immunity.

Conclusions:Marseille-4shouldbeconsideredasamajorSARS-CoV-2variant.Itssuddenappearance

pointstowardsananimalreservoir,possiblymink.Theprotectiveroleofpastexposureandcurrent

vaccinesagainstthisvariantshouldbeevaluated.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(

http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

The severeacuterespiratorysyndromecoronavirus 2 (SARS-CoV-2)epidemicthatstartedinWuhan,ChinainDecember2019 hasspreadrapidlyaroundtheworld(https://coronavirus.jhu.edu/ map.html). At the Méditerranée Infection Institute (IHU) in Marseille,routinediagnosisofSARS-CoV-2byPCRwassetupin January2020(Lagieretal.,2020;Colsonetal.,2020a).Theﬁrst SARS-CoV-2-infected patient was diagnosed at the IHU on February27,2020(Colsonetal.,2020c)( https://www.mediterra-nee-infection.com/covid-19/). Since then, more than 450 000 SARS-CoV-2 PCR testshavebeenperformed atIHU,2000virus

isolates have been obtained by cell culture, whole genome sequencinghasbeenperformedon2000 isolates, and carehas beengivento14000SARS-CoV-2-positivepatients.

InEurope,SARS-CoV-2circulationhassofarbeencharacterized bytwomajorepisodes.Theﬁrstone,referredtohereinasphase1, startedinFebruaryandalmostendedinMay(Colsonetal.,2020d;

Colsonetal.,2021).However,asecondphase(phase2)suddenly occurredattheendofJune,exhibitinganatypicalepidemiccurve, which ledustosuspect that thetwoepisodes werecaused by distinctviralvariants.Hence,wholegenomesequencingof SARS-CoV-2 strains was performed over time to characterize their geneticdiversity.Thisenabledustoidentify10distinctgenomic patternsthatsuccessivelyorconcomitantlyspreadintheMarseille area (Colson et al., 2021; Fournier et al., 2021). Of these, two variantswereidentiﬁedat highfrequencyin thepopulationof individualsdiagnosedattheIHU.TheMarseille-1variantcaused mildinfections inyoungerpatientsand predominatedfromthe endofJunetotheendofJuly2020(Colsonetal.,2021).Evidence

*Correspondingauthorsat:IHU–MéditerranéeInfection,19–21BoulevardJean Moulin,13005Marseille,France.

E-mailaddresses:pierre-edouard.fournier@univ-amu.fr(P.-E.Fournier),

didier.raoult@gmail.com(D.Raoult).

https://doi.org/10.1016/j.ijid.2021.03.068

1201-9712/©2021TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

International

Journal

of

Infectious

Diseases

(3)

wasaccumulatedindicatingthatthisvariantoriginatedinAfrica andwasbroughttoMarseillebyferryboattravellersandsailors fromNorthAfrica.InFrance,itdidnotspreadoutsideMarseilleand itvanishedrapidly.OnJuly29,2020,anewvariantwasidentiﬁed andnamedMarseille-4(Figures1and2).Theaimofthisstudywas toexaminethevirological,clinical,andepidemiological character-isticsofthisvariant.

Materialsandmethods Genomesequencing

Viralgenomeswereobtainedfromnasopharyngealswabﬂuid usingnext-generationsequencing(NGS)andtheIlluminaNextera XTpaired-endstrategyonaMiSeqinstrument(IlluminaInc.,San Diego, CA, USA), as described previously (Colson et al., 2021). Genomeconsensussequenceswereassembledbymappingonthe SARS-CoV-2genomeofGenBankaccessionnumberNC_045512.2 (Wuhan-Hu-1 isolate)using CLCGenomicsworkbenchv.7,with thresholdsof80%fornucleotidesequencecoverageand90%for

nucleotidesimilarity.SARS-CoV-2sequencesobtainedinthestudy institute have beensubmitted tothe GISAID database(https:// www.gisaid.org).

Genomeanalysis

The1038SARS-CoV-2wholegenomesequences obtainedin ourlaboratorywereanalysedusingtheNextcladetool(https:// clades.nextstrain.org/) (Hadfield et al., 2018) and an in-house scriptwritteninPython.Viralcladesweredefinedonthebasisof at least five available genomes sharing the same pattern of mutations. Phylogenetic trees were reconstructed using the nextstrain/ncov tool (https://github.com/nextstrain/ncov) and visualized with Auspice software (https://docs.nextstrain.org/ projects/auspice/en/stable/). In addition, the SARS-CoV-2 genomesobtainedinourlaboratorywereintegratedintoanother phylogeneticanalysistogetherwithsequencesfromtheGISAID database (https://www.gisaid.org) that were recovered from humans and mink. All of these genomes were aligned using MAFFT v.7 (Katoh and Standley, 2013). Then, phylogeny

Figure1.SchematicdiagramoftheevolutionoftheSARS-CoV-2Marseille-4variantinEurope.

P.-E.Fournier,P.Colson,A.Levasseuretal. InternationalJournalofInfectiousDiseases106(2021)228–236

(4)

Figure2. EvolutionoftheMarseille-4variantovertime.(a)WeeklynumberofgenomesoftheMarseille-4variantworldwide.(b)Weeklyfrequencynormalizedto100%of thecountrieswheregenomesoftheMarseille-4variantwereobtained.(c)TimedistributionofthedailynumberofgenomesoftheMarseille-4variantpercountry.(d) WeeklynumberofgenomesoftheMarseille-4variantinFrenchregions.(e)Weeklyfrequencynormalizedto100%oftheFrenchregionswheregenomesoftheMarseille-4 variantwereobtained.

(5)

reconstructionwasperformedusingIQ-TREEsoftwarewiththe GTRModeland1000ultrafastbootstraprepetitions(http://www. iqtree.org)(Minhetal.,2020),andthetreewasvisualizedwith iTOL (Interactive Tree Of Life) software (https://itol.embl.de/) (LetunicandBork,2016).

PCRdetectionoftheSARS-CoV-2Marseille-4variant

A qPCR system was designed that targetsthe nsp4 gene at nucleotidepositions9460–9543inreferencetogenomeGenBank accession number NC_045512.2(Wuhan-Hu-1 isolate). The

primers and probe are described in Supplementary material Table S1.ThisqPCR was run onan LC480 thermocycler(Roche Diagnostics, Mannheim, Germany). The reaction mixture con-tained5

m

lof4XTaqManFastVirus1-StepMasterMix(Thermo FisherScientiﬁc,GrandIsland,NY,USA),0.5

m

lofforwardprimer (10pmol/

m

l),0.5

m

lofreverseprimer(10pmol/

m

l),0.4

m

lofprobe (10pmol/

m

l),and8.6

m

lofwater,anditwascompletedwith5

m

lof extractedviral RNA.PCR conditions were as follows:a reverse transcriptionstepfor10minat50_C,_then₂₀_s_at₉₅_C_followed_by

40cyclescomprisingadenaturationstepat95Cfor15sanda hybridizationandelongationstepat60Cfor60s.

Figure3.Genomesequence-basedphylogenetictreesshowingtheevolutionofSARS-CoV-2Marseille-4variantstrains.(a)Time-scalephylogenetictree.(b)Phylogenetic treebasedonmutationalevents.

Full-length genomesequencesobtainedinthisstudywerecomparedtothoseavailableintheGISAIDdatabase(https://www.gisaid.org/).Phylogenetictreeswere reconstructedandvisualizedusingtheNextstrainpipeline(https://github.com/nextstrain/ncov/)(Hadﬁeldetal.,2018).

(6)

Comparisonsofepidemiologicalandclinicalfeaturesofpatients diagnosedduringphases1and2

Thedemographicandclinicalfeaturesofpatientsinfectedwith the Marseille-4 variant were compared to those of patients infectedwithclade20Astrainsduringphase1,betweenMarchand May 2020. Statisticalanalyses were conductedusing R version 4.0.2. (R Core Team, R Foundation for Statistical Computing, Vienna,Austria,2020;https://www.Rproject.org/).

Results

IdentiﬁcationandcirculationoftheMarseille-4variant

The highly transmissible SARS-CoV-2 Marseille-4 variant identiﬁed in Marseille at theend of July 2020rapidly became predominant, reaching 100% of identiﬁed viral strains in the geographicalareaonNovember2,2020.Usinggenomesequences availablethroughtheGISAIDdatabase(https://www.gisaid.org/),

the outbreaks of this variant were traced back in different countries.ThefirstcaseofinfectionwiththeMarseille-4variant, named 20A.EU2 in the Nextstrain classification (https://clades. nextstrain.org/)(Hodcroftetal.,2020),wasdetectedinaGerman patientonMarch24,2020.Then,twocasesweredetected ona Balearicisland, Spain,onMay29 andJune18, 2020.Additional casesweredetectedinSouthwesternFrancefromJuly9,thenin Denmark, and from August1 in otherEuropean countriesand otherregionsofFrance(Figures1and2;Supplementarymaterial FigureS1).TheMarseille-4variantwasdetectedfromSeptember inNorthAmerica(Canada,thenUSA),Australia,andNewZealand, fromOctoberinAsia(Thailand,HongKong,Singapore,andSouth Korea)andAfrica(TunisiaandMorocco),andfromDecemberin Israel. In Marseille, 269 Marseille-4 complete genomes were sequencedfrominfectedpatients,andaMarseille-4-specificqPCR (Supplementary material Table S1) was designed that enabled rapididentificationofanadditional1579cases.Overall,thisvariant caused2106casesandaccountedforabouttwo-thirdsofall SARS-CoV-2virusestestedfromSeptember2020toJanuary2021atIHU.

Table1

(7)

Genomicfeatures

TheMarseille-4variantevolvedfromclade20Astrains(Figure 3) and is characterized by a combination of 20 mutations compared to the Wuhan-Hu-1 strain. Among these mutations, 13arehallmarksofthisvariant(C4543T,G5629T,G9526T,C11497T, G13993T, G15766T, A16889G, G17019T, G22992A, C25710T, T26876C,G28975C,andG29399A)(SupplementarymaterialFigure S2).TheMarseille-4variantwasprovisionallysubdividedinto11 subgroups(Marseille-4-A1toMarseille-4-J),witha geneticdrift rangingfrom21to24mutationscomparedtotheWuhan-Hu-1 strain(Table1).Strikingly,comparativegenomicsshowedthatthe setof13hallmarkmutationsappearedaltogether.Theyarelosses ofaGinsevencasesandofaCinthreecases,andarescattered along the viral genome. Seven (46%) are non-synonymous mutations, including two located in the RNA-dependent RNA polymerase(RdRP)(nsp12;A176SandV767L),twointheNTPase/ helicase(nsp13;K1141RandE1184D),twointhenucleocapsid(N; M234IandA376T),andoneinthespikeglycoprotein(S;S477N). Fifteenadditionalmutationswereobservedin5viralgenomes obtainedinthestudyinstitute(C222U,C503U,G2600U,A2647G, C8937U, G18105U, C23191U, G25534U, U26442C, G26720U, G27877U,C27942U,G28086U,G29701A, andG29511U).Overall, 283 nucleotide positions were found to be mutated in 1 Marseille-4genomes,mostlyinthensp3andSgenes.Theywere mostfrequentlyC>U(36%),G>U(25%),U>C(8%),G>A(6%),and A>G(5%)mutations,andU> deletions(6%).Phylogenetically, theMarseille-4variantwasfoundtofallwithinagroupofviruses fromEuropeonly(SupplementarymaterialFigureS3).

TheMarseille-4variantharbourstheS477Nsubstitutionwithin thereceptorbindingdomain(RBD)ofthespikeglycoprotein.This RBDattachestheviriontothecellmembranebybindingtothe viralreceptorACE2,andmediatesviralentry(Lanetal.,2020).The spike isa major targetofneutralizing antibodies(Barnesetal., 2020)andthecurrentvaccines(DaiandGao,2020)(Figure4).The S477Nsubstitutionhasbeenreportedtobeassociatedwithbroad resistancetomonoclonalneutralizingantibodies(Liuetal.,2020). Thesedatacouldexplainthelackofresistancetoinfectionbythis Marseille-4 variant among people previously infected with differentstrainsthat circulatedearlier, duringtheﬁrstphase of the2020pandemic.Thissubstitutionliesbetweensubstitutions

observedinvirusesinfectinghumansandothersseeninviruses infecting mink (Figure 4) (Garry, 2021). It adds to the D614G substitutionthat wasreportedtoincreasethestability ofspike trimersandtoconfergreaterafﬁnityforACE2(Korberetal.,2020). Itis worthnotingthattheﬁrstgenomeavailablein theGISAID database(EPI_ISL_7079562020-03-24),originatingfromGermany on March24, 2020, does not harbour this S477Nsubstitution, whichmayexplainwhyitdidnotapparentlyspreadfurther.Other criticalmutationsmaybesubstitutionQ57HinORF3a,aviroporin that formsion channels and wasreportedas requiredfor viral replication,virulence,andrelease,andisalsopredictedtobea pro-apoptotic protein (Bianchi et al., 2021; Law et al., 2005), and substitutionsA176Sin theRdRPand K1141RandE1184Din the NTPase/helicase.

InsearchoftheoriginoftheMarseille-4variant

TheoriginoftheMarseille-4variantiscurrentlyunknown.It emerged abruptlywithitsblock ofspecificmutations, withno known intermediate form, at a time when the SARS-CoV-2 epidemichad almostendedinFranceand Europe(Figures1–3). ThisapparentlydiscontinuousevolutionofSARS-CoV-2genomes isabnormal,particularlyifweconsiderthatafteritsfirstdetection thisvariantshowedasubsequentmutationratesimilartothatof other lineages (e.g., mutation in the RdRP did not alter the polymerase fidelity). Although the existence of a missing intermediatethathasnotsofarbeensequencedfromcoronavirus disease2019(COVID-19)patientscannotbeexcluded,thiscould alsosuggestthatthereisanoverlookedreservoirinwhichthevirus wassubmittedtoaselectionpressurethatfavouredaparticular increaseinmutationaccumulation.

Interestingly,amongthe10516sequencesfromtheMarseille-4 variant in theGISAID database (on January 24, 2021), the272 genomes from our laboratory had close relatives with those originatingfromNorthernEurope,mostlyDenmark(n=3366),the UK(n=2652),andSwitzerland(n=1147)(Supplementarymaterial Figure S1). A phylogenetic tree was constructed that included genomesfromminkandhumanSARS-CoV-2strains.Minkstrains weredividedintofourandsixmaingroupsforthesamplesfrom theNetherlandsandDenmark,respectively(Figure5).Acommon phylogenetic node between mink strains, the Marseille-4,

Figure4.Three-dimensionalstructureofthespikeproteinshowingtheaminoacidsubstitutionsinthereceptor-bindingmotifoftheMarseille-4variantandofothervariants detectedinhumansand/ormink.

ThestructurewaspredictedusingthePhyre2webportal(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index)(Kelleyetal.,2015)andvisualizedusingthePymol toolv.1.8(https://pymol.org/2/)(JansonandPaiardini,2020).Aminoacidswhereasubstitutionwasobservedinhumansareshowninred,thosewhereasubstitutionwas observedinminkareshowninyellow,andthosewhereasubstitutionwasobservedinhumansandminkareshowninorange.

(8)

Marseille-5, Marseille-6 variants, and the20 H/501Y.V2 variant wasobserved.ThisnodepointedtothecommonmutationQ57Hin ORF3adescribedabove.

The rapid emergence of the Marseille-4 variant during the summerof2020,aftertheendoftheﬁrstepidemicphase,may pointtowardsananimalreservoir.Minkfarmswereidentiﬁedas reservoirsandsourcesofSARS-CoV-2mutantsintheNetherlands inApril(OudeMunninketal.,2021)andinDenmarkinJune2020 (Hammeretal.,2021).InFrance,oneofthefourminkfarmswas infectedandanimalswereculled.SARS-CoV-2isanepizooticagent that caused anoutbreak inhumansbeforebeingtransferredto minkinwhichitspreadrapidlythroughdenselycagedanimalsand subsequentlybecameasourceforhumaninfection.Todate,more than800humaninfectionsfromminkhavebeenreported(Oude Munninketal.,2021).Onehypothesiscouldbethatahuman SARS-CoV-2frominfectedcaregiversinfectedmink,thenthefrequency of viralmutationschanged intheminkdue toa differenthost selection pressure, and this mink-adapted virus (with multiple mutations)becameanewviralsourcetoinfecthumans.

ThegenomeobtainedfromaGermanpatientsampledonMarch 24,2020(EPI_ISL_7079562020-03-24)isatypicalasitisdevoidof theS477Nsubstitution,oneoftheMarseille-4hallmarkmutations, butharboursmoremutations(n=31)thantheotherMarseille-4 strains, includingin the Nsp2, Nsp3, S, and N proteins,and in ORF1b,particularlytheNsp14exonuclease,whichhas proofread-ingactivity(Shannonetal.,2020).Theevolutionaryrelationships

ofthisgenomewithotherMarseille-4genomeswarrantsfurther investigationwiththeavailabilityofothergenomesobtainedfrom samplescollectedduringthesameperiod.

Clinicalﬁndings:theMarseille-4variantmayescapeimmunity conferredbyaﬁrstSARS-CoV-2infection

Comparedtotheclade20Astrainsthatpredominatedduring phase1betweenMarchandMay2020,theMarseille-4variantwas associatedwithalowerfrequencyofcough,rhinitis,andolfactory and gustatory disorders (Table 2).By contrast, hypoxemia was morefrequentinpatientsinfectedwiththeMarseille-4variant.It hasbeenreportedthatdifferencesobservedinCOVID-19severity mayinpartbeassociatedwiththedysfunctionofcellularimmune responsestoSARS-CoV-2and/oraweaknessoftheneutralizing humoralresponse(Moderbacheretal.,2020).

WediagnosedtwosuccessivecasesofCOVID-19,separatedby more than 4 months, in 11 patients. The ﬁrst infection was diagnosedbeforeJune2020whenMarseille-4wasnotcirculating in Marseille (Colson et al., 2020b; Brouqui et al., 2021), and genomicorqPCR(oneand10patients,respectively)conﬁrmation thatthesecondepisodewascausedbytheMarseille-4variantwas obtained.Thissuggestseithershortprotectiveimmunity(onlya few weeks or months), as observed previously with seasonal coronaviruses(Edridgeetal.,2020),oralackofcross-immunity betweendifferentSARS-CoV-2 variants,allowing Marseille-4 to

Figure5. PhylogenetictreebasedonSARS-CoV-2full-lengthgenomes.

Atotalof744genomesofSARS-CoV-2wereintegratedinaphylogeneticanalysis.AllgenomeswerealignedusingMAFFTversion7(KatohandStandley,2013).The phylogenetictreewasreconstructedusingIQ-TREEwiththeGTRmodel,with1000ultrafastbootstraprepetitions(Minhetal.,2020),andwasvisualizedwithiTOL (InteractiveTreeOfLife;https://itol.embl.de/)(LetunicandBork,2016).DK,Denmark;NTH,TheNetherlands.

(9)

evadeimmuneprotectionelicitedbyanotherearliervariant.This mayberelatedtotheS477Nmutation,whichcouldchangethe afﬁnityofRBDforACE2anddecreasethesensitivityofthevariant virustoanti-RBD-speciﬁcneutralizingantibodies(Andreanoetal., 2020).

Discussion

TherecentevolutionoftheSARS-CoV-2epidemicsreﬂectsthe generation of new variants in different ecosystems that have spreadwithglobalizationandhavereplacedtheoriginalvariants arisingfromWuhan.Somecanbeassociatedwithdifferentclinical features,asinthecaseoftheMarseille-4variant.Theecosystems allowingthisselectionmayconsistofhumangroupsisolatedfora while, or animal reservoirssuch asmink in large farms.Large concentrations of farmed mink have been infected by human SARS-CoV-2(OudeMunninketal.,2021).Undertheseconditions, sub-speciation mayoccur(Darwin,1859).The re-connection of isolated ecosystems (either countries and/or farmed animals) where different variants have developed, has generated new outbreaksincountriesthatwereexposedtoincomingpopulations suchastravellers.

Severalreasonsledustobelievethatminkwerethesourceof the Marseille-4 variant. First, this variant carries a new set of severalmutationsthatseemstohaveappearedsuddenlybasedon the analysis of all the genomes available worldwide, and not gradually. This suggeststhat this brutalgenome evolution was overlooked.Secondly,therewasnoSARS-CoV-2epidemicinFrance atthetimeoftheemergenceofthisvariant,exceptinaregionnear thecityofLaval(Mayenne,WesternFrance)locatedbetweenthe mostdenseareaforwildmink(Brittany)andaminkfarm (Eure-et-Loire)where30%ofminkwereprovedtobeSARS-CoV-2-positive by qPCR and 97% had antibodies against the virus. As a consequence, the entire mink population of the farm was slaughtered ( https://www.plateforme-esa.fr/article/covid-19-et-animaux-mise-a-jour-au-05-01-2021) (Fenollar et al., 2021). Progressively,thisSARS-CoV-2epidemicspreadinFranceduring the summer, and we observed the ﬁrst cases of Marseille-4 infectionsinMarseillewhenFrenchtouristsarrivedinourregion. Forunknownreasons,thesequenceofthevirusobtainedfromthe farmedminkinfectedinmid-Novemberisnotyetavailable.

Inconclusion,overallwebelievethatthesegregationofviral strainsinisolatedgeographicalareasandinanimalreservoirsmay contributetoexplainthedifferencesobservedamongtheepidemic curves around the world. This would help to understand the mechanismofthesecondepisodeofSARS-CoV-2circulationthat developedinMarseille,initiallycausedbyanAfricanvariantthat disappeared (Colson et al., 2021), and then by emerging new variantslinkedtodifferentareasofEurope,includingthosehosting hugeminkfarms.Finally,theroleofthetreatmentsofCOVID-19 withremdesivirorhyperimmuneplasma(Choietal.,2020;Kemp et al., 2020) in generating and selecting variants should be consideredastheymayalsohavecontributedtothenewoutbreaks observedinthemostdevelopedcountries.

Sincetheﬁnalacceptanceof thisarticle,thesequenceofthe SARS-CoV-2genomeobtainedfromafarmminksampledthe15th

ofNovember,2020inEure-et-Loirewaseventuallyreleasedthe 29th_of_March,₂₀₂₁_{(EPI_ISL_1392906).}_As_we_suspected_and_stated

in the present article, this genome is strictly identical to the genomeofa Marseille-4variantconﬁrmingourhypothesisofa commonsourceofthisvariantbetweenFrenchminksandhumans. Funding

ThisworkwassupportedbytheFrenchGovernmentunderthe “InvestmentsfortheFuture”program managedbytheNational Agency for Research (ANR), Méditerranée-Infection10-IAHU-03, andwasalsosupportedbyRegionProvence-Alpes-Côted’Azurand EuropeanfundingFEDERPRIMMI(FondsEuropéende Developpe-mentRegional-PlateformesdeRechercheetd’Innovation Mutua-liséesMediterranéeInfection),FEDERPA0000320PRIMMI. Ethicalapproval

The study was approved by the Ethics Committee of the MéditerranéeInfectionInstitute(ReferenceNo.2020-016-3). Availabilityofdataandmaterials

Data underlying the study are available from the GISAID database (https://www.gisaid.org/) or from the corresponding authoruponrequest.

Table2

Demographics,outcomes,andclinicalsymptomsinpatientsinfectedwithdifferentSARS-CoV-2variants.

Demographicsandoutcomes(N=759) 20A(n=339) Marseille-4(n=420) P-valuea

n % n %

Sex 0.059

Female 188 55.5 204 48.6

Male 151 44.5 216 51.4

Age(years),meanSD 50.222.3 48.923.1 0.41

Hospitalization 53 15.6 68 16.2 0.835

Transfertointensivecareunit 5 1.5 10 2.4 0.44

Death 10 2.9 16 3.8 0.52

Symptoms(N=444) 20A(n=254) Marseille-4(n=190) P-valuea

n % n % Cough 123 48.4 73 38.4 0.036* Rhinitis 106 41.7 37 19.5 <0.0001* Anosmia 76 29.9 35 18.5 0.006* Ageusia 71 27.9 34 18.0 0.015* Dyspnoea 72 28.3 42 22.1 0.136 SpO2<96% 37 14.6 42 22.1 0.04*

SD,standarddeviation;SpO2,oxygensaturation.

a_Chi-square_test_or_Fisher’s_exact_test_for_qualitative_variables;_Student_t-test_for_quantitative_variables. *_{Statistically}_{signiﬁcant.}

(10)

Conﬂictofinterest

The authorshavenoconﬂictsofinteresttodeclare.Funding sourceshadnoroleinthedesignandconductofthestudy,inthe collection,management,analysis,andinterpretationofthedata,or inthepreparation,review,andapprovalofthemanuscript. Authorcontributions

Conceivedanddesignedtheexperiments:DR,PEF,PCandPG. Contributedmaterials/analysistools:PEF,PC,AL,CD,PG,MB,JD, LB,LP,JCLandFF.Analysedthedata:PEF,PC,AL,PG,JD,JCL,FFand DR.Wrotethepaper:PEF,PC,CD,PGandDR.Allauthorsapproved theﬁnalversionofthemanuscript.

Acknowledgements

WearegratefultoOliviaArdizzoni,VincentBossi,Madeleine Carrera,VeraEsteves-Vieira,LaurenceThomas,PriscillaJardot,and Raphael Tola for their technical help, and to Audrey Giraud-GatineauandLéaLucianifortheirhelpwiththedataanalysis.The manuscripttexthasbeeneditedbyanativeEnglishspeaker. AppendixA.Supplementarydata

Supplementarymaterialrelatedtothisarticlecanbefound,inthe onlineversion,atdoi:https://doi.org/10.1016/j.ijid.2021.03.068. References

AndreanoE,PicciniG,LicastroD,JohnsonNV,PacielloI,MonegoSD,etal. SARS-CoV-2escapeinvitrofromahighlyneutralizingCOVID-19convalescentplasma. bioRxiv2020;,doi:http://dx.doi.org/10.1101/2020.12.28.424451.

BarnesCO,WestJr.AP,Huey-TubmanKE,HoffmannMAG,SharafNG,HoffmanPR, et al. Structures of human antibodiesbound to SARS-CoV-2 spike reveal commonepitopesandrecurrentfeaturesofantibodies.Cell2020;182:828–42. BianchiM,BorsettiA,CiccozziM,PascarellaS.SARS-Cov-2ORF3a:mutabilityand

function.IntJBiolMacromol2021;170:820–6.

BrouquiP,ColsonP,MelenotteC,HouhamdiL,BedottoM,DevauxC,etal.COVID-19 re-infection. Eur J Clin Invest 2021;(March)e13537, doi:http://dx.doi.org/ 10.1111/eci.13537Epubaheadofprint.

ChoiB,ChoudharyMC,ReganJ,SparksJA,PaderaRF,QiuX,etal.Persistenceand evolutionof SARS-CoV-2in animmunocompromised Host.N EnglJ Med 2020;383:2291–3.

ColsonP,Esteves-VieiraV,Giraud-GatineauA,ZandottiC,FilosaV,ChaudetH,etal. Temporal and age distributions of SARS-CoV-2 and other coronaviruses, southeasternFrance.IntJInfectDis2020a;101:121–5.

ColsonP,FinaudM,LevyN,LagierJC,RaoultD.EvidenceofSARS-CoV-2re-infection withadifferentgenotype.JInfect2020b;(November),doi:http://dx.doi.org/ 10.1016/j.jinf.2020.11.011S0163-4453(20)30706-4.Onlineaheadofprint. ColsonP,LagierJC,BaudoinJP,BouKJ,LaScolaB,RaoultD.Ultrarapiddiagnosis,

microscopeimaging,genomesequencing,andcultureisolationofSARS-CoV-2. EurJClinMicrobiolInfectDis2020c;39:1601–3.

ColsonP,LevasseurA,DelerceJ,ChaudetH,BossiV,BenKhedherM,etal.Dramatic increaseintheSARS-CoV-2mutationrateandlowmortalityrateduringthe secondepidemicinsummerinMarseille.IHUPre-prints;2020,doi:http://dx. doi.org/10.35088/68c3-ew82.

ColsonP,LevasseurA,GautretP,FenollarF,HoangVT,DelerceJ,etal.Introduction intotheMarseillegeographicalareaofamildSARS-CoV-2variantoriginating fromsub-SaharanAfrica.TravelMedInfectDis2021;40:101980.

DaiL,GaoGF.ViraltargetsforvaccinesagainstCOVID-19.NatRevImmunol2020;1– 10.

DarwinC.Ontheoriginofspecies.London:JohnMurray;1859.

EdridgeAWD,KaczorowskaJ,HosteACR,BakkerM,KleinM,LoensK,etal.Seasonal coronavirusprotectiveimmunityisshort-lasting.NatMed2020;26:1691–3. FenollarF,MediannikovOY,MaurinM,DevauxCA,ColsonP,LevasseurA,etal.Mink,

SARS-CoV-2,andthehuman-animalinterface.FrontMicrobiol2021;(April), doi:http://dx.doi.org/10.3389/fmicb.2021.663815inpress.

FournierPE,ColsonP,LevasseurA,GautretP,BedottoM,FilosaV,etal.Genome sequenceanalysis enableddecipheringtheatypicalevolutionofCOVID-19 epidemicsinMarseille,France. IHUPre-prints;2021,doi:http://dx.doi.org/ 10.35088/kmct-tj43.

GarryRF.MutationsarisinginSARS-CoV-2spikeonsustainedhuman-to-human transmissionand human-to-animalpassage.Virological org;2021.https:// virological.org/t/mutations-arising-in-sars-cov-2-spike-on-sustained-human-to-human-transmission-and-human-to-animal-passage/578.

HadﬁeldJ,MegillC,BellSM,HuddlestonJ,PotterB,CallenderC,etal.Nextstrain: real-timetrackingofpathogenevolution.Bioinformatics2018;34:4121–3. HammerAS,QuaadeML,RasmussenTB,FonagerJ,RasmussenM,MundbjergK,

etal.SARS-CoV-2transmissionbetweenMink(Neovisonvison)andhumans, Denmark. Emerg Infect Dis 2021;27:547–51, doi:http://dx.doi.org/10.3201/ eid2702.203794Epub2020November18.

HodcroftEB,ZuberM,NadeauS,ComasI,GonzalezCandelasF,SeqCOVID-SPAIN Consortium,etal.EmergenceandspreadofaSARS-CoV-2variantthrough Europeinthesummerof2020.medRxiv2020;,doi:http://dx.doi.org/10.1101/ 2020.10.25.20219063.

JansonG,PaiardiniA.PyMod3:acompletesuiteforstructuralbioinformaticsin PyMOL.Bioinformatics2020;(October)btaa849,doi:http://dx.doi.org/10.1093/ bioinformatics/btaa849Onlineaheadofprint.

KatohK,StandleyDM.MAFFTmultiplesequencealignmentsoftwareversion7: improvementsinperformanceandusability.MolBiolEvol2013;30:772–80. KelleyLA,MezulisS,YatesCM,WassMN,SternbergMJ.ThePhyre2webportalfor

proteinmodeling,predictionandanalysis.NatProtoc2015;10:845–58. KempSA,CollierDA,DatirR,FerreiraI,Gayed S,Jahun A,etal.Neutralising

antibodiesinSpikemediated SARS-CoV-2adaptation.medRxiv 2020;,doi:

http://dx.doi.org/10.1101/2020.12.05.20241927.

KorberB,FischerWM,GnanakaranS,YoonH,TheilerJ,AbfaltererW,etal.Tracking changesinSARS-CoV-2Spike:evidencethatD614Gincreasesinfectivityofthe COVID-19virus.Cell2020;182:812–27.

LagierJC,MillionM,GautretP,ColsonP,CortaredonaS,Giraud-GatineauA,etal. Outcomes of 3,737 COVID-19 patients treated with hydroxychloroquine/ azithromycinandotherregimensinMarseille,France:aretrospectiveanalysis. TravelMedInfectDis2020;36:101791.

LanJ,GeJ,YuJ,ShanS,ZhouH,FanS,etal.StructureoftheSARS-CoV-2spike receptor-bindingdomainboundtotheACE2receptor.Nature2020;581:215–20. LawPTW,WongCH,AuTCC,ChuckCP,KongSK,ChanPKS,etal.The3aproteinof severeacuterespiratorysyndrome-associatedcoronavirusinducesapoptosisin VeroE6cells.JGenVirol2005;86:1921–30.

LetunicI,BorkP.Interactivetreeoflife(iTOL)v3:anonlinetoolforthedisplayand annotationofphylogeneticandothertrees.NucleicAcidsRes2016;44:W242–5. LiuZ,VanBlarganLA,BloyetLM,RothlaufPW,ChenRE,StumpfS,etal.Landscape analysis of escape variants identiﬁes SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization. bioRxiv 2020;, doi:http://dx.doi.org/10.1101/2020.11.06.372037.

MinhBQ,SchmidtHA,ChernomorO,SchrempfD,WoodhamsMD,vonHA,etal. IQ-TREE2:newmodelsandefﬁcientmethodsforphylogeneticinferenceinthe genomicera.MolBiolEvol2020;37:1530–4.

ModerbacherCR,RamirezSI,DanJM,GrifoniA,HastieKM,WeiskopfD,etal. Antigen-speciﬁcadaptiveimmunitytoSARS-CoV-2inacuteCOVID-19and associationswithageanddiseaseseverity.Cell2020;183:996–1012. Oude Munnink BB, Sikkema RS, Nieuwenhuijse DF, Molenaar RJ,Munger E,

MolenkampR,etal. TransmissionofSARS-CoV-2onminkfarmsbetween humansandminkandbacktohumans.Science2021;371:172–7.

ShannonA,LeNT,SeliskoB,EydouxC,AlvarezK,GuillemotJC,etal.Remdesivirand SARS-CoV-2: structural requirements at both nsp12 RdRp and nsp14 exonuclease active-sites. Antiviral Res 2020;178:104793, doi:http://dx.doi. org/10.1016/j.antiviral.2020.104793.:104793.