2015 an observational, laboratory-based study of outbreaks of middle east respiratory syndrome...

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©TheAuthor2014.PublishedbyOxfordUniversityPressonbehalfoftheInfectiousDiseasesSocietyofAmerica.Allrightsreserved.ForPermissions,pleasee‐mail:[email protected].

Anobservational,laboratory‐basedstudyofoutbreaksofMERS‐Coronavirusin

JeddahandRiyadh,KingdomofSaudiArabia,2014

ChristianDrosten1,2,*,#,DoreenMuth1,*,VictorCorman1,2,*,RaheelaHussain4,7,*,

MalakiAlMasri3,WaleedHajOmar7,OlfertLandt5,AbdullahAssiri3,Isabella

Eckerle1,AliAlShangiti7,JaffarA.Al‐Tawfiq6,AliAlbarrak8,AlimuddinZumla3,9,

AndrewRambaut10,ZiadMemish3,11,+

1InstituteofVirology,UniversityofBonnMedicalCentre,Bonn,Germany

2GermanCentreforInfectionResearch

3GlobalCentreforMassGatheringsMedicine(GCMGM),MinistryofHealth,Riyadh,KingdomofSaudiArabia(KSA)

4JeddahRegionalLaboratory,Jeddah,KingdomofSaudiArabia

5Tib‐Molbiol,Berlin

6JohnsHopkinsAramcoHealthcare,SaudiAramco,Dhahran,KingdomofSaudiArabiaandIndianaUniversitySchoolofMedicine,Indianapolis,IN(USA)

7RegionalLaboratory,MinistryofHealth

8PrinceSultanMilitaryMedicalCity,Riyadh,KSA

9DivisionofInfectionandImmunity,UniversityCollegeLondon,andNIHRBiomedicalResearchCentre,UniversityCollegeLondonHospitalsNHSFoundationTrust,London,UnitedKingdom

10InstituteofEvolutionaryBiology,UniversityofEdinburgh,CentreforInfection,ImmunityandEvolution,UniversityofEdinburgh,UK,andFogartyInternationalCenter,NationalInstitutesforHealth,USA

11AlfaisalUniversity,Riyadh,KingdomofSaudiArabia

+Correspondingauthor:ZiadA.MemishEmail:[email protected]

#Alternatecorrespondingauthor:ChristianDrostenEmail:drosten@virology‐bonn.de

*equalcontribution

Clinical Infectious Diseases Advance Access published October 16, 2014 at W

ashington State University L

ibraries on October 21, 2014

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Summary

Inspring2014,anexplosiveoutbreakofMERS‐CoronavirusinJeddahcaused

conjecturesaboutchangesinviraltransmissibility.Functionalexaminationof

circulatingvirusesaswellasanalysesofdiagnosticlaboratorydatasuggestcausationby

nosocomialtransmissionofabiologicallyunchangedvirus.

Abstract

Background

Inspring2014,asuddenriseinthenumberofnotifiedMERS‐Coronavirusinfections

occurredacrossSaudiArabiawithafocusinJeddah.Hypothesestoexplaintheoutbreak

patternincludeincreasedsurveillance,increasedzoonotictransmission,nosocomial

transmission,changesinviraltransmissibility,aswellasdiagnosticlaboratoryartifacts.

Methods

DiagnosticresultsfromJeddahRegionalLaboratorywereanalyzed.Virusesfromthe

JeddahoutbreakandvirusesoccurringduringthesametimeinRiyadh,Al‐Kharj,and

Madinahwerefullyorpartiallysequenced.Asetoffoursinglenucleotide

polymorphismsdistinctivetotheJeddahoutbreakweredeterminedfromadditional

viruses.VirusesfromRiyadhandJeddahwereisolatedandstudiedincellculture.

Resultsandconclusions

Upto481sampleswerereceivedperdayforRT‐PCRtesting.Alaboratoryproficiency

assessmentsuggestedpositiveandnegativeresultstobereliable.Forty‐ninepercentof

168positive‐testingsamplesduringtheJeddahoutbreakstemmedfromKingFahd

Hospital.AllvirusesfromJeddahweremonophyleticandsimilar,whilevirusesfrom

Riyadhwereparaphyleticanddiverse.Ahospital‐associatedtransmissioncluster,to

whichcasesinIndiana/USAandtheNetherlandsbelonged,wasdiscoveredinRiyadh.

OneJeddah‐typeviruswasfoundinRiyadh,withmatchingtravelhistorytoJeddah.

VirusisolatesrepresentingoutbreaksinJeddahandRiyadhwerenotdifferentfrom

MERS‐CoVEMC/2012inreplication,escapeofinterferonresponse,andserum

neutralization.Detectionratesandaveragevirusconcentrationsdidnotchange

significantlyovertheoutbreakinJeddah.Theseresultssuggesttheoutbreakstohave

beencausedbybiologicallyunchangedvirusesinconnectionwithnosocomial

transmission.

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Introduction

TheMiddleEastrespiratorysyndromecoronavirus(MERS‐CoV)wasdiscoveredin2012

andhassincebeenfoundtocausesporadiccasesandsmallcaseclustersofsevereacute

respiratoryillness[1].AllpatientsoccurredintheArabianpeninsulaorhad

epidemiologicallinkstotheregion.Thetotalnumberofnotifiedcasessince2012was

199asof25March2014[2].FromtheendofMarchthroughApril2014anexponential

increaseofnewcasesoccurredinSaudiArabiawithafocusinJeddah,causing

conjecturesaboutpotentialchangesinfundamentalepidemiologicalparameters[3].

Hypothesestoexplaintheoutbreakpatternincludeincreasedsurveillance,increased

zoonotictransmission,increasingnosocomialtransmission,changesinviral

transmissibility,aswellasfalsepositiveresultsduetolaboratoryerrors.Thelatter

optioncausedconcernaboutthevalidityoftheoverallcasecountnotifiedtoWHO[3].

TofullyappreciatetheextensiveoutbreakinJeddah,itwillbenecessarytoreconstruct

transmissionchainsanddissecttheepidemiologyinsuchawaythatfundamental

epidemiologicalparameterscanbeinferred.Whiletheseanalysesmaytakeconsiderable

time,healthauthoritiesareinurgentneedofinformationtoguidepotentialalterations

ofpreventivemeasuresandtravelrecommendations.Virologicalstudiescanprovide

valuableinsightintovirulenceandtransmissibilityeveninabsenceofdetailedclinicalor

epidemiologicalinformation.Moreover,thetrendinnumbersandnatureofrequests

receivedinthediagnosticlaboratorycanprovidehelpfulinsightintothegeneral

situationatpointofcare.

DuringtheoutbreakinJeddah,allRT‐PCRtestingwascentrallyperformedbyJeddah

RegionalLaboratory(JRL).JRLisareferencefacilitywithinthelaboratorynetworkof

theSaudiMinistryofHealththatservestheJeddahregionandprovidesconfirmatory

MERS‐CoVtestingforallMinistryofHealthlaboratoriesacrosstheKingdom.Herewe

providedirectinsightintolaboratoryresultsfromJRLandperformedathorough

analysisoftheoutbreak‐associatedvirusalongwithfunctionalstudiesofvirulenceand

immuneescapeincellculture.WecompareJeddah‐derivedviruseswithviruses

occurringelsewhereinthecountryduringthesametime.




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MaterialsandMethods

RT‐PCRandsequencing

Allproceduresfollowedprotocolsdescribedpreviously[5‐7].JRLusedLightMixkits

(TIBMolbiol)containingpre‐mixedprimersandprobefortheupEandORF1Aassaysto

minimizetheriskofreagent‐basedcontaminationanddetectionartifacts[5].Primers

forviralgenomesequencingareavailableuponrequest.

Virusisolation

SampleswereinoculatedinVeroB4cellsseededat3x105cells/mLin24wellplates16h

priortoinfection,for1hat37°C.Cellswereincubatedat37°Candcheckeddailyfor

cytopathogeniceffects.Every2days,cellculturesupernatantwassampledandtestedby

real‐timeRT‐PCRforincreaseofMERS‐CoV‐specificviralRNA.PCRpositivewellswere

harvestedandusedfortheproductionofvirusstocks.Virusstockswerequantifiedby

plaquetitrationonVeroB4cellsasdescribedearlier[8].

Virusgrowthkinetics

A549cells(ATCCCCL‐185)wereseededat2x105cells/wellin24wellplates16hprior

toinfection.At1,8,24,48and72hpostinfection,supernatantsweresampledandthe

increaseofMERS‐CoV‐specificviralRNAquantifiedbyreal‐timeRT‐PCR[8].

Plaquetitrationandneutralizationassay

VeroB4cellswereseededat1.5x105cells/wellin24wellplates16hpriortotitration.

Cellswereoverlaidafterinfectionwith500µLAvicel(FCMBioPolymer)atafinal

concentrationof1.2%inDMEM[9].Threedayspostinfection,cellswerefixedin6%

formaldehydeandstainedwithcrystalvioletsolution.Forneutralizationassay[10,11],

25plaqueformingunitsofMERS‐CoVwerepre‐incubatedwithdilutedserumforone

hourat37°C.




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Results

Laboratoryperformanceandoveralldiagnosticresults

CaseidentificationandnotificationduringtheoutbreakinJeddahwasmainlybasedon

laboratorytesting.Toobtaininsightintolaboratorytestingduringtheoutbreak,the

samplereceptionlistinJRLwasanalyzed(Figure1).Therewasastrikingincreaseof

diagnosticrequestsduringAprilwhichwasmainlycausedbysamplesfromJeddah

(Figure1A).FromJanuary1sttoApril28th,JRLreceived6,285samplesforRT‐PCR

testingforMERS‐CoV.5,828ofthesesampleswerereceivedonlysinceMarch26th,the

datewhenthefirstcaseintheJeddahoutbreakwastested.Thissuggestsa36.8‐fold

increaseofthemonthlyworkloadinApril.Themaximalnumberofsamplesreceivedina

singledaywas481.AlmosthalfofallpositivetestingsamplesduringtheJeddah

outbreak(82of168)stemmedfromKingFahdHospital.Therateofsampleswith

positivetestsfromKingFahdHospitalseemedtoincreaseearlierthaninotherhospitals

inthecity(Figure1B).OverthecourseoffourweeksinApril,thefractionofpositive

RT‐PCRresultsinsamplesfromJeddahaswellassamplesfromallcitiesdidnotvary

significantly(SupplementaryTable1).Whilethelaboratoryentrylistdidnotidentify

thesymptomsstatusofpatients,itindicatedbypresenceofapatientidentifiercode

whethercaseswereinhospitalorlikelypartofacontactinvestigation(Table1).There

wasamarkedincreaseofcontactinvestigationsinJeddahversusotherlocations.

Expectedly,theproportionofsampleswithlowviralloads(indicatedbyhighCtvalues)

washighincontactinvestigations(Figures1CandD).

StudiesofreliabilityoflaboratoryproceduresaspresentedinSupplementarydataset

1didnotrevealanyevidenceforgenericbackgroundcontaminationinthelaboratory.

Viralgenomesequenceandphylogeny

SevenvirusesfromtheJeddahoutbreakwereentirelysequencedandcomparedwith

full‐lengthorsubtotalgenomesequencesavailableinApril2014inGenBank

(SupplementaryTable2).Ananalysisofmajorreadingframesacrossthegenome

takingintoaccountadditionalspikegenesequences(KM027263‐KM027276)suggested

nouniqueaminoacidchangesinrelevantproteindomains(SupplementaryDataset2).

AllvirusespertainingtotheJeddahoutbreakclusteredinonephylogeneticclade

(Figure2).Seventeenpartialgenomesequencesweredeterminedfromsamples




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obtainedfromRiyadh,Al‐Kharj,andMadinahduringMarchandApril2014for

comparison.Thesepartialsequencescomprisedtheentirestructuralproteingenesof

theMERS‐CoVgenomes,ca.8.7kBinlength.AsshowninFigure2,virusesfromRiyadh

fellinto6differentpositions,oneofwhich(clade2)mayconstituteahuman‐to‐human

transmissionclustertowhichalsotheexportedcasestoIndiana/USAaswellasthe

Netherlandsbelong(SupplementaryTable3)[12,13].AnothervirusfromRiyadh

clusteredwithJeddah‐typeviruses.ThispatientoriginatedfromJeddahandhadvisited

hissicksoninKingFahdHospitalinJeddahbeforehistriptoRiyadh.

TobetterevaluatethediversityofvirusescirculatinginJeddah,singlenucleotide

polymorphisms(SNP)werestudied(Table2).Allsamplesexceptonehadthesame

combinationofSNPs.TheonedeviatingsamplewastakenonApril22ndandhada

doublepeakinoneSNPthatwasconfirmedtwicebyrepetitionofRT‐PCRand

sequencing.Furtherpartialsequencingofthisvirusdidnotyieldanyotherdouble

peaks,suggestingtheongoingformationofquasispeciesasdescribedbefore[14],rather

thansimultaneousinfectionwithtwoviruses.ThesequencesfromaUScaseandacase

inRiyadhwithknowntravelhistoriestoJeddahhadJeddah‐typicalSNPpatterns(Table

2).Incontrast,virusesdetectedinJeddahonemonthand5monthsbeforetheoutbreak

didnotclusterwiththeJeddah‐typeoutbreakviruses.AvirusdetectedinRiyadh

(SA2014_158)wasrelatedtocamelvirusessharingarecentcommonancestorwith

Jeddah‐typeoutbreakviruses,butwasdistinctinitsSNPpattern.

Virusinfectionstudies

Tostudypotentialalterationsinvirusfunctions,16clinicalsamplesfromJeddahwith

projectedviralloadsof5x106copiespersampleorhigherwereselectedandinoculated

inVeroB4cells.Fiveviralisolateswereobtained.Becausethereplicationphenotypeof

allviruseswashighlysimilarinpreliminaryexperiments,oneisolatetermedMERS‐CoV

Jeddah_10306wasfullysequencedandchosenforfurtherstudy(GenBankNo

KM027260,SupplementaryTable2).Forcomparison,viruswasisolatedfrompatients

inahospital‐associatedclusterinRiyadhandanisolatetermedMERS‐CoVRiyadh_683

waschosenandsequenced(GenBankNoKM027262,SupplementaryTable3).The

originalviralisolateEMC/2012[1]wascomparedaswell.




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SinglestepgrowthcurvesweredoneonVerocellsbyinoculationwithhigh

multiplicitiesofinfection(MOI)of1infectiousdosepercell,whichwillrevealgross

differencessuchasintheviruses´capacitytoentercells.AsshowninFigure3A,there

werenorelevantdifferencesinreplicationbetweenthethreeviralstrains.BecauseVero

cellsderivedfromrhesusmonkeykidneytissuemightnotoptimallyreflectthetarget

tissueofMERS‐CoVinfection,A549cellsderivedfromahumanalveolarepithelial

carcinoma(nonsmallcelllungcancer)wereusedinparallel.Resultsofone‐stepgrowth

curveswerehighlysimilar(Figure3B).

Becausedifferencesintheviruses´adaptationtoreplicateinprimatecellsmaynot

becomeobviousinone‐stepgrowthcurves,replicationtrialswererepeatedinparallel

inbothcelllinesusingareducedMOIof0.01thatcausesaprolongedcourseof

replicationwithmultipleroundsofinfectioninculture.Norelevantdifferencein

replicationwasseenbetweenall3viralisolatesinVeroandA549cells(Figure3Cand

D).

ThetypeIinterferonsystemisamongthemostefficientinnateantiviraldefenses.As

MERS‐CoVEMC/2012wasshowntobehighlysusceptibleagainsttypeIinterferon,

infectiontrialsweredoneinVerocellspre‐treatedwithinterferonalphatoinducean

antiviralstatepriortoinfectionincellsatMOI=0.01.EventhoughVerocellsareknown

toinduceanefficientantiviralstateuponexternalIFNstimulus,nodifferencesbetween

thethreeviralstrainswereseen(Figure3E).

Antibodyfunctionsprovidealaboratorycorrelateofadaptiveimmunity.Asvirusesmay

differintheirrobustnessagainstneutralizingantibodies,allthreeviruseswere

subjectedtoplaquereductionneutralizationassaysusingserumofaMERSpatientwith

knownantibodytiter[7].Norelevantdifferencesinthereductionofviralplaques

dependingonserumdilutionwereseenwithanyvirus(Figure3F).

Viralloads

Viralloaddatareflectclinicalvirusexcretion,whichcannotbemodeledincellculture.Ct

valuesasasurrogateofviralloadswerecomparedbetweensamplesfromJeddahand

othercities(Figure4AandB).MeanCtvaluesinJeddahandelsewherewerenot

significantlydifferent(30.4and31.4,respectively).However,thefrequency




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distributionsandmedianvaluessuggestedapronunciationoflowerviralloadsamples

inJeddah.WithinJeddah,CtvaluesinKFH(n=82)werenotdifferentfromthoseinany

otherhospitals(n=108).AllsamplesfromJeddahtestedduringAprilwerecategorized

byweekofreceptionandplottedasshowninFigure4C.Therewasasubjectivetrend

towardlowerCtvaluesbythethirdweek.However,thesepointswereidentifiedas

outliervaluesandmeanviralloadsdidnotdiffersignificantlyinanyoftheweeksof

AprilaccordingtoANOVAanalysis(F=0.82,p=0.48).Oneofthoseoutliersampleswitha

verylowCtvalueencounteredonApril20th,2014yieldedtheisolateofMERS‐CoV

C10306,whichhasbeenentirelysequencedwithoutanyevidenceforsignificant

mutations,andwhichwasstudiedinabove‐describedcellcultureexperimentswithout

anyevidenceforincreasedvirulence.

Discussion

TheunprecedentedincreaseinnewcasesofMERS‐CoVinfectionsduringspring2014

hascausedconcerninthepublichealthcommunityworldwide.Ourinitialsequence

analysescommunicatedduringtheongoingoutbreakprovidedapreliminaryideaofthe

molecularepidemiologywithoutbreakvirusesformingahomogeneous,monophyletic

clade[4].Paraphylyofconcurrentvirusesisexpectedwheninfectionsare

independentlyacquiredfromadiversifiedsourcepopulationsuchasexpectedinanimal

reservoirs.InRiyadh,concurrentlycirculatingviruseswereindeeddistributedacrossat

leastsixdifferentclades,suggestingtheseinfectionstoresultfromincreasedzoonotic

activityorintroductionofhumanvirusesfromotherregions.Onelargerviruscluster

wasobservedinRiyadh,associatedwithonespecifichospitalsuggestingnosocomial

transmission(clade2).ThecaseexportedtoIndiana/USAhadworkedinthishospital

whilethecasesintheNetherlandswerehospitalizedinMadinahbutnotRiyadh[12,13].

Thissuggestsunnoticedtransmissionlinkssuchasinfectedpatientstransferred

betweenhospitals,oracquisitionfromcommonzoonoticsources.

Interestingly,oneofthevirusesseeninRiyadhresembledcamelvirusesinclose

relationshiptoJeddah‐typestrains.Thesevirusesmayhavebeenwidelydistributedin

camelsbylate2013toearly2014,astheyweredetectedinTaifsouthwestofJeddahand

inQatarontheeasternArabianPeninsula[14,15].VirusesencounteredinJeddah

shortlybeforetheoutbreaksuchasJeddah‐1orJeddah_C6664wereclearlydistinct,

suggestingthattheoutbreakmighthavebeeninitiatedbytheintroductionofJeddah‐




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typevirusesintocamelsintheregion.Themonophylyandsimilarityofoutbreakviruses

favorstheideathatthesubsequenttransmissiontookplaceinhumans.Theregional

restrictionofoutbreakvirusesmatchesourearlierobservationoflowtransmissibility

betweenhumansinnon‐nosocomialsettingssuchashouseholdcontactclusters[16].In

spiteofadocumentedtransmissionfromJeddahtothecapitalRiyadh,therewasno

evidenceoffurtherhuman‐to‐humanspreadinRiyadh.FromtheanalysisofSNP

patternsitwasconcludedthatallJeddah‐typeviruseswerehomogenouswithout

evidenceforconcomitantcirculationofotherstrainsduringtheoutbreak.Nevertheless,

ourpreliminarysequencingstudiesfoundnorelevantgeneticchangessufficientto

explainanalteredepidemicpattern[4].Aswehavenowbeenabletoisolateliveviruses,

wecanprovideafirstside‐by‐sidecomparisonofdifferentviralstrainsofMERS‐CoV.Of

note,thesevirusisolateswererepresentativeoftwolikelynosocomialoutbreaksin

JeddahandRiyadh,bothcausinginternationalspreadofthevirustotheUSA,the

Netherlands,aswellasGreece.Cellcultureexperimentsyieldednoevidenceforchanges

inviralreplicationorimmuneescape.Theabsenceofdifferencesinserum

neutralizationdisfavorsantigenicvariabilityasapromoteroftransmissibility.Asthe

selectedvirusesrepresentmajorbranchesoftheknownMERS‐CoVtree,thesedata

additionallysuggesttheabsenceofserotypesinMERS‐CoV,whichisreassuring

regardingtheprospectstodevelopimmunizationapproaches.

BytheendoftheoutbreaklateinApril2014,theaccumulationoflaboratorydataatJRL

allowedfirstinsightsintosheddingpropertiesofcirculatingvirus,whichcompensates

fortheinabilityofcellculturetoreflectvirustransmissibility.Wehaveobtainedno

evidencesuggestingthatconcentrationsofshedvirusmighthavechanged.Asubjective

trendtowardhigherpeak(butnotaverage)concentrationslaterintotheoutbreakmay

beexplainedbyincreaseddiseaseawarenessinhospitalsleadingtoanearlier

investigationofsuspectedcases.SimilarobservationsweremadeduringtheSARS

epidemicinHongKongwherecasesweredetectedearlieraftersometimeintothe

outbreak[17].Theabsenceofchangesinaveragevirusconcentrationsmakesitunlikely

forthevirustohavechangeditstransmissibilityandvirulenceoverthecourseofthe

outbreak.

ThereasonfortheexplosivenatureoftheepidemicinJeddahmaythusbefound

elsewhere,suchasintherateofhuman‐to‐humancontact.Inthislight,ouranalysisof




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laboratorystatisticsishighlysuggestiveofanoutbreakfuelledbythehealthcaresetting.

Notonlydidabouthalfofallpatientswithapositivediagnosispertaintooneparticular

hospital,butalsothefirstpeakcasecountsinthishospitalpredatedincreases

elsewhere,andnewpeakswerefollowedbypeaksofcasesinotherhospitals.This

patternishighlysuggestiveofanepidemiologicalhotspotwherethevirusisamplified

andfromwherelimitedtransmissionchainsareseeded.Indeed,KingFahdHospitalis

thelargestcommunalhospitalinJeddahservingastheprimarycarecenterforall

patientsattendingtheMOHhealthcaresystem,aswellasforalargefractionof

expatriateworkersinthecity.ItisreassuringthatthenumberofnewcasesinKingFahd

Hospitalcamedowntowardtheendofthestudyperiod.Thistrendstartedevenbefore

changessuchastheclosureofemergencyroomsandthetransferofinfectedpatients

wereimplemented,pointingtothepossibilitythattransmissionmayhavebeenlimited

mainlybyheightenedawarenessofthediseaseamonghealthcareworkersandpatients.

Again,asimilareffecthasbeendocumentedduringtheSARSepidemicinHongKong

[17].

Animportantobservationincasenotificationsduringtheoutbreakwastheincreaseof

casesnotifiedas"asymptomatic"or"mild"intheJeddahcasestatistics.Asshowninour

assessmentofsamplereceptions,thehugeamountoflaboratoryrequestsduringpeak

phasesoftheepidemiccausedanoverloadonlaboratorycapacitieswithoutasignificant

increaseofthefractionofrequeststhatwereconfirmedvirus‐positive.Alowpredictive

valueofclinicalsuspicioniscausedbyaninsufficientcasedefinitionorlackof

adherencetothecasedefinition,suchassuggestedbyahighfractionoftestsincases

withoutproperhospitalregistrationnumber.UnjustifiedRT‐PCRtestingraisesthe

likelihoodofhumanerror.Asfaraspossible,wehaveassessedthetechnicalcapabilities

ofJRLandfoundnogeneralissuesofcross‐contamination.Nevertheless,wecannot

excludeissueselsewhereinthelogisticschain,suchasnearthebedsidewhere

diagnosticsamplesmayhavebeenhandledinbulk.ThehighsimilarityofallJeddah‐type

viruseswillmakeitimpossibletoresolvepotentialcontaminationsources

retrospectivelybysequencingofstoredsamples.Nevertheless,acertainrateofpositive

testresultsinasymptomaticpersonsmightbeconsideredplausibleasunnoticed

replicationhasbeenshownforSARS‐CoVwhoseRNAwasdetectedinexposed

healthcareworkerswithnoormildsymptoms,aswellasinourrecentstudyon




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householdcontactsofMERS‐CoVcases[16,18].Suchreplicationmaybetransient,and

thelowviralloadsseenincontactsmightnotsufficetoestablishinfectionchains.

Inconclusion,ourinvestigationssuggestapredominanceofhuman‐to‐human

transmissionduringtheJeddahoutbreakwithoutevidenceformodificationofviral

shedding,replication,andimmuneescape.AcoincidentincreaseofcasesinRiyadhwas

theresultofmultiple,independent,sourceswithsomephylogeneticevidenceof

nosocomialspread.ContacttracingbyRT‐PCRshouldberestrictedtodefinedgroupsof

patientstoavoidanoverloadonthehealthcaresystem.Retrospectiveserologicaltests

mayprovideavalidalternativetoRT‐PCRtestingofcontacts[16].

ACKNOWLEDGEMENTS:WearegratefultoallstaffoftheMinistryofHealth,Saudi

Arabia.

FINANCIALSUPPORT:ChristianDrostenacknowledgessupportfromtheEuropean

Commission(EMPERIE;www.emperie.eu/emp/;contractno.223498)andANTIGONE

(contractno.278976),infrastructuralsupportfromtheGermanCentreforInfection

Research,theGermanMinistryforResearchandEducation,andtheGermanResearch

Council(grants01KIO701andDR772/3‐1).IsabellaEckerle,DoreenMuth,andVictor

CormanacknowledgegrantsupportfromEuropeanCommission,MinistryofResearch

(Germany),andGermanResearchCouncil(DFG).AlimuddinZumlaacknowledges

supportfromtheUniversityCollegeLondonHospitalsNHSFoundationTrust,the

NationalInstituteofHealthResearch,BiomedicalResearchCentre,UCLHospitals,the

EDCTPandtheEC‐FW7(RiD‐RTI).AndrewRambautacknowledgessupportbythe

EuropeanCommissionundertheprojectPREDEMICS(contractno.278433).

CONFLICTSOFINTEREST:OlfertLandtisCEOofTibMolbiol,acompanyproviding

someoftheRT‐PCRreagentsusedinthisstudy.Heorhiscompanyhadnoinfluencein

thedecisiontousethesereagents.Theworkdoesnotmakeanycomparisonsofthese

reagentswithproductsprovidedbycompetingcommercialornoncommercialentities.

Allotherauthorsdeclarenoconflictsofinterest.




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Table1.Testsinsampleswithandwithouthospitalnumber,byCity

City Testswithhospitalnumber Testswithouthospitalnumbera Ratio

Jeddah 3739(4%positive) 1056 (1.7%positives) 28%

Non‐Jeddah 1072(2.9%positive) 59(0positives) 5.5%

a:Thesecaseswereenlistedwithnohospitalnumberbutcarriedeitherofthefollowing

identifiers:"Contact","HCW",orhadacellphonenumberenteredintheidentifierfieldthatthe

laboratorywasaskedtocallincaseofself‐initiateddiagnostictestsbyphysiciansortheirfamily

members(n=41).




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Table2.SinglenucleotidepolymorphismsinJeddah‐typevirusesandreferenceviruses

SNP position in EMC/2012 genomeSample ID Sample/patient origin Sampling date 737 17836 23953 2877868 samples from JRLa Jeddah, Makkah 26 Mar to 23 Apr 2014 C T G A Human|2014SA_693b Riyadh 22 Apr 2014 C T G A Human|Florida/USA-2/Jeddah Jeddah 10 May 2014 C T G A Human|C10829 Jeddah 22 Apr 2014 C T G A/T Camel|Qatar_2|KJ650098 Qatar 16 Feb 2014 C C A T Human|C6664c Jeddah 18 Feb 2014 T C ? T Human| 2014SA_158d Riyadh 20 Mar 2014 T C A T Camel|Jeddah_1_2013|KJ556336e Jeddah 6 Nov 2013 T C A T Camel|KSA-505|KJ713295 Taif Nov 2013 T C A T Camel|KSA_378|KJ713296 Taif Nov 2013 T C A T Human|2014SA_683 Riyadh 21 Apr 2014 T C A T Camel|KSA-503|KJ713297 Taif Nov 2013 T T A T Camel|KSA-363|KJ713298 Taif Nov 2013 T T A T Human|EMC/2012|JX869059 Bisha Jun 2012 T C A T

a:Mediansamplingdateon14.April.The68samplesrepresented40%ofallpositivesamplesidentifiedatJRLinJeddahpatients

b:ThispatienthadatravelhistorytoKingFahdHospitalinJeddahwithinoneincubationtimebeforeonsetofsymptoms

c:ThiswasthelastpatientdetectedandsequencedinJeddahbeforetheonsetoftheoutbreakendofMarch.TheSNPatposition23953

couldnotbesequencedbecausethediagnosticsamplecontainedonlyminuteamountsofRNAandhadbeenstoredat‐20°Cforprolonged

time.

d:Thispatienthadnotravelhistory.Virus2014_SA158clustersamongstcamelvirusesinancestralrelationshiptoJeddah‐typehuman

viruses,suchasCamel_Qatar2_KJ650098.

e:ThisviruswastransmittedfromacamelinJeddah,October/November2013

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LegendstoFigures

Figure1.SummaryoffeaturesoftheoutbreakasderivedfromJRLlabfiledata.A,

overalldiagnosticrequests;B,positivecases(yscale=casesperday)inKingFahd

Hospitalversusallotherhospitals,recording3‐dayintervalsstartingonMarch26and

endingonApril28.CandD:DistributionofCtvaluesin1056samplespertainingto

investigationsincaseswithouthospitalnumberinJeddah(n=18positivesamples),

versus3799sampleswithhospitalnumber(n=150positivesamples).AverageCtvalues

incasesandcontactswere30and33.1,respectively(2‐tailedt‐test,p<0.009).

Figure2.PhylogenetictreeinferredusingMrBayes[20]fortheconcatenatedcoding

regionsof105MERS‐CoVgenomesorpartialgenomessampledfromhumansand

camels.Weemployedacodon‐position‐specificGTRsubstitutionmodelwithgamma‐

distributedratesamongstsites.Displayedisthemajority‐consensusof10,000trees

sampledfromtheposteriordistributionwithmeanbranchlengths.Posteriorsupportis

shownfornodeswherelessthan0.90.Sequencessampledfromcamelsaredenoted

withayellowcircle,thosefromhumanswithagreencircle.Sequencesnewtothisstudy

arelabelledinbold.TheclustercomprisingvirusesisolatedfromtheJeddah/Makkah

hospitalsinApril2014arehighlightedwitharedboxandthosefromthePrinceSultan

MilitaryMedicalCity,RiyadhinMarch,April2014arehighlightedinblue.For

comparisontheAl‐Hasa2013hospitaloutbreak[21]ishighlightedinyellowandthe

2013Hafr‐Al‐Batincommunityoutbreak[22]ingreen.

Figure3:GrowthkineticsofMERS‐CoVEMC/2012,Jeddah_10306,andRiyadh_683in

cellculture.VeroB4andA459cellswereinfectedatMOI1(AandB,respectively)orMOI

0.01(CandD,respectively).Samplesfromthesupernatantweretakenatindicatedtime

pointsandvirusgrowthwasmeasuredbyreal‐timeRT‐CPR.VeroB4cellsinfectedat

MOI1(A)showedtotalcytopathogeniceffect48hpostinfection,terminatingthe

experiment.A459cellsdidnotshowanyCPEevenwheninfectedatMOI1at72hp.i.

(B).E,effectofpretreatmentofcellcultureswithtypeIinterferonatloworhighdosage.

D,virusneutralizingeffectofhumanserumwithknownanti‐MERS‐CoVneutralizing

antibodytiteratdifferentdilutions.




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Figure4.Virussheddinginpatients.CtvaluesduringtheoutbreakinJeddah.AandB,

frequencydistributionofCtvaluesinJeddahversusothercities;C,Ctvaluesduringthe

outbreakinJeddahbyweek,startingonMarch26th,2014.




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2015 an observational, laboratory-based study of outbreaks of middle east respiratory syndrome...

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