Ebola virus disease diagnosis by real-time RT-PCR: A comparative study of 11 different procedures

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Article

Reference

Ebola virus disease diagnosis by real-time RT-PCR: A comparative study of 11 different procedures

CHERPILLOD, Pascal, et al.

Abstract

The diagnosis of Ebola virus disease relies on the detection of viral RNA in blood by real-time reverse-transcription PCR. While several of these assays were developed during the unprecedented 2013-2015 Ebola virus disease outbreak in West Africa, few were applied in the field.

CHERPILLOD, Pascal, et al . Ebola virus disease diagnosis by real-time RT-PCR: A

comparative study of 11 different procedures. Journal of Clinical Virology , 2016, vol. 77, p.

9-14

DOI : 10.1016/j.jcv.2016.01.017 PMID : 26874083

Available at:

http://archive-ouverte.unige.ch/unige:85381

Disclaimer: layout of this document may differ from the published version.

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ContentslistsavailableatScienceDirect

Journal of Clinical Virology

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j c v

Ebola virus disease diagnosis by real-time RT-PCR: A comparative study of 11 different procedures

Pascal Cherpillod

^a,b,c,∗,1

, Manuel Schibler

^b,c,∗

, Gaël Vieille

^a,b,c

, Samuel Cordey

^a,b,c

, Aline Mamin

^b,c

, Pauline Vetter

^b,c

, Laurent Kaiser

^a,b,c

aSwissNationalReferenceCentreforEmergingViralDiseases,GenevaUniversityHospitals,Geneva,Switzerland

bLaboratoryofVirology,GenevaUniversityHospitals,Geneva,Switzerland

cUniversityofGenevaMedicalSchool,Geneva,Switzerland

a r t i c l e i n f o

Articlehistory:

Received13October2015

Receivedinrevisedform4December2015 Accepted27January2016

Keywords:

Ebolavirus Diagnosis Real-timeRT-PCR Analyticalsensitivity RNAquantiﬁcation

a b s t r a c t

Background:ThediagnosisofEbolavirusdiseasereliesonthedetectionofviralRNAinbloodbyreal- timereverse-transcriptionPCR.Whileseveraloftheseassaysweredevelopedduringtheunprecedented 2013–2015EbolavirusdiseaseoutbreakinWestAfrica,fewwereappliedintheﬁeld.

Objectives:Tocomparetechnicalperformancesandpracticalaspectsof11Ebolavirusreal-timereverse- transcriptionPCRprocedures.

Studydesign:Weselectedthemostpromisingassaysusingserialdilutionsofculture-derivedEbolavirus RNAanddeterminedtheiranalyticalsensitivityandpotentialrangeofquantiﬁcationusingquantiﬁed invitrotranscribedRNA;viralloadvaluesintheserumofanEbolavirusdiseasepatientobtainedwith theseassayswerereported.Finally,easeofuseandturnaroundtimesofthesekitswereevaluated.

Results:Commercialassayswereatleastassensitiveasin-housetests.Fiveoftheformer(Altona,Roche, Fast-trackDiagnostics,andLifeTechnologies)wereselectedforfurtherevaluation.Despitedifferences inanalyticalsensitivityandlimitsofquantiﬁcation,allofthemweresuitableforEbolavirusdiagnosis andviralloadestimation.TheLifetechLyophilizedEbolaVirus(Zaire2014)assay(LifeTechnologies) appearedparticularlypromising,displayingthehighestanalyticalsensitivityandshortestturnaround time,inadditiontorequiringnoreagentfreezing.

Conclusions:Commercialkitswereatleastassensitiveasin-housetestsandpotentiallyeasiertousein theﬁeldthanthelatter.Thisqualitativecomparisonofreal-timereversetranscriptionPCRassaysmay serveasabasisforthedesignoffutureEbolavirusdiseasediagnostics.

1. Background

As of September, 2015, more than 28,000 cases and over 11,000deathshave occurredduringtheWestAfricanepidemic of Ebola virus disease (EVD) (http://apps.who.int/ebola/ebola- situation-reports),whichiscausedbytheMakonavariantofthe Zaireebolavirus(EBOV)species.Itsgenomeisa19kblongsingle- stranded,negative-senseRNA.Rapiddiagnosisisacriticalinfection controlmeasure,particularlyin lightofEVD’searlysymptoma- tology,which is indistinguishablefromthat of other infections including malaria [1]. Ebola virus diagnostics have improved

∗ Correspondingauthorat:LaboratoryofVirology,DivisionofInfectiousDis- eases,GenevaUniversityHospitals,4RueGabrielle-Perret-Gentil,1211Geneva 14/Switzerland.Fax:+41223724097.

E-mailaddress:manuel.schibler@hcuge.ch(M.Schibler).

1 Contributedequally(co-ﬁrstauthors).

considerablyfollowingthedevelopmentofreal-timereversetran- scriptionPCR(real-timeRT-PCR)assayscapableofrapidlydetecting viralRNAinbloodspecimens[2–7].Theirabilitytoestimateviral loadsinblood,whichcorrelatewithclinicaloutcome[8–11],aswell asinvariousbodyﬂuids[12–20],makesthemusefultoolsinthe post-diagnosticphase.Therecentimplementation,thoughsome- whatcontroversial,ofanegativebloodreal-timeRT-PCRresultas adischargecriterionhasonlyincreasedtheseassays’importance [21,22].

Yetcommercialmoleculardiagnosticassaysweremadeavail- ableonlyin2014.Validationresultsofreal-timeRT-PCRassays recentlyusedintheﬁeldinWestAfricaandinothercountriesfor screeningarelimitedandnotpubliclyavailable,andnoassayshave yetbeencomparedsystematically.

http://dx.doi.org/10.1016/j.jcv.2016.01.017

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10 P.Cherpillodetal./JournalofClinicalVirology77(2016)9–14 2. Objectives

Weundertookthepresentcomparativestudytoevaluatethe performanceofseveralcommercialandin-houseEBOVreal-time RT-PCRassaysinordertoinformfutureselectionsanduseofthese toolsbothintheﬁeldandinhigh-resourcesettings.

3. Studydesign

ThestudyﬂowchartisshowninFig.1.

3.1. EBOVRNAs

C5 (GenBank no. KJ660348) and C15 RNAs (GenBank no.

KJ660346) extracted from Vero-cell cultures after inoculation withserum from patient C5 in Guéckédou and patient C15 in Kissidougou [23] were kindly provided by the Swiss Institute for NBC-Protection (Spiez Laboratory, Biology–Virology Group, Spiez, Switzerland). Alignment of the two sequences revealed 5nucleotidechanges(99%nucleotidesequencehomology): two werelocatedintheNgene atpositions 2124and2185,one in theGPgeneatposition6909,andtwointheLgeneatpositions 13,856and15,660.Noneofthesepositionsaretargetedbyanyof thereal-timeRT-PCRassaystestedinourstudy,exceptpossiblyfor theposition13,856nucleotidechange,whichcouldbeinvolvedin thetargetsequenceoftheRealStar^® FilovirusScreenRT-PCRKit 1.0,theRocheLightMix^®ModularEbolaVirusZaireassay,and/or theFTD^®Ebolareal-timeRT-PCR.

Inordertodetermineboththeanalyticalsensitivityandthelin- earrangeofselectedreal-timeRT-PCRassays,weusedtwoRNA transcripts990bases long(TriLink BioTechnologies, SanDiego, USA)atknownconcentrations.OnespanstheLgene(nt12,792to nt13,781ofthegenomicstrand,GenBankref:KM233117)andthe othertheNPgene(nt374tont1363ofthegenomicstrand,GenBank ref:KM233117);togetherthesecoverallreal-timeRT-PCRtargets usedinthisstudy.WealsousedRNAextractedfromtheserumof anEVDpatientmanagedinourinstitution[19].AllRNAinthesame experiment,eitherextractedortranscribedinvitro,underwentthe samefreeze–thawcycle.

3.2. RNAextraction

RNA extractionwas performedwith the EasyMagautomate (NucliSENS^®EasyMAG,bioMérieux,Geneva,Switzerland)follow- ingthemanufacturer’sinstructions.400␮lofspecimen(EbolaZaire positiveserum)wereinactivatedby1mloflysisbuffer(EasyMAG^® LysisBuffer),followedbynucleicacidextractionwithaﬁnalelution volumeof50␮l.

3.3. rRT-PCRassays

Thefollowingsixcommercialassayswereselectedbasedon theiravailabilityonthemarketinSwitzerlandatthetimeofthis studyand theirability tobeconductedonopen platformsand adaptedtothespeciﬁcationsofanaverageclinicalmicrobiology Laboratory:RealStar^®FilovirusScreenRT-PCRKit1.0(ref:441013, Altona,Hamburg,Germany)andtheZaireEbolavirusassayfrom theRealStar^®FilovirusTypeRT-PCRKit1.0(ref:451003,Altona, Hamburg,Germany),RocheLightMix^®ModularEbolaVirusZaire (ref:40-0666-96,Roche,Rotkreuz,Switzerland),FTD^®Ebola(ref:

FTD-71-64,Fast-trackDiagnostics(FTD),Sliema,Malta), Lifetech EbolaVirus(Zaire2014)andLifetechLyophilizedEbolaVirus(Zaire 2014)(ref:4489990,LifeTechnologies,Waltham,USA).Thesetests arereferredtoasAltonaScreen,AltonaType,Roche,FTD,Lifetech andLifetechL,respectively,throughoutthemanuscript.

Accordingtothemanufacturer’sinstructions,thefollowingreal- timePCRplatformsweresuitableforeachcommercialkit:

Altonascreen:

Mx3005P^TMQPCRSystem(Stratagene),VERSANT^®kPCRMolec- ularSystemAD (Siemens),ABIPrism^® 7500SDSand7500Fast SDS(AppliedBiosystems),LightCycler^®480InstrumentII(Roche), Rotor-Gene^® 3000/6000 (Corbett Research), Rotor-Gene^® Q5/6 plexPlatform(QIAGEN),andCFX96^TM/DxReal-TimeSystem(BIO- RAD).

Altonatype:

VERSANT^®kPCRMolecularSystemAD(Siemens),ABIPrism^® 7500SDSand7500FastSDS(AppliedBiosystems),LightCycler^®480 InstrumentII(Roche),Rotor-Gene^®3000/6000(CorbettResearch), Rotor-Gene^®Q5/6plexPlatform(QIAGEN),andCFX96^TM/DxReal- TimeSystem(BIORAD).

Roche:

LightCycler^®480IIInstrument(Roche),andCobasz480(Roche).

FTD:

ABI Prism^® 7500 SDS, 7500 Fast SDS, and ABI ViiA7 (Applied Biosystems),CFX96^TM/Dx Real-Time System(BIORAD), LightCycler^® 480IIInstrument(Roche),Rotor-Gene^®3000/6000 (CorbettResearch),andSmartCycler^®(Cepheid).

LifetechandLifetechL:

7500FastSDS,ABIViiA7,andQuantStudioreal-timePCRsys- tems(AppliedBiosystems).

Inaddition,wetested5in-housereal-timeRT-PCRassays:The real-timeRT-PCRdesignedbyGibbandcolleaguesin2001[2],as wellasa modiﬁedversion, whichwe adaptedaccording tothe viralcirculatingsequencesin2014;amodiﬁedversionoftheEbola Zaire-TMassaydesignedin2010[7],whichwasadaptedaccording to2014EBOVsequencesbytheSwissInstituteforNBC-Protection (Spiez Laboratory, Biology–Virology Group,Spiez, Switzerland);

andtwoin-housereal-timeRT-PCRassays,whichweredesigned accordingtopubliclyavailableEbolasequencesthroughOctober 2014,the“EBOV-GP-GE-14”andthe“EBOV-L-GE-14”real-timeRT- PCRs,targetingtheGPandLgenesrespectively.Thesein-house assaysarereferredtoasEBOGP-1D,EBOGP-1D14,EbolaZaire-TM, EBOV-L-GE-14 and EBOV-GP-GE-14, respectively. Primers pairs andprobesweredesignedusingPrimerExpresssoftwareversion 3.0.

TheRoche,AltonaScreenandAltonaTypeassayswereusedwith theLightCycler 480thermocycler (Roche Diagnostics, Rotkreuz, Switzerland)whiletheFTD,LifetechandLifetechLassayswererun withtheViiA7thermocycler(LifeTechnologies,Waltham,USA), accordingtothemanufacturer’sinstructions.Allnon-commercial real-timeRT-PCRswererunusingtheStepOnePlusthermocycler (LifeTechnologiesWaltham,USA).Characteristicsofthe11eval- uatedreal-time RT-PCRassays, includingcycling conditionsand primers’andprobes’sequences,whenavailable,aresummarized inTableS1.

3.4. Lowerlimitsofdetectionandquantiﬁcation,andEBOVRNA quantiﬁcation

Foreachselectedreal-timeRT-PCRassay,knownconcentrations ofinvitrotranscribedRNAs(seeabove)wererunintriplicateintwo separateexperimentstoestablishstandardcurvesanddeﬁnethe limitofdetection(LOD)andthelowerlimitofquantiﬁcation(LOQ).

TheLODwasdefinedasthelowestRNAconcentrationdetectedin allofthesixreplicates.TheLOQwasdefinedbasedonbothtrip- licateexperimentsasthelowestRNAconcentrationthatcouldbe plottedonastandardcurvewithaslopebetween−3.1and−3.6 (correspondingtoaPCRefficiencybetween90%and110%),anr² valueabove0.95,andvisuallylimiteddispersionaroundthecurve.

ForEBOVRNAquantiﬁcationintheclinicalspecimen,astandard

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Fig.1. Flowchartofthestudy.LOD:limitofdetection;LOQ:lowerlimitofquantiﬁcation.

curvewasestablishedusingserialdilutionsofquantiﬁedinvitro transcribedRNAineachexperiment.

4. Results

4.1. Analyticalsensitivitycomparisonofsixcommercialandﬁve in-houserRT-PCRassays

Each of the six commercial and ﬁve in-house assays was evaluated by end-point dilution using 5-fold dilutions of two culture-derivedfull-lengthEBOVgenomeRNAs,relatedtoisolates collectedinGuineainMarch2014(namedC5andC15RNAs)(Fig.1 andTableS2).

Whileall11assayscoulddetectRNAfrombothisolates,the commercialkitstendedtobemoresensitive.Sincethein-house real-timeRT-PCRsprovidednoadvantagesintermsofperformance oreaseofuse,theywerenotselectedforfurtherevaluation.Wedid notretaintheLifetechEbolaVirus(Zaire2014)assayforfurther testingeither,asitwaslesssensitivethanitslyophilizedcounter- partandlackeditspracticaladvantages.

TheLODwasdeterminedfortheﬁvemostpromising assays (AltonaScreen,AltonaType,Roche,FTD,andLifetechL),usingserial dilutionsofquantiﬁedinvitrotranscribedRNAs(Fig.2,Table1).As canbeseeninFig.2,whichrepresentstwoseparateexperiments performedsevenmonthsapart,theinter-assayvariabilityisvery small.

4.2. Lowerlimitofquantiﬁcation

Thefiveselectedassayswerefurthertestedfortheirquantifica- tionabilities(Fig.2,Table1).Ofnote,thehighestRNAconcentration testedinouranalysiswas6.25E9RNAcopies/ml,whichdidnot reachtheupperlimitofquantificationforanyassay.

4.3. EBOVRNAquantiﬁcationandend-pointdilutionusingserum ofanEVDpatient

Theﬁvecommercialreal-timeRT-PCRswerealsorunindupli- cateon3-folddilutionsof RNAextractedfroma serumsample withdrawnfromanEVDpatientmanagedinourinstitution[19]

(Table1).TheEBOVRNAquantiﬁcationvaluesobtainedwiththe variousassaysintheundilutedserumwereasfollows:1.16E6RNA copies/ml(Altona Screen),1.13E5RNA copies/ml(AltonaType), 9.5E4RNAcopies/ml(Roche),4.0E4RNAcopies/ml(FTD),and2.5E4 RNAcopies/ml(LifetechL).

4.4. Easeofuseandturnaroundtime

The complexity of technical manipulations was comparable betweentheﬁveassays,exceptfortheLifetechLyophilizedsystem, whichdoesnotrequireanyreal-timeRT-PCRmixpreparation.For eachassay,weestimatedseparatelythehandlingtimefromRNA extractiontoreal-timeRT-PCRandthereal-timeRT-PCRcycling time,andthetotalturnaroundtime(Table1).

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12 P.Cherpillodetal./JournalofClinicalVirology77(2016)9–14

Fig.2. Graphicalrepresentationofstandardcurve,linearityrange,lowerlimitofquantification(LOQ)andlimitofdetetction(LOD)foreachEBOVrRT-PCRassay.Eachplot showsvaluesobtainedfromtwoseparateexperimentsinwhichserialdilutionsofinvitrotranscribedEBOVRNAwererunintriplicate.Reddiamondsrepresentthefirst experimentandbluediamondsthesecond.ThelowestRNAconcentrationforwhichthelinepassesthroughrepresentstheLOQ.Theverticalgreydashedlinerepresentsthe LOD.Foreachassay,thecyclingnumberrecommendedbyeachmanufacturerisrepresentedbythehighestnumberindicatedontheyaxis.Diamondssituatedabovethe maximumcyclelinerepresentRNAdilutionsthatwerenotdetected.Slopeandr²valuesareindicatedforeachcurve.(Forinterpretationofthereferencestocolorinthis figurelegend,thereaderisreferredtothewebversionofthisarticle.)

Table1

ComparisonofﬁveselectedEBOVrRT-PCRassays.

Assay LOD(invitro transcribedRNA copies/ml)

LOQ(invitro transcribedRNA copies/ml)

Highestpatient RNAdilution detected

Handlingtime fromextraction torRT-PCR^a (min)

rRT-PCRcycling time(min)

Totalturnaround time(min)

Storagetemperature

AltonaScreen 1250 12,500 27× 70–100 120 190–220 −20^◦C

AltonaType 6250 625,000 27× 70–100 120 190–220 −20^◦C

Roche 1250 6250 27× 70–105 80 150–185 −20^◦C

FTD 625 1250 81× 70–100 75 145–175 −20^◦C

LifetechL 62.5 625 243× 55–80 50 105–130 2–8^◦C

aNucleicacidextraction,rRT-PCRmixpreparation,platepipettingLOD:limitofdetection;LOQ:lowerlimitofquantiﬁcation.

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5. Discussion

We providetheﬁrstsystematic comparisonof severalEBOV real-timeRT-PCRassays,sixofwhicharecommerciallyavailable, usingserialdilutionsoffull-lengthEBOVRNAsfromthe2013–2015 EVDoutbreak.In-houseassaysdidnotmatchtheperformanceof thecommercialkits evenafter primer adaptations in linewith recentlyisolatedEBOVsequences.Thiscouldbeexplainedinpart bythelackofextensivereagentoptimization.

The ﬁve most promising assays, all commercially available (Altona Screen, Altona Type,Roche, FTD, and Lifetech L),were selectedforfurtherevaluationusingquantiﬁedRNAtranscripts.

TheLifetechLyophilizedassaydisplayedthebestanalyticalsen- sitivitywithanLODof62.5RNAcopies/ml,andthelowestLOQ(625 RNAcopies/ml),whichmaybeexplainedinpartbythekit’srela- tivelyhighRNAeluatevolume.Indeed,25␮lofeluateisrequired;

thisis2.5to5timesmorethanthatrequiredbytheRoche,FTDand bothAltonaassays.

Turnaroundtimesrangedfromtwo (LifetechLyophilized)to 3.5h (both Altonaassays). The differences are relatively small, however,andshouldhavelittleimpactonpatientmanagement.

Complexityofuseandreagentstorageconstraintsarelikelytobe moresigniﬁcantissuesintheﬁeld.

Altogether,despite differences in analytical sensitivities and LOQs,theAltonaScreen,Roche,FTD,andLifetechLassaysalldis- playedanalyticalsensitivitieshighenoughtoensureaccurateEVD diagnosisandlinearquantiﬁcationrangessufﬁcienttoassessviral kinetics,atleastwhendealingwithhighRNAconcentrations.

TheZaireEbolavirusassayfromtheRealStar^®FilovirusTypeRT- PCRKit1.0wastheleastsensitiveandtheleastsuitedforEBOVRNA quantiﬁcation.Thisisaminorissue,however,iftheassayisused primarily,asitsnameimplies,totypetheZaireebolavirusspecies.

Nevertheless,thedifferentsensitivitiesexhibitedbythereal- time RT-PCR kits can have considerable clinical impact. For instance,astheviralRNAloaddecreasesduringtherecoveryphase, thevariousreal-timeRT-PCRassaysmayyieldundetectableresults atdifferenttimepoints,potentiallyresultinginprolongingorshort- eningofpatientisolationtime.Thisissuewasexempliﬁedduring themanagementofanEVDinfectedpatientinourcenter,aswe obtaineddifferentreal-timeRT-PCRresultswiththeRocheand Altonaassaysrunonthesamesamplesduringtheconvalescent phase[19].However,one canarguethatwhena patientshows signsofclinicalcure,thelow-leveldetectionofviralRNAproba- blyreﬂectsgenomicremnantsratherthanresidualinfectiousvirus [24].Therefore,highCtvaluesobtainedduringtherecoveryphase shouldprobablynotbeoveremphasized.

EBOVRNAquantiﬁcationbyreal-timeRT-PCRisclinicallyuse- ful,sincehighviremiaiscorrelatedwithahighermortality[8–11].

PreciseEBOVRNAquantiﬁcationwillalsobeavaluabletoolintrials evaluatingantiviralagents.

It should be noted, however, that in this study we did not perform a complete real-time RT-PCR quantification validation, but rather assessed theirrespective quantification potential. A thoroughvalidation according totheMinimum Informationfor Publication of Quantitative Real-time PCR Experiments (MIQE) guidelines[25]wouldbeessentialforanyformalassessmentof EBOV-RNAquantificationperformance.Moreover,asshownwith thequantificationofEBOVRNAintheserumofanEVDpatient,RNA copies/mlvaluesvariedconsiderablyaccordingtothereal-timeRT- PCRassay,from4.41to6.07log10RNAcopies/ml.Thisvariability canhaveanimpactonviralloadquantificationresultsinclinical investigations,andhenceontheconclusionsdrawnbysuchstud- ies.Therefore,inordertocomparequantificationvaluesobtained withdifferenttests,theyshouldbeexpressedininternationalunits (IU)permillilterusinganinternationalquantificationstandard,as hasbeendoneforotherviruses.

TheLifetechLandFTDassaysdisplayedremarkablylowLOQs, 625and1250RNAcopies/ml,respectively,whichcouldbeinterest- inginEBOVRNAquantiﬁcationinaresearchcontext.TheLifetechL assay,whichtothebestofourknowledgedidnothavetheopportu- nitytobetestedintheﬁeldduringthe2013–2015EVDoutbreak, displaysmanyinterestingfeatures.Theabsenceofreal-timeRT- PCRmixpreparationandfreezerrequirements,shortturnaround time,highsensitivityandlowLOQrenderthisassayparticularly suitableinthecontextofanoutbreakinlimited-resourcesettings, inwhichcoldchainmanagementcanbechallenging.

Ourstudyhasseverallimitations.Theﬁrstisthelackofsequence data regardingthe primersand probes includedin commercial assays, which is a consequence of the restrictive data-sharing policies applied by various agencies. Second, our study is not exhaustive,aswemadeaselectionofavailableassaysatagiven timeandataspeciﬁcplace,andthatwereadaptedtoourlabora- tory.Third,itshouldbenotedthatweusedanucleicacidextraction procedure(EasyMag)nottypicallyusedinWestAfrica,andthree differentthermocyclersaccordingtothedifferentassays.Finally, thepresentanalysisisfocusedonthe2013–2015EVDepidemicin WestAfrica,andourconclusionsarethusrelevanttotheMakona EBOVvariantcurrentlycirculating.Indeed,whiletheAltonaScreen andRocheassaysperformedwellonRNAfromthe2001EBOVepi- demicinGabon,thesamewasnot truefortheFTDassay(data notshown).Thisillustratestheneedtoadaptreal-timeRT-PCR assaysaccordingtopotentialviralsequenceevolution,asstressed bySozhamannanetal.[26].

Inconclusion,among11EBOVreal-timeRT-PCRassays,com- mercialkitsweresuperiortoin-houseassays;amongtheformer, theLifetechLassaydisplayedthehighestsensitivityandeaseof useaswellasthelowestturnaroundtime.

Funding

TheSwissNationalReferenceCentreforEmergingViralInfec- tionsis supportedbytheSwissFederal Ofﬁceof PublicHealth.

AdditionalﬁnancialsupportwasprovidedbytheSwissNational FoundationGrant32003B-146993toLaurentKaiser.Thefunders hadnoroleinstudydesign,datacollectionandinterpretation,or thedecisiontosubmittheworkforpublication.

Competinginterests Nonedeclared.

Acknowledgements

WeareindebtedtoFelixBaezforkindlydonatinghisbloodfor multipleinvestigations.WearegratefultoOlivierEnglerandChris- tian Beuretfromthe Spiez Laboratory,Biology–Virology Group, Spiez,Switzerland,forprovidinguswithMakonaEBOVC5andC15 RNAs.WealsothankourcolleaguesSabineYerlyfromtheLabora- toryofVirology,GenevaUniversityHospitals,Geneva,Switzerland, fortheconstructivediscussions,andAngelaHuttner,InfectionCon- trolProgram,GenevaUniversityHospitals,Geneva,Switzerland,for editingthemanuscript.

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