Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay

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Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay

CORDEY, Samuel, et al.

CORDEY, Samuel, et al . Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay. Journal of Virological Methods , 2011, vol. 177, no. 1, p. 118-122

DOI : 10.1016/j.jviromet.2011.06.018 PMID : 21763351

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Journal of Virological Methods

jo u r n al h om epa ge :w w w . e l s e v i e r . c o m / l o c a t e / j v i r o m e t

Short communication

Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay

Samuel Cordey

, Roland Sahli

, Marie-Laurence Moraz

, Christine Estrade

,

Laurence Morandi

, Pascal Cherpillod

, Remi N. Charrel

, Stefan Kunz

, Laurent Kaiser

aSwissNationalReferenceCentreforEmergingViralDiseases,UniversityofGenevaHospitals,Geneva,Switzerland

bLaboratoryofVirology,UniversityofGenevaHospitals,Geneva,Switzerland

cUniversityofGenevaMedicalSchool,Geneva,Switzerland

dInstituteofMicrobiologyoftheCentreHospitalierUniversitaireVaudoisandUniversityofLausanne,Lausanne,Switzerland

eUnitédesVirusEmergents(UMR190),UniversitédelaMéditerranée– InstitutdeRecherchepourleDéveloppement,Marseille,France

Articlehistory:

Received9March2011

Receivedinrevisedform24May2011 Accepted21June2011

Available online 6 July 2011

Keywords:

Lymphocyticchoriomeningitisvirus Real-timeRT-PCR

Cerebrospinalfluid Prevalence

a b s t r a c t

Lymphocyticchoriomeningitisvirus(LCMV)isararecauseofcentralnervoussystemdiseaseinhumans.

Screeningbyreal-timeRT-PCRassayisofinterestinthecaseofasepticmeningitisofunknownetiology.

AspecificLCMVreal-timeRT-PCRassay,basedonthedetectionofgenomicsequencesoftheviral nucleoprotein(NP),wasdevelopedtoassessthepresenceofLCMVincerebrospinalfluids(CSF)sentfor viralscreeningtoaSwissuniversityhospitallaboratory.

A10-folddilutionseriesassayusingaplasmidcontainingthecDNAoftheviralNPoftheLCMVisolate Armstrong(Arm)53bdemonstratedthehighsensitivityoftheassaywithalowestdetectionlimitof

≤50copiesperreaction.HighsensitivitywasconfirmedbydilutionseriesassaysinapoolofhumanCSF usingfourdifferentLCMVisolates(Arm53b,WE54,TraubandE350)withobserveddetectionlimitsof

≤10PFU/ml(Arm53bandWE54)and1PFU/ml(TraubandE350).

Analysisof130CSFshowednocasesofacuteinfection.Theabsenceofpositivecaseswasconfirmed byapublishedPCRassaydetectingallOldWorldarenaviruses.

Thisstudyvalidatesaspecificandsensitivereal-timeRT-PCRassayforthediagnosisofLCMVinfections.

ResultsshowedthatLCMVinfectionsareextremelyrareinhospitalizedpatientswesterninSwitzerland.

© 2011 Elsevier B.V. All rights reserved.

Lymphocytic choriomeningitis virus (LCMV), the prototypic memberoftheArenaviridaefamily,wasisolatedfromafatalcase ofasepticmeningitisduringtheSt.Louisencephalitisepidemicin 1933(ArmstrongandLillie,1934).

In nature, LCMV is maintained by congenital transmission withininfectedpopulationsofthemousespeciesMusdomesticus andMusmusculus.LCMVinfectsreadilyotherrodents,including hamstersandguineapigs.TheLCMVcarrierstateischaracterized bypersistentinfectionwithhighvirusloadsinserum andsev- eralorgansintheabsenceofavirus-specificimmuneresponseand overtpathology.Humansareaccidentalhostsandthemainroute oftransmissionisbycontactwithinfectedrodentsthatshedlarge

∗Correspondingauthorat:LaboratoryofVirology,DivisionofInfectiousDis- eases,UniversityofGenevaHospitals,4RueGabrielle-Perret-Gentil,1211Geneva 14,Switzerland.Tel.:+41223724079;fax:+41223724097.

E-mailaddress:samuel.cordey@hcuge.ch(S.Cordey).

1 Theseauthorscontributedequallytothiswork.

quantitiesofvirusinnasalsecretions,saliva,milk,semen,urine, andfeces.

AlthoughhumanLCMVcasesareobservedthroughouttheyear, diseaseincidenceincreasesinwinter,dueprobablytothemove- mentofinfectedrodentsindoors,thusincreasingtheriskofhuman exposure(Bartonetal.,1993).In immunocompetentadultindi- viduals, LCMV infection is either asymptomatic or results in a self-limitingfebrileillnessassociatedrarelywithfatalities.Signs andsymptomsarelargelynon-specific,includingfever,myalgia, and malaise. Central nervous system involvement manifestsas headacheandphotophobia,associatedwithnauseaorvomiting.

In mostcases, infectedindividuals recover completely,but this mayrequireseveralmonths.Severeasepticmeningitisormenin- goencephalitis is observed only in a minority of cases (Barton etal.,1993).Human-to-humantransmissionhasbeendocumented through organ donation associated with severe disease in the immunocompromisedrecipient(Fischeretal.,2006),andcongeni- talinfectionsleadingtosevereandirreversiblebrain(Bartonetal., 1993;BartonandMets,2001;Bonthiusetal.,2007;Wrightetal., 0166-0934/$–seefrontmatter© 2011 Elsevier B.V. All rights reserved.

doi:10.1016/j.jviromet.2011.06.018

1997)andretinal(Bonthiusetal.,2007;Metsetal.,2000)injury havebeendescribed.

AcutehumanLCMVinfectionsarediagnosedgenerallybyvirus isolationfromCSFandtheuseofserologicaltestingorclassicalPCR assays(Parketal.,1997;Emonetetal.,2007)offerthepossibility todetectpre-andpostnatalLCMVinfections.Morerecently,SYBR Green-basedquantitativePCRassaysweredevelopedtoquantify LCMVininfectedmice(McCauslandandCrotty,2008;Emonetetal., 2007).Duetotheirhighersensitivityandspecificitycomparedto bothviralcultureandimmunofluorescence-baseddetectionmeth- ods, RT-PCR viral assays are favored for the diagnosis of viral infectioninmostroutinelaboratories.

A real-time RT-PCR assay with specific primers and probes adaptedtopublishedLCMVsequencesavailablewasdevelopedto screenacuteLCMVinfectionsinCSF.Theuseofaspecificprobehas themainadvantagetoincreasetheLCMV-specificitycomparedto SYBRGreenPCRmethods.Overaperiodoftwoyears,allhospital- izedpediatriccasesandadultslessthan25-years-oldforwhoma screeningforviralmeningoencephalitiswasrequiredbythephysi- cianattheUniversityofGenevaHospitalswerescreenedforLCMV infectionsusingthisreal-timeRT-PCR.

Toidentifyprimersandprobes,conservedregionsspecificto LCMVwerescreenedbasedonanextensivealignmentofallLCMV and Old World arenavirus’s sequences available in Genbank in August2010,includingallrecentlydiscoveredarenaviruses(Briese etal.,2009;Guntheretal.,2009;Palaciosetal.,2008).Thisalign- ment allowed to pinpoint a conserved region within the viral nucleoprotein (NP) gene. Primer pairs were designed tocorre- spondto theleast number of combinationsmatching perfectly eachdistinctLCMVsequence.Therefore,theprimersusedinthe PCR are constituted of a mix of 9 and 8 selected forward and reverseprimers,respectively(Fig.1).Primersandprobe(Eurogen- tech,Seraing,Belgium)werescreenedbyNCBInucleotideBLAST (Altschuletal.,1990)toexcludeanycross-reactionswithhuman cellularsequencesand distantly relatedviruses.The probewas labelledatthe5endwiththe6-carboxyfluorescein(FAM)andat the3endwiththeBHQ1blackholequencher.Inbrief,theviral genomewasextractedindividuallyfrom400␮lofsamplesusing theNucliSENSeasyMAG(bioMérieux,Geneva,Switzerland)nucleic acidkit, according to the manufacturer’s instructions. In addi- tion,20␮lofstandardizedcaninedistempervirus(CDV)ofknown concentrationwasaddedtoeachsamplebeforeextractiontocon- trolforintra-andinter-assayvariabilityaspreviouslydescribed (Cordey et al., 2010). Toremove a maximum of DNA contami- nantineluates,aDNAsetreatmentwasdoneusingtheDNA-free kit(Ambion,Rotkreuz,Switzerland), accordingtothemanufac- turer’sinstructions.Thisstepwasnecessaryforoptimalsensitivity inthepresenceofmorethan5ng/mlofhumanDNAinthesam- ple.ThesynthesisofcDNAwasperformedwithrandomhexamers (Roche,Rotkreuz,Switzerland) at 42^◦C using theSuperscript II ReverseTranscriptase(Invitrogen,Basel,Switzerland),according tothemanufacturer’s instructions.cDNA wasamplifiedusing a TaqMan^®7500(AppliedBiosystems,Rotkreuz,Switzerland)ther- mocyclerunderthefollowingcyclingconditions:95^◦Cfor9min;50 cyclesof15sat95^◦Cand1minat58^◦C.Afterassessmentofoptimal primers/probeconcentrations,thereactionwasperformedin20␮l containing1×TaqMan^®UniversalPCRMasterMix(AppliedBiosys- tems),0.2␮MofeachindividualLCMVforwardprimer,0.2␮Mof eachindividualLCMVreverseprimer,0.2␮MofLCMVprobeper reaction,and5␮lofcDNA.ResultswereanalyzedusingtheSDS1.4 program(AppliedBiosystems).

Theanalytical sensitivityof theLCMVreal-time RT-PCRwas determined using the mammalian expression plasmid pcAGGS containingthe cDNA of theNP derived fromthe LCMVisolate Armstrong(Arm)53bin10-foldserialdilutions.Theexperiment showeda linear range between50 and 5×10⁴ copies of input

with thelowest limit of detection being ≤50 copies per reac- tion (Fig. 2). Of note, 5 plasmid copies were detectedonly in oneofthetriplicatewells.Sensitivitywasassessedfurtherusing crude stocksof theLCMVisolates LCMV-Arm53b,LCMV-WE54, LCMV-Traub, and LCMV-E350 grown in BHK21 cells (Table 1) whichweredilutedseriallyinapoolofhumanCSFnegativefor LCMV.ThefourLCMVisolatesshowedlowlimitsofdetectionin plaque-forming units (PFU) determined by immunofocus assay (≤10PFU/ml for Arm53b, ≤10PFU/ml for WE54, ≤1PFU/ml for Traub,and≤1PFU/mlforE350).Similarly,dilutionseriesassays performedin phosphate bufferedsalineshoweda reproducible detectionlimitof≤1PFU/mlforeachLCMVisolate,thussuggest- ingthepresenceofdefectiveinterferingparticles,awell-known phenomenonwithLCMV(MartinezPeraltaandLehmann-Grube, 1983; Francisand Southern,1988; Meyerand Southern, 1997).

Inaddition,therecentlyidentifiedLCMV-Marseillestrainsuper- natant(Emonetetal.,2007)hasalsobeentestedpositive.Toassess thepotentialspecificityofthisreal-timeRT-PCR,thegenetically moredistantNewWorldarenaviruses,Juninvirusand Pichinde virusisolates,aswellasindividualpcAGGSplasmidscontaining the cDNA of the NP derived from Lassa virus, Machupo virus, Tacaribe virus, Latinovirus, andWhitewater Arroyoviruswere examinedalso(Table1).Consistentwiththeirphylogeneticdis- tance fromLCMV, noneof these arenaviruseswas detectedby theassay,exceptLassavirusforwhichaweakcross-reactionwas observedat a veryhighconcentration,although a minimumof 1and4mismatchesarepresentintheforwardand thereverse primersets,respectively.Thiscross-reactioncanbeexplainedby theextremelyhighsequencehomologybetweenLassaandLCMV.

Therefore,cross-reactioneventsshouldbeconsideredforsamples withCT (thresholdvalue)values close tothelimit ofpositivity determinedat40C_T.Finally,alargepanelofunrelatedvirusisolates, includingherpessimplexvirus,varicellazostervirus,Epstein–Barr virus,JCvirus,measlesvirus,enterovirusandparechovirus,belong- ingtoothervirusfamiliesknowntoleadpotentiallytomeningitis ormeningoencephalitiscomplications,remainedundetectedwith theLCMVreal-timeRT-PCR.

Allhospitalizedpediatriccasesandadultslessthan25yearsold whounderwentaCSFscreeningwithnegativeresultsforselected viralinfections (enterovirus,parechovirus,herpessimplexvirus, varicellazostervirus,Epstein–Barrvirus,JCvirus,and/ormeasles virus)betweenOctober2008andSeptember2010wereassessed forLCMVwiththenewlydevelopedreal-timeRT-PCRassay.

Ofatotalof130CSFsamples(56female[43%];74male[57%];

medianage,2yearsold),51(39%)werecollectedininfantsless than1-year-old.Theclinicalsyndromesrangedfrommeningitis, encephalitis,convulsions,andothercentralnervoussystemdis- eases.Theanalysiscoverstwowinterseasonsknowntorepresent thepeakwindowperiodofinfection.

Noneofthe130CSFsampleswasfoundpositiveforLCMV.Since thedesignofthespecificLCMVprimersandprobewasbasedonthe availablesequenceinformationinGenbankatthattime,thepres- enceofyetunknownstrainsofLCMVcirculatinginrodentsand transmittedtohumanscannotberuledout.Toaddressthisissue, apreviouslypublishedpan-arenavirusRT-PCR(Viethetal.,2007) capableof detecting allcurrently knownOld World arenavirus species,includingLCMV,Lassavirus,andMobalavirus,wasused.

Whenappliedtothe130CSFsamples,nonewaspositiveforLCMV, similartotheresultsobtainedwiththespecificLCMVreal-time RT-PCRassaydescribedabove.

ThisreportdescribestheanalyticalvalidationofaLCMVreal- timeRT-PCRassayallowingrapiddetectionofLCMVstrainsthat havebeenshowntoinfecthumans.

Despite importanteffortsto contactmostinvestigators with documentedcases ofhumanLCMVinfectionsover thelast few years,itwasnotpossibletoobtainsuchrareclinicalsamplesdue

Fig.1.PrimersandprobeforLCMVreal-timeRT-PCRassay.(a)NineteenLCMVNPreferencesequencesindicatedbytheirGenbankaccessionnumbers(EU480452,EU480450, DQ868487,DQ118959,M22138,DQ868485,DQ286931,AB261990,AB261991,FJ895883,FJ895884,FJ895882,AF325215,AF325214,DQ868483,DQ361065,AY847350, M20869.1andNC004294)werealignedwiththeMAFFTalgorithmintheGeneioussoftwarepackage.Consensuswasestablishedwith100%identicalresidueateach position.ConservedresiduesareindicatedbydotsandpolymorphicresiduesbytheiractualIUPACcode.Themostconservedarea,shownhereasdoublestrandedcDNA,was usedasthetargetforthereal-timeRT-PCRassay.Theprimersequencesareboxedinstrand-specificarrows.Theforwardprimers(F1–F9)areonthecoding(upper)strand andthereverseprimers(R1–R8)areonthecomplementary(lower)strand.Theprobeisshowninboldfontsintheupperstrandoftheconsensussequence;capitalletters indicateLNAresiduesusedtoincreaseitsmeltingtemperature.LNAresidueswerepositionedwithinthishighlyconservedareatolimittheriskoffalsenegativitywith unknownLCMVsequences.Numbersontoprefertothe5endoftheprimer/probepositionswithintheNPopenreadingframeusingtheArmstrongstrainsasreference.

LCMVphylogenyisdepictedbythetreeontheleft-handsidepointingtoeachindividualreferencesequence.Primercombinationadaptedtotheamplificationofeach genotypeisshowntotherightoftheaccessionnumberandreferstotheprimernamesindicatedin(b)(F,For;R,Rev).

totheverylimitedamountofCSFcollectedinitially.Nevertheless, serialdilutionsofbothArm-derivedplasmidanddifferentLCMV strainsculturesinapoolofCSFconfirmedthehighanalyticalsen- sitivityof thenewassaywithlimits ofdetection reaching≤50 copiesperreactionand≤10or1PFU/ml,respectively,indicating apotentialhighclinicalsensitivity.

Irrespectiveof thePCR assay used (LCMV specific real-time RT-PCR and classical broad range arenavirus PCR), LCMV was not detected in any CSF samples over the study period. These

results show the extremely low incidence of LCMV infections leading tohospital admission for acutemeningoencephalitis in western Switzerlandand confirm previous prospectivesurveil- lanceinvestigationsconductedelsewhere.Forinstance,Parketal.

(1997) found nopositive case for LCMVamong a total of 813 CSF samples collected over a one-year period at two Birming- ham(UK)hospitals.Onlyextensiveserologicalstudiesatcountry level may reveal the prevalence of LCMV within the human population.

Table1

PerformanceoftheLCMVreal-timeRT-PCRforthedetectionofdifferentLCMVandotherarenavirusstrains.

Strain LCMVreal-timeRT-PCR Limitofdetection Slope R²

LCMV-ARM53b Positive ≤10PFU/ml(inCSF) 3.62 1

LCMV-WE54 Positive ≤10PFU/ml(inCSF) 3.91 0.99

LCMV-Traub Positive ≤1PFU/ml(inCSF) 3.87 1

LCMV-E350 Positive ≤1PFU/ml(inCSF) 3.65 1

LCMV-Marseille Positive ND ND ND

Lassa Weakpositivecross-reaction(CT>39) Testedfor10⁴plasmidcopies/PCR

Tacaribe Negative Testedfor10⁴plasmidcopies/PCR

Machupo Negative Testedfor10⁴plasmidcopies/PCR

Latino Negative Testedfor10⁴plasmidcopies/PCR

WhitewaterArroyo Negative Testedfor10⁴plasmidcopies/PCR

Junin Negative Testedfor10⁵copies/ml(inplasma)

Pichinde Negative Testedfor10⁵copies/ml(inplasma)

Fig.2.AnalyticalsensitivityoftheLCMVreal-timeRT-PCRassay.10-Foldserial dilutionsofArmstrong-derivedplasmid(5×10⁴–5×10¹copiesperreaction)were testedbyreal-timeRT-PCRassay.Thelogofcopynumbersisplottedversusthe thresholdcycle(CT).Eachdotrepresentstheaverageofthreeindependentexper- iments.Ofnote,adilutionwith5×10⁰plasmidcopiesshowedpositivedetection inonlyonesingletriplicate.Itwasnotconsideredforslopecalculation.Errorsbars indicatestandarddeviations.

Thereal-timeRT-PCRassaydescribedhereprovidesasensitive andadaptedtoolforspecializedclinicallaboratories.

Althoughthecurrentstudydidnotrevealthepresenceofacute LCMVinfectionin childrenand youngadultsover thepast two yearsinwesternSwitzerland,potentialinfectionsshouldnotbe overlookedasclinicalcomplications relatedtoLCMVhavebeen observed in sporadic cases throughout theworld (Asnis et al., 2010;Ceianuetal.,2008;Emonetetal.,2007;Sosaetal.,2009).

Inaddition,thisreal-timeRT-PCRassaycouldbeusedinrodents, particularlyinpetshops, astheyrepresent theprincipalLCMV reservoir.

Competinginterests Nonedeclared.

Ethicalapproval Notrequired.

Acknowledgements

WewouldliketothankProf.StephanGüntherfromtheBern- hardNochtInstitute,Hamburg,Germany,forprovidinguswiththe OldWorldPan-arenavirusupdatedPCRassay.Wearealsograteful

toRosemarySudanfor editorialassistance.Thisstudywassup- portedbytheSwissFederalOfficeofPublicHealth(L.K.)andby fundsfromtheUniversityofLausanne(S.K.).

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