Development of tailored real-time RT-PCR assays for the detection and differentiation of serotype O, A and Asia-1 foot-and-mouth disease virus lineages circulating in the Middle East

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ContentslistsavailableatScienceDirect

Journal

of

Virological

Methods

jou rn a l h om ep a 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

Development

of

tailored

real-time

RT-PCR

assays

for

the

detection

and

differentiation

of

serotype

O,

A

and

Asia-1

foot-and-mouth

disease

virus

lineages

circulating

in

the

Middle

East

Scott

M. Reid

a,1

_,

_Valerie

_Mioulet

a

_,

_Nick

_J.

_Knowles

a

_,

_Nazeem

_Shirazi

b

_,

Graham

J. Belsham

c

_,

_Donald

_P.

_King

a,∗

a_Pirbright_Institute,_Ash_Road,_Pirbright,_Woking,_Surrey_GU24_0NF,_United_Kingdom b_Khorasan_Razavi_Central_Veterinary_Laboratory,_Iran_Veterinary_{Organization,}_Iran

c_National_Veterinary_Institute,_Technical_University_of_Denmark,_Lindholm,₄₇₇₁_Kalvehave,_Denmark

Articlehistory: Received6April2014

Receivedinrevisedform26June2014 Accepted1July2014

Availableonline9July2014 Keywords:

Foot-and-mouthdiseasevirus FMDVserotyping

FMDVreal-timeserotypingRT-PCRassay FMDVmultiplexreal-timeserotyping RT-PCRassay

a

b

s

t

r

a

c

t

Rapidandaccuratediagnosisis essentialforeffectivecontroloffoot-and-mouthdisease(FMD).In

countrieswhereFMDisendemic,identiﬁcationoftheserotypesofthecausativevirusstrainsisimportant

forvaccineselectionandtracingthesourceofoutbreaks.Inthisstudy,real-timereversetranscription

polymerasechainreaction(rRT-PCR)assaysusingprimer/probesetsdesignedfromtheVP1codingregion

ofthevirusgenomesweredevelopedforthespeciﬁcdetectionofserotypeO,AandAsia-1FMDviruses

(FMDVs)circulatingintheMiddleEast.Theseassayswereevaluatedusingrepresentativeﬁeldsamples

ofserotypeOstrainsbelongingexclusivelytothePanAsia-2lineage,serotypeAstrainsoftheIran-05

lin-eageandserotypeAsia-1virusesfromthreerelevantsub-groups.WhenRNAextractedfromarchivaland

contemporaryﬁeldstrainswastestedusingone-ortwo-steprRT-PCRassays,allthreeprimer/probesets

detectedtheRNAfromhomotypicvirusesandnocross-reactivitywasobservedwithheterotypicviruses.

Similarresultswereobtainedusingbothsingle-andmultiplexassayformats.Usingplasmidstandards,

theminimumdetectionlevelofthesetestswasfoundtobelowerthantwocopies.Theresultsillustrate

thepotentialoftailoredrRT-PCRtoolsforthedetectionandcategorizationofvirusescirculatinginthe

MiddleEastbelongingtodistinctsubgroupsofserotypesO,AandAsia-1.Theseassayscanalsoovercome

theproblemofserotypingsampleswhicharefoundpositivebythegenericrRT-PCRdiagnosticassaysbut

negativebyvirusisolationandantigen-detectionELISAwhichwouldotherwisehavetobeserotypedby

nucleotidesequencing.AsimilarapproachcouldbeusedtodevelopserotypingassaysforFMDVstrains

circulatinginotherregionsoftheworld.

(http://creativecommons.org/licenses/by/3.0/).

1. Introduction

Foot-and-mouthdisease(FMD)isahighlycontagiousvesicular diseaseofwildanddomesticcloven-hoofedanimals,particularly cattle,sheep,pigsandgoats.It isthemosteconomically impor-tantviraldiseaseofdomesticatedlivestockthroughouttheworld today,beingendemicinmanycountriesofAfrica,AsiaandSouth

∗ Correspondingauthor.Tel.:+4401483231131.

E-mailaddresses:[email protected],[email protected]

(D.P.King).

1 _Present_address:_Department_of_Avian_Virology,_Animal_Health_and_Veterinary

LaboratoriesAgency-Weybridge,WoodhamLane,NewHaw,Addlestone,Surrey KT153NB,UK.

America.However,outbreakscanalsooccurincountriesthatare normallyfreeofFMD:includingrecentlyJapanandKorea(2000 and2010),theUnitedKingdom(UK:2001and2007),Franceand TheNetherlands(2001)plusBulgaria(2010–2011).Thecausative agent,FMDvirus(FMDV),isasingle-strandedpositive-senseRNA virusofaround8.4kilobasesinlength,belongingtothegenus Aph-thoviruswithinthefamilyPicornaviridae(Belsham,2005).There aresevenimmunologicallydistinctserotypes:O,A,C,SAT (South-ernAfricanTerritories)1,SAT2,SAT3andAsia-1whichencompass adiverseantigenicspectrumofvirusstrains.Thesevenserotypes arenotdistributedequallyaroundtheworld(KnowlesandSamuel, 2003).SerotypesOandAarewidelydisseminatedbuttypeCviruses havenotbeendetectedsince2004,whileAsia-1,despitehaving beendetectedinGreece,ismainlyconﬁnedtotheAsiancontinent. http://dx.doi.org/10.1016/j.jviromet.2014.07.002

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TheSATserotypesarenormally,butnotexclusively,restrictedto sub-SaharanAfrica.

IncountriesnormallyfreeofFMD,rapidconfirmationofFMDV asthecausativeagentinmaterialfromsuspectdiseasecasesis theprimarygoaloffieldandlaboratoryinvestigations.Anumber of generic (pan-serotypic)real-time reverse transcription poly-merasechainreaction(rRT-PCR)assayshave beendevelopedto targethighlyconservedregionsoftheRNAgenomeofFMDV(Reid et al., 2002; Callahan et al., 2002; Moniwa et al., 2007) which efficiently detect all sevenserotypes in clinicalsamples. These assays have undergoneextensiveevaluation;parallel testing of sampleshasshownthatthesensitivityofthesemolecularassays is atleast equal tothat ofthe current“gold standard”method ofvirusisolationincellculture(Shawetal.,2004; Ferrisetal., 2006;Kingetal.,2006;Reidetal.,2009).However,alimitationof therRT-PCRprocedurestargetingconservedsequencesin untrans-lated regions of the genome or those encoding non-structural proteinsisthattheycannotdeterminetheserotypeofthecausative FMDV.Therefore,usingtheseassaysitisnotpossibletoidentify theserotypeof virussamplesthat are positive byrRT-PCR but virusisolation (VI)/antigen-detectionELISA(Ag-ELISA)negative. Serotypingofthesesamplesmayonlybeaccomplishedbytheuseof serotype-specificprimersandprobesand/ornucleotide sequenc-ing.Serotype-specificprimer/probesetsmayalsobespecifically beneficialtothosecountriesthatcurrentlyuseonlythegeneric rRT-PCRassaysoracombinationofgenericrRT-PCRandAg-ELISA butnotVI.

In FMD-endemiccountries, identificationoftheserotypes of thecausativevirusstrainsisimportantforvaccineselection, dis-ease containment and for tracing thesource of theoutbreaks. ConventionalRT-PCRproceduresusingprimerscorrespondingto theVP1 (1D)codingregionandtothe2A/2B codingregionsfor serotyping of FMDV have beenreported (Vangrysperre and De Clercq,1996;CallensandDeClercq,1997).However,subsequent studies have shown that these agarose gel-based assays have relativelypoorsensitivityandspecificityduetothegenetic diver-sitywithinall FMDVserotypes (Reid etal., 1999,2001).While theseprocedurescanbeusedinconjunctionwithAg-ELISAand VItoprovideadditionalinformation,theyareinsufficiently sen-sitivetoreplacethemforprimarydiagnosisofFMD(Reidetal., 1999).AlternativeRT-PCRassaysreportedbySuryanarayanaetal. (1999)andAlexandersenetal.(2000)forserotype-specific diag-nosis were cumbersome and unsuitable for routine use in the eventofanepidemic.AconventionalRT-PCRprocedurefor dif-ferentiation of FMDV serotypes native to India using multiple primersbasedmostlyonnucleotidesequencesofviruses circulat-inginthatgeographicalareawasdescribed byGiridharanetal. (2005).These studiesdemonstrated the potentialfor using tai-loredmoleculartoolstoidentifyspecificserotypesasanalternative oradditiontopan-serotypicassaysfordetectionofFMDV.More recently,thedevelopmentand evaluationofareal-timereverse transcription-loop-mediated isothermal amplification assay for rapidserotypingofFMDVhasbeenreported(Madhanmohanetal., 2013).

Theobjective of this studywas todevelop tailoredrRT-PCR methods for identiﬁcation of FMDV serotypes. In addition to usingthesetoolsinendemiccountries,suchanapproachwould overcome the problem of serotyping samples which are posi-tive by pan-serotypic rRT-PCR but cannot be sequenced using standardprotocols.ThiscollaborativestudybetweentheWRLFMD (ThePirbrirhgt Institute, UK), Technical University of Denmark (DTU) and Khorasan Razavi Central Veterinary Laboratory in Iran(CVL)describesthedevelopmentand evaluationoftailored serotype-speciﬁcrRT-PCRassaysforthedetectionofarchivaland contemporarystrainsofFMDVserotypesO,AandAsia-1circulating intheMiddleEast.

2. Materialsandmethods

2.1. Virusisolatesandsamplepreparation

FMDVisolateswereselectedfromarchivalstocksheldinthe WRLFMD(PirbrightInstitute, UK)repositoryat−20◦_C_and

con-temporarysubmissionsfromwithintheserotypeOPanAsia-2and serotype A Iran-05 lineages, respectively, serotype Asia-1 sub-groups 1,2 and6, plusrepresentative FMDV-GenomeDetected samplesofundeterminedserotype(FMDV-GD:Ag-ELISAandVI negative). To check the diagnostic specificity of the serotype-specific primer/probe sets, archival and currently circulating serotype O and A viruses belonging to other topotypes (geo-graphicallyandgeneticallydistinctevolutionarylineages;Knowles and Samuel,2003)and serotypeAsia-1 virusesfromother sub-groupswerealsotested.Afurthercheckonthespecificityofthe primer/probesetswasmadebytestingFMDVstrainsbelongingto serotypesC,SAT1andSAT2plusrepresentativestrainsofswine vesiculardiseasevirus(SVDV)whicharecapableofcausing clini-callyindistinguishable(‘look-alike’)diseasetothatcausedbyFMDV inpigsandsomesamples,designated‘NVD’,fromwhichnovirus wasdetectedbyeitherAg-ELISA,VIorpan-serotypicrRT-PCR.Most samplescomprised∼10%(w/v)originalsuspensionsofvesicular epithelium(OS;FerrisandDawson,1988)whichhadbeentested byAg-ELISAandVI(FerrisandDawson,1988)atthetimeofreceipt. Additionalclinicalsamplessuchassalivasamplesorhomogenates generatedfromhearttissuewerealsotested,andcellculture super-natantsofselectedstrainsweretestedwhenOSwasunavailable.All thesamplestestedarelistedinthesupplementarydata—Annexes AandB.

2.2. Totalnucleicacidextraction

Total nucleic acid was extracted from the samples by an automated procedure (Shaw et al., 2007). Priorto nucleic acid extraction,0.2mlofOS,salivaorcellculture-grownviruseswere addedper0.3mlofLysis/BindingBuffer(Roche)or0.2mlofOSor cellculture-grownviruswasaddedto1.0mlofTRIzol®_Reagent

(Invitrogen)andstoredat−70◦_C._Nucleic_acid_extraction_was_also

performedmanuallyusingaQIAampViralRNAMiniKit(Qiagen) accordingtothemanufacturer’sinstructions.

2.3. Primerandprobedesign

GenBank sequences (http://www.ncbi.nlm.nih.gov/) were aligned from the VP1 coding region of the FMDV genome for serotypeOPanAsia-2,serotypeAIran-05andAsia-1strainsof sub-groups1,2and6,respectively,usingBioEdit(http://www.mbio. ncsu.edu/bioedit.html).Hydrolysis(TaqMan®₎_probes_and_primers

were designed from conserved sequences using PrimerExpress version3.0software(AppliedBiosystems).

Inpilotexperiments,forserotypesOandA,twocandidate for-wardprimers,tworeverseprimersandoneprobeweredesigned for evaluation. Four forward primers, two reverse primers and fourprobeswereevaluatedinallcombinationsfordetectionof Asia-1 virus sequences of thethree targeted subgroups of this serotype.Thebest-performingprimer/probesetsforeachserotype were selected after all sets were tested by one-step rRT-PCR usinganampliﬁcationprotocoladapted forroutinediagnosisof FMDV (Shaw et al.,2007)withtemplate nucleicacidextracted fromserotypeOPanAsia-2,serotypeAIran-05andAsia-1viruses (subgroups 1, 2 and 6, includingthe recently circulating Asia-1/Sindh-08lineage),respectively(datanotshown).Table1lists thesequencesofthebest-performingprimer/probesets.

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Table1

FMDVserotype-speciﬁcityandsequenceoftheprimers/probesets.

Primer/probename Orientation FMDVserotypespeciﬁcity Sequence(5–3)

TypeOforprimer1 Forward Oa _{CCGAGACAGCGTTGGATAACA}

TypeOrevprimer1 Reverse O CCATACTTGCAGTTCCCGTTGT

PanAsiaOprob349 Forward O FAM-CCGACTTGCACTGCCTTACACGGC-TAMRA

TypeAforprimer2 Forward Ab _{ACGACCATCCACGAGCTYC}

TypeArevprimer2 Reverse A RCAGAGGCCTGGGACAGTAG

AIran05probe Forward A FAM-CGTGCGCATGAAACGTGCCG-TAMRA

Asia1forward3 Forward Asia-1c _{GCAGTWAAGGCYGAGASCATYAC}

Asia1reverse2 Reverse Asia-1 GCARAGGCCTAGGGCAGTATG

Asia1probe4 Forward Asia-1 FAM-AGCTGTTGATCCGCATGAAACGYGCG-TAMRA

a_FMDV_serotype_O_PanAsia-2_lineage. b _FMDV_serotype_A_Iran-05_lineage. c _FMDV_serotype_Asia-1_subgroups_1,₂_and_6.

2.4. EvaluationoftheFMDVserotype-speciﬁcprimer/probesets inassaysonclinicalsamples

Clinical samples were tested at the WRLFMD (Pirbright Institute, UK) by one-step rRT-PCR assays using the serotype-speciﬁcprimer/probesetsruninparallelwiththegenericFMDV primer/probesetstargetingthe5 UTR(Reidetal.,2002)and3D codingregion(Callahanetal.,2002)usingtheprotocolofShaw etal. (2007). Thepan-serotypic assays,targeting independently conservedregionsoftheFMDVgenome,alsoservedtodetermine thepresence of FMDV RNA. AllrRT-PCR reactions wereset-up manuallyandampliﬁcationsperformedinaStratageneMx3005P thermalcycler.

AtDTU(Lindholm,Denmark), two-steprRT-PCRassayswere performedwithrandomprimersforcDNApreparation followed byrRT-PCRampliﬁcationtargetingthe5UTR(Reidetal.,2002), 3Dcoding region(Callahan etal., 2002)orusing the serotype-speciﬁcprimer/probesets.Fiftycyclesofreplicationwereusedin aStratageneMx3005Pthermalcycler.

TheCVL(Iran)methodologyusedbothone-andtwo-step rRT-PCRprotocolswiththebest-performingserotypeO,Aand Asia primers/probesetsfromTable1.AmpliﬁcationbyrRT-PCRwas performed in a Lightcycler 480 (Roche) or ABI 7500 (Applied Biosystems)thermalcyclerswiththefollowingampliﬁcation pro-gramme:anRTstepof50◦Cfor30min;denaturationat95◦Cfor 15min,1cycle;94◦Cfor30s,60◦Cfor1min,50cycles.

2.5. EvaluationofFMDVserotype-speciﬁcprimer/probesetsin multiplexassays

Theindividualserotype-speciﬁcassayswerecombinedtogether inamultiplexformatthatallowedsingleRNAsamplestobe simul-taneouslytested forallthreetargetswithinthesametube.The reactionsweresetupasamultiplexassaywiththeprobetargeting FMDVserotypeOlabelledwith5-Cy5and3-BlackHoleQuencher (BHQ)2,theprobetargetingFMDVserotypeAlabelledwith5 -FAMand3-BHQ1andtheprobetargetingFMDVserotypeAsia-1 labelledwith5-Cy3and3-BHQ2(Sigma-Aldrich,UK).Cycling con-ditionsusedintheseassayswereidenticaltothoseusedforthe individualassays. The diagnosticperformance of themultiplex

assaywascomparedtotheindividualcomponent(singleplex) rRT-PCRassays.

2.6. PreparationofRT-PCRproductssuitableasstandardsforthe serotype-speciﬁcrRT-PCRassays

RT reactions(in 20␮l) were set up containing 4␮l of RNA fromO/IRN/8/2005,A/IRN/1/2005andAsia1/IRN/10/2004using SuperScriptTM_III_reagents_(Invitrogen,_Paisley,_UK)_according_to_the

manufacturer’sinstructionswith10␮Meachofthereverseprimers (Table2).Primers,RNA,wateranddNTPswereﬁrstcombinedand themixtureheatedto65◦Cfor5min,followedbychillingonice. Theremainingreagentsweresubsequentlyaddedandheatedat 50◦Cfor60minfollowedbya15mininactivationstepat95◦C.PCRs werepreparedusing a Platinum® _Taq_system_(Invitrogen,

Pais-ley,UK)accordingtothemanufacturer’sinstructionswith10␮M eachoftheprimers(Table2).Amplificationresultedinthe produc-tionoffragmentsof231,168and197bpforserotypesO,Aand Asia-1,respectively,correspondingtotheassaytargetregion.The PCRcyclingprotocol(Bio-RadLaboratories,Herts,UK)included30 cyclesconsistingof30seachofdenaturationat94◦C,annealing at55◦Candextensionat68◦C.Oncompletionofcycling,an addi-tionalextensionstepof10minat68◦Cwascarriedoutfollowed byafinalholdstepat4◦C.ResultantPCRproductswerevisualised on2%(w/v)agarosegels(Sigma-Aldrich,UK),beforebeingexcised fromtheagarosegelandpurifiedusingaQiaquickGelExtractionKit (Qiagen,Crawley,UK)accordingtothemanufacturer’sinstructions. 2.7. Preparationofplasmidstandardsfordeterminationof

analyticalsensitivityofserotype-speciﬁcrRT-PCRassays

TheamplifiedassaytargetregionswereclonedintoapGEMT Easyvector(Promega,UK)accordingtothemanufacturer’s instruc-tions.Anumberofpositivecoloniesweregrownovernightin3mlof LB-brothcontaining50␮g/mlofampicillin.Plasmidswerepurified usingaQiagenplasmidminikit(Qiagen,Crawley,UK)according tothemanufacturer’sinstructionsandtestedforthepresenceof theassaytargetregionbyPCRwiththesameprimers.Thecorrect sequenceandorientationwasconfirmedbysequencingwithM13 forwardandreverseprimers(Promega,UK)ona3730DNA anal-yser(AppliedBiosystems,Warrington,UK)bydideoxy-sequencing

Table2

FMDVserotypespeciﬁcity,sequenceandampliconsizewiththeprimersetsusedtomakestandards.

Primername Sequence(5_–3₎ _FMDV_serotype_speciﬁcity _Size_(bp)

SeroOquantfor TCGCAGACCTCGAGGTGG O(IRN8/2005) 231

SeroOquantrev CTTGCAGGTCACCTCTCACG O(IRN8/2005)

PEXSeroAquantfor GGGGTGACCTGGGGCCTCTTGCG A(IRN1/2005) 168

PEXSeroAquantrev TGTCCTGCGACAACACTTCCAC A(IRN1/2005)

PEXSeroAsia1quantfor GGAAACAACCTCACGGCGTGGTG Asia1(IRN10/2004) 197

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usingaBigDye® _Terminator_v3.1_Cycle_Sequencing_Kit_(Applied

Biosystems)followingthemanufacturer’sinstructions.

RNAwastranscribedfrom0.4␮goflinearisedpGEMTplasmids

fromtheT7promotersiteusingaMEGAscript®_T7_in_vitro

transcrip-tionkit(Ambion,Warrington,UK)accordingtothemanufacturer’s

instructions except that transcription wascontinued overnight.

TranscribedRNAwasquantiﬁedusingaNanodrop(Thermo

Scien-tiﬁc,UK)andveriﬁedbyrunning1␮lRNAonaBioAnalyser(Agilent,

UK)usingtheRNA6000Nanokit(Agilent,UK).

2.8. Determinationofthedynamicrangeandanalytical

sensitivityoftheserotype-speciﬁcassaysusingtheRNAand

plasmidstandards

DilutionsoftheRNAtranscriptscontainingbetween1012_and

100 _copies/_␮l _were_used_as_target_material_to_test _the_dynamic

rangeandsensitivityofthesingle-andmultiplexassays.Real-time

RT-PCRwascarriedoutusingSuperscriptIIIPlatinum®_One-Step

qRT-PCRreactionmixand0.5␮lenzyme(Invitrogen,Paisley,UK).

The ﬁnal reaction volume (25␮l) included 1.5␮l of probe at

5pmol/␮l;2␮lofeachprimerat10pmol/␮l,12.5␮l2×reaction

mix, 1.5␮l of DEPC-water (Ambion, Warrington, UK)per

reac-tionand5␮loftargetRNA.Real-timeRT-PCRreactions,including

standardRNAmaterialofbetween1012_and₁₀0_copies/_␮l,_were

carried out in quadruplicatein an Mx3005preal-time thermal

cycler(Stratagene,UK).Fortheseexperiments,thethermalcycling

conditionsincludedasingle60◦CcDNAsynthesisstepfor30min

followedbya95◦Cdenaturationstepof10minbeforePCRcycling

whichincluded50cyclesconsistingof15seachofdenaturation

at95◦C,andannealing/extensionat60◦Cfor1min(aspreviously

described;Shawetal.,2007).

3. Results

3.1. Diagnosticsensitivityandspeciﬁcityofserotype-speciﬁc rRT-PCRprimer/probeassaysinsingleplexandmultiplexassay formats

3.1.1. SingleplexrRT-PCRassayformat

RNA samplesderived from knownor suspect cases of FMD were assayed initially in singleplex rRT-PCR assays. Threshold cycle (CT)values wereassigned to each reaction as previously

described(Reidetal.,2002).Asforroutinediagnosisusingthe pan-serotypicone-steprRT-PCRassays,aCTvalueof32.0wasusedas

thepositive/negativecut-offpoint(Shawetal.,2007;Reidetal., 2009).Adeﬁnitivepositive/negativecut-offwasnotassignedto theserotype-speciﬁcrRT-PCRassays;thestrongestresult(i.e. low-estCTvalue)generatedforeachsamplefromeithertheserotypeO,

AorAsia-1-speciﬁcprimer/probesetwasusedtodesignateanO, AorAsia-1serotypeforthatsample.

SingleplexassaydatageneratedfromWRLFMD,DTUandCVL are summarisedin Table2 and theresults fromtheindividual samplesarelistedinsupplementarydata—AnnexA.Assayswith thepan-serotypicprimersandprobesconfirmedthepresenceof FMDVRNAwithineachsample.Asexpected,bothpan-serotypic assays (targeting the 5 UTR and 3D coding regions) demon-stratedabroaderspectrumofreactivitythantheserotype-specific primer/probesetsanddetectedthegenomes ofserotypeC,SAT 1 and SAT 2 viruses, as wellas being broadlyreactive against serotypeO,AandAsia-1virusesacrossalltopotypes/lineages.All threeserotype-specificprimer/probesetstestedwerestrongly pos-itiveusingRNAextractedfromhomotypicserotypeO,AandAsia-1 virusesofthetargetedlineageorsubgroup.Therewereonlythree instances,allinvolvingtheserotypeO-specificprimers/probeset,

whenhomotypicvirusesofthenon-targetedlineage,weredetected (virusesbelongingtothetopotypesEA-3,EA-4andCATHAY).

Thespecificprimer/probesets weresuccessfulinidentifying fourofthe12FMDV-GDsamplestestedasserotypeOandsevenof thesesampleswereserotypeA.Ofthe12FMDV-GDsamplestested bythesingleplexformat,9and10samples,respectively,were pos-itivebythe5UTRand3Dpan-serotypicPCRassays,however,two samplestestingpositiveusingtheserotypeA-specificprimer/probe setwerenegativeinassayswithpan-serotypicprimer/probesets. No cross-reactivity was observed when the specific primer/probe sets were tested with heterotypic type O, A or Asia-1 isolates except when testing the serotype O PanAsia-2 sample O/PAK/4/2006which alsoproduced aweakreaction (of higher CT value)against the serotypeA-specific primers/probe

set.ThissamplemayhavebeenadualserotypeO/Ainfectionbut asa stronger CT value (28.33)wasproduced withtheserotype

O primer/probe set than that resulting from the heterologous serotype A set (CT value of 31.55), a serotype O identiﬁcation

could be correctly deduced for this sample. Furthermore, no cross-reactivity was observed when all three serotype-speciﬁc primer/probe sets were tested against the heterologous FMDV serotypesC,SAT1andSAT2orwithSVDVisolates(Table3).

Atwo-stepsingleplexrRT-PCRformatwasalsousedatDTUand atCVLinparallelwiththeone-stepformat(datasummarisedin Table3).Liketheone-stepapproach,thetwo-stepassayswiththe speciﬁcprimer/probesetswerestronglyreactivewiththe target-tedtypeOstrainsofPanAsia-2lineage,typeAstrainsoftheIran-05 lineageandAsia-1virusesfromsubgroups1and2,respectively, butoverallassayperformancewasbetterwiththeone-step for-mat(datanotshown).Nocross-reactivitywasobservedwhenthe serotype-speciﬁcprimer/probesetsweretestedusingthetwo-step formatassaysagainsttheheterologousserotypeC,SAT1,SAT2and SAT3FMDVs.

3.1.2. MultiplexrRT-PCRassayformat

Thepositive/negativeacceptancecriteriaadoptedforthe pan-serotypicandserotype-speciﬁcrRT-PCRassaysusingthesingleplex formatweresimilarlyappliedforthemultiplexassaysperformed attheWRLFMD.MultiplexassaydataaresummarisedinTable3 and theCT valuesfromindividualsamplesarelistedin

supple-mentarydata—AnnexB.Speciﬁcityofdetectionusingtheserotype O,AandAsia-1primer/probesetsisalsoapparentfromtheheat maps(Fig.1a–c,respectively).Theseclearlydemonstratethestrong speciﬁcityofrecognitionoftheprimer/probesetsagainstthe tar-gettedvirussequencesoftheisolatesbelongingtotheserotype OPanAsia-2lineage(Fig.1a),serotypeAIran-05lineage(Fig.1b) andAsia-1subgroups1and2(Fig.1c),respectively.However,one sample(O/BAR/1/2008)didnotgenerateapositiveresultsforthe multiplexassayalthoughthesingleplexversionofthistestgavea positivesignal.

The samples Asia-1/PAK/32/2011 and Asia-1/PAK/109/2011 wereamongﬁveAsia-1virusesintheAsia-1/Sindh-08lineagenot detectedbytheAsia-1-speciﬁcprimers/probesets.Bothviruses hadtwomismatchesintheprobe-bindingregionresultingin false-negativerRT-PCRresults.Table4showsthesequencesofselected Asia-1viruses,highlightingmismatchesintheprimerandprobe regions.AllotherAsia-1viruseslistedinTable4weredetectedby themultiplexassay.

Themultiplexassayformatwasalsosuccessfulinassigningthe serotypicidentity tosamplesdesignatedasFMDV-GD(Table3, supplementarydata—AnnexBandheatmap;Fig.1d).Eighty-ﬁve FMDV-GDsamplesweretested(77and82hadtestedpositiveby the 5 UTRand 3D assays, respectively) and of these, serotype O identity wasassigned to 33 samples,17 were designatedas serotypeAandfoursamplesweredesignatedasserotypeAsia-1.

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Table3

SummaryoftherRT-PCRresultsusingserotypeO-,A-andAsia-1-speciﬁcandpan-serotypic5_UTR_and_3D_{primers/probe}_sets_in_multiplex_and_singleplex_assay_formats.

FMDVserotype orother vesicularvirus FMDV topotypea FMDV lineageb

Ratioofnumberofsamplespositivetototal numbertestedbyrRT-PCRc

Multiplexassayformatd _Singleplex_assay_formate,f

Og _Ah _Asia-1i ₅_UTR _3D _Og _Ah _Asia-1i ₅_UTR _3D

O ME-SA PanAsia-2 12/13 0/13 0/13 13/13 13/13 26/26e_5/5f _1/26e_0/5f _0/26e_0.5f _23/26e_5/5f _24/26e_5/5f

ME-SA PanAsia-2ANT-10 _19/19 _0/19 _0/19 _18/19 _19/19 _17/18e _0/18f _0/18f _NTj _18/18f

ME-SA PanAsia-2FAR-09 _2/2 _0/2 _0/2 _2/2 _2/2 _NT _NT _NT _NT _NT

ME-SA PanAsia 5/5 0/5 0/5 5/5 5/5 NT NT NT NT NT ME-SA Unnamed 1/1 0/1 0/1 1/1 1/1 NT NT NT NT NT ME-SA Ind-2001d 1/1 0/1 0/1 1/1 1/1 NT NT NT NT NT SEA Unnamed 0/2 0/2 0/2 2/2 2/2 0/1e _0/1e _0/1e _1/1e _1/1e SEA Mya-98 1/10 0/10 0/10 10/10 10/10 NT NT NT NT NT EA-1 Unnamed 0/1 0/1 0/1 1/1 1/1 0/1e _0/1e _0/1e _1/1e _1/1e EA-2 Unnamed 0/2 0/2 0/2 2/2 2/2 0/2e _0/2e _0/2e _2/2e _2/2e EA-3 Unnamed 0/1 0/1 0/1 1/1 1/1 1/2e _0/2e _0/2e _2/2e _2/2e EA-4 Unnamed 0/1 0/1 0/1 1/1 1/1 1/1e _0/1e _0/1e _1/1e _1/1e WA Unnamed 0/1 0/1 0/1 1/1 1/1 0/1e _0/1e _0/1e _1/1e _1/1e ISA-1 Unnamed 0/1 0/1 0/1 1/1 1/1 0/1e _0/1e _0/1e _1/1e _1/1e ISA-2 Unnamed 0/1 0/1 0/1 1/1 1/1 0/1e _0/1e _0/1e _1/1e _1/1e CATHAY Unnamed 0/3 0/3 0/3 3/3 3/3 1/2e _0/2e _0/2e _2/2e _2/2e

A ASIA Iran-05AFG-07 _0/16 _16/16 _0/16 _15/16 _16/16 _NT _NT _NT _NT _NT

ASIA Iran-05FAR-09 _0/1 _1/1 _0/1 _1/1 _1/1 _NT _NT _NT _NT _NT

ASIA Iran-05BAR-08 _0/4 _4/4 _0/4 _4/4 _4/4 _NT _NT _NT _NT _NT

ASIA Iran-05 0/17 17/17 0/17 17/17 17/17 0/22e_0/9f _22/22e_8/9f _0/22e_0/9f _22/22e_5/9f _22/22e_8/9f

ASIA Iran-05EZM-07 _0/1 _1/1 _0/1 _1/1 _1/1 _NT _NT _NT _NT _NT

ASIA Iran-05QAZ-11 _0/2 _2/2 _0/2 _2/2 _2/2 _NT _NT _NT _NT _NT

ASIA Iran-05ESF-10 _0/1 _1/1 _0/1 _1/1 _1/1 _NT _NT _NT _NT _NT

ASIA Iran-87 0/1 0/1 0/1 1/1 1/1 0/1e _0/1e _0/1e _1/1e _1/1e ASIA Iran-96 NT NT NT NT NT 0/3e_0/2f _0/3e_0/2f _0/3e_0/2f _2/3e_2/2f _3/3e_2/2f ASIA Sea-97 0/4 2/4 0/4 4/4 4/4 NT NT NT NT NT AFRICA G-VIII 0/2 1/2 0/2 2/2 2/2 0/2e _1/2e _0/2e _2/2e _2/2e EURO-SA A24 0/1 1/1 0/1 1/1 1/1 0/1e 1/1e 0/1e 1/1e 1/1e EURO-SA A12 0/1 1/1 0/1 1/1 1/1 0/1e 1/1e 0/1e 1/1e 1/1e AFRICA G-VII 0/2 1/2 0/2 2/2 2/2 0/2e _1/2e _0/2e _2/2e _2/2e AFRICA G-II 0/1 0/1 0/1 1/1 1/1 1/1e _0/1e _0/1e _1/1e _1/1e AFRICA G-VI 0/2 0/2 0/2 2/2 2/2 0/2e _0/2e _0/2e _2/2e _2/2e AFRICA G-V 0/1 1/1 0/1 1/1 1/1 0/1e _1/1e _0/1e _1/1e _1/1e AFRICA G-IV 0/1 0/1 0/1 1/1 1/1 NT NT NT NT NT

Asia-1 ASIA Unnamed 0/4 0/4 4/4 4/4 4/4 0/1e _0/1e _0/1e _1/1e _1/1e

ASIA Sindh-08 0/12 0/12 7/12 12/12 12/12 NT NT NT NT NT ASIA Group1 NT NT NT NT NT 0/4e_0/1f _0/4e_0/1f _4/4e_1/1f _4/4e_1/1f _4/4e_1/1f ASIA Group2 0/2 0/2 2/2 2/2 2/2 0/6e_0/6f _0/6e_0/6f _6/6e_6/6f _6/6e_1/2f _6/6e_6/6f ASIA Group4 NT NT NT NT NT 0/1e _0/1e _1/1e _1/1e _1/1e ASIA Group5 NT NT NT NT NT 0/1e _0/1e _1/1e _1/1e _1/1e ASIA Group6 NT NT NT NT NT 0/2e _0/2e _2/2e _2/2e _2/2e C NT NT NT NT NT 0/1e_0/1f _0/1e_0/1f _0/1e_0/1f _1/1e_1/1f _1/1e_1/1f SAT1 0/3 0/3 0/3 2/3 3/3 0/4e_0/1f _0/4e_0/1f _0/4e_0/1f _2/4e_1/1f _4/4e_1/1f SAT2 0/3 0/3 0/3 3/3 3/3 0/4e_0/4f _0/4e_0/4f _0/4e_0/4f _4/4e_1/1f _4/4e_4/4f SAT3 NT NT NT NT NT 0/1f _0/1f _0/1f _1/1f _1/1f FMDV-GDk _33/85 _17/85 _4/85 _77/85k _82/85k _4/12e _7/12e,m _0/12e _9/12e,m _10/12e,m SVDVn _0/3 _0/3 _0/3 _0/3 _0/3 _0/3e _0/3e _0/3e _0/3e _0/3e NVDo _0/4 _0/4 _0/4 _0/4 _0/4 _NT _NT _NT _NT _NT

a_FMDV_topotypes:_ME-SA,_Middle_East-South_Asia;_SEA,_South_East_Asia;_EA-1,_EA-2,_EA-3_and_EA-4,_East_Africa_1–4;_WA,_West_Africa;_ISA1,_ISA-2,_Indonesia₁_and_2. b _{http://www.wrlfmd.org/fmd}_{genotyping/prototypes.htm}_.

c _For_{pan-serotypic}_rRT-PCR_assays,_samples_producing_a_C

Tvalue<32.0wereconsideredpositive;withallserotype-speciﬁcrRT-PCRassays,thelowestCTvaluegenerated

amongsttheprimers/probesetsuseddeﬁnedtheserotype.Resultsinboldtyperepresentapparentanomalousresultswherepositiveresultswereachievedbythespeciﬁc primer/probesetusingRNAextractedfromhomotypicserotypeO,AandAsia-1viruseshomotypicvirusesofthenon-targetedlineagelineageorsubgroup.

d _All_multiplex_data_were_generated_at_the_WRLFMD.

e_Singleplex_data_generated_at_the_WRLFMD_using_one-step_rRT-PCR_assays.

f _Singleplex_data_generated_from_DTU_and_CVL_using_either_one-_or_two-step_rRT-PCR_assays. g_{Primers/probe}_set:_{TypeOforprimer1/TypeOrevprimer1/PanAsiaOprob349.}

h _{Primers/probe}_set:_{TypeAforprimer2/TypeArevprimer2/AIran05probe.} i _{Primers/probe}_set:_{Asia1forward3/Asia1reverse2/Asia1probe4.} j _NT,_not_tested.

k _FMDV-GD,_FMDV-genome_detected_samples,_i.e._samples_of_undetermined_serotype_that_are_positive_by_rRT-PCR_but_{VI/Ag-ELISA-negative.} l_Thirty-six_samples_were_FMDV-genome_detected_(by_at_least_one_generic_{primers/probe}_set)_but_negative_using_{serotype-speciﬁc}_{primers/probe}_sets.

m _Two_samples_were_positive_using_{TypeAforprimer2/TypeArevprimer2/AIran05probe}_set_were_negative_using_{pan-serotypic}_primer/probe_sets. n _SVDV,_swine_vesicular_disease_virus.

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Fig.1.Diagnosticsensitivityandspeciﬁcityofthelineage-speciﬁcrRT-PCRassaysinmultiplexedformat(togetherwithcomparativedataforthepan-serotypicrRT-PCR assays)todetectrepresentativeFMDviruspositivesamplesfromserotypeOPanAsia-2lineage(a),serotypeAIran-05lineage(b)andAsia-1subgroups1and2(c).Dataare presentedasaheat-mapforCTvaluesgeneratedwiththedifferentrRT-PCRassays(scaleshown).Neighbor-joiningtreesshownrepresentgeneticrelationshipsbetween

VP1sequencesfortheindividualviruses.Individualdatavaluesarepresentedinsupplementaldata.

Table4

SequencesofserotypeAsia-1FMDV sampleshighlightingmismatches withprimers andprobesequences. SamplesAsia-1/PAK/32/2011and Asia-1/PAK/109/2011werenotdetectedbythemultiplexrRT-PCRassay.

21

Asia-1 FMDV sample Forward primer (5’ -3’) Probe (5’ –3’) Reverse primer 3’ -5’) GCAGTWAAGGCYGAGASCATYAC AGCTGTTGATCCGCATGAAACGYGCG CATACTGCCCTAGGCCTYTGC Asia-1/PAK/89/2011 ...A.A... ...C... ...C... Asia-1/AFG/48/2011 ...A.A... ...C... ...C... Asia-1/AFG/51/2011 ...A.A... ...C... ...C... Asia-1/AFG/55/2011 ...A.A... ...C... ...C... Asia-1/BAR/3/2011 ...A.A... ..T..C... ...T..C... Asia-1/BAR/4/2011 ...A.A... ..T..C... ...T..C... Asia-1/IRN/33/2011 ...A.A... ..T..C... ...C... Asia-1/IRN/38/2011 ...A.A... ..T..C... ...C... Asia-1/IRN/43/2011 ...A.A... ..T..C... ...C... Asia-1/IRN/46/2011 ...A.A... ..T..C... ...C... Asia-1/PAK/90/2011 ...A.A... ..T..C... ...C... Asia-1/PAK/32/2011 ...A.A... ...C....A... ...C... Asia-1/PAK/109/2010 ...A.A... ...C....A... ...C...

3.2. Analyticalsensitivity/minimumdetectionlevelof

serotype-speciﬁcrRT-PCRprimers/probeassays

Fig. 2a–c shows the wide dynamic range of the single-plex assays using plasmid standards with the serotype O-, A-and Asia-1-speciﬁc primer/probe sets. Using DNA templates,

the three assays had a limit of detection of 1.78, 2.58 and 2.74 copies, respectively, thus demonstrating the high ana-lytical sensitivity of these tests. The ampliﬁcation efﬁciencies for these three assays were estimated to be 94.9, 93.3 and 99.2%, for the serotype O, A and Asia-1 assays, respectively. Of the two formats, the singleplex assay format had a lower

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Fig.2.AnalyticalsensitivityanddemonstrationofthedynamicrangeoftherRT-PCRassaysusingplasmidstandardswiththeserotypeO-(a),A-(b)andAsia-1-(c)speciﬁc primer/probesets.

minimum detection level than themultiplex version (data not shown).

4. Discussion

Whilepan-serotypicrRT-PCRassaysfordetectionofFMDVhave beenextensivelyevaluatedforroutineuse(Callahanetal.,2002; Shawetal.,2004;Moniwaetal.,2007),severalchallengesfacethe designofserotype-specificprimer/probesetsforrRT-PCR.Insilico analysescanbeusedtotrytoidentifyconserved sequencesfor primer/probebindingsitesthatallowthespecificrecognitionofall viruseswithinaparticularserotype,butdonotcross-reactwith virusesofotherserotypes.Thisprocessischallengingduetothe highvariabilityoftheFMDVgenomeandlackofsequencesthat areconservedwithin,butrestrictedto,aparticularserotype.For thesereasons,effortsherehaveconcentratedondetectingviruses ofparticularserotypesfromwithindistinctgeographicalregions orlineagesratherthanacrossallstrainsgloballywithinaserotype. ThiscurrentstudyutilisedVP1coding sequencestodesign and establishserotype-specificrRT-PCRassays;VP1isoneofthecapsid proteinswhichdisplaysepitope(s)onthevirussurfacetothehost’s immunesystem.VP1iswidelyacceptedasbeingthemost anti-genicallysignificantofthecapsidproteinsanditssequencecanbe usedtoidentifytheserotypeofthestrain.TheVP1codingsequence hasbeenthemostcommonlysequencedandhenceitisnowthe mostinformativeregionoftheFMDVgenomefordeterminingthe relationshipbetweendifferentstrainsforepidemiologicalanalyses. Initial screening of clinical samples in three different lab-oratories using singleplex assay formats showed that the serotype-specificprimer/probessets appropriately detectedthe FMDVgenomeofserotypeOPanAsia-2,serotypeAIran-05 and Asia-1strainsofsubgroups1,2and6asintended(Table3and sup-plementarydata—AnnexA).Furthermore,theassaysdiscriminated betweenvirusesbelongingtotheselineagesfromvirusesofother lineagesorsubgroupswithinserotypesO,AandAsia1,e.g.from otherregionsoftheworld.Nocross-reactivitywasdemonstrated intheassayswithheterotypicserotypesofFMDV.Equally impres-siveassayspecificitywasdemonstratedwhentheprimer/probe setswereusedinamultiplexassayformat(Table3,supplementary data—AnnexBandFig.1).FiveserotypeAsia-1virusesbelonging totheSindh-08lineagewerenotdetectedwiththemultiplex for-mat.Sequencingoftwooftheseisolatesshowedthatthiswasdue tothepositioningoftwomismatchesintheprobe-bindingregion (Table 4). Using either assay format, the specific primer/probe setswerealsoabletoidentifytheserotypeofsamplesdesignated FMDV-GD,therebyprovidingimportantadditionalepidemiological

informationforthesesamplesintheabsenceofanyserotypingdata providedbythecombineduseofAg-ELISAandVI.

Theanalyticalsensitivityofthespecificprimer/probesetsas determinedbytestingserialdilutionsofRNAandplasmid stan-dards demonstrated the wide dynamic range of theassays. As withanyotherRNAviruswitha rapidlyevolvinggenome, con-tinuous monitoringof the primer and probe sequences of the serotype-specific sets against the corresponding sequences of newlycirculatingandemergingviruseswillbeessentialtoensure thattheassaysmaintainfitnessforpurpose.However,theresults fromthisstudyclearlydemonstratethepotentialofrRT-PCRfor routineandrobustdetectionanddiscriminationofFMDVisolates belongingtogeographicallydistinct lineagesand sub-groupsof serotypesO,AandAsia-1currentlycirculatingintheMiddleEast. Previously,alineage-specificrRT-PCRhasbeendevelopedfor useinNorthAfricancountriestospecificallydetectSAT2viruses thatemergedfromsub-SaharanAfricaduring2012andto differ-entiatethesevirusesfromendemicFMDvirusstrainsthatmight bepresentinthesecountries(Ahmedetal.,2012).Thesefindings supporttheuseofthisapproachtodevelopassaystodetectother topotypesofFMDVserotypesO,AandAsia-1,andalsoforother serotypes,especiallySAT2asvirusesofthisserotypehavemore sequence variation in theVP1 coding sequence compared with virusesofserotypesO,AandC.Thiscurrentstudyisthefirstto describetheuseofmultiplelineage-specificrRT-PCRsforthe detec-tionanddiscriminationofFMDVin clinicalsamplesinendemic settings.Theseassaysrepresentthefirststeptowardsthe develop-mentofasuiteofmoleculartools(moleculartoolbox)fordifferent countriesandregions.Asimilarapproachcouldthereforebeused todevelopFMDV-serotypingassaysforothergeographicalregions oftheworld.

Acknowledgements

TheauthorsthankDr.NigelFerrisandGeoffreyHutchingsofthe WRLFMD,fortheinitialpropagation,characterisationandsupplyof thevirusesusedinthestudy.TheworkperformedatThePirbright InstitutewasfundedbyDefra,UK(Projectnumbers:SE1124and SE1127)whilefundingforDTUandCVLwasprovidedbyEuFMD.

AppendixA. Supplementarydata

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jviromet.2014.07.002.

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