Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa

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

and

evaluation

of

tailored

specific

real-time

RT-PCR

assays

for

detection

of

foot-and-mouth

disease

virus

serotypes

circulating

in

East

Africa

Katarzyna

Bachanek-Bankowska

a,∗

,

Herieth

R.

Mero

b

,

Jemma

Wadsworth

a

,

Valerie

Mioulet

a

,

Raphael

Sallu

c

,

Graham

J.

Belsham

d

,

Christopher

J.

Kasanga

b

,

Nick

J.

Knowles

a

,

Donald

P.

King

a

a_{ThePirbrightInstitute,AshRoad,Pirbright,Woking,SurreyGU240NF,UK}

b_{DepartmentofMicrobiologyandParasitology&SouthernAfricanCentreforInfectiousdiseasesSurveillance,SokoineUniversityofAgriculture,Morogoro,}

Tanzania

c_{TanzaniaVeterinaryLaboratoryAgency,P.O.Box9252,DaresSalaam,Tanzania}

d_{NationalVeterinaryInstitute,TechnicalUniversityofDenmark,Lindholm,KalvehaveDK-4771,Denmark}

Articlehistory:

Received26April2016

Receivedinrevisedform26July2016

Accepted1August2016

Availableonline27August2016

Keywords: Foot-and-mouth-diseasevirus Serotype-specific Real-timeRT-PCR EastAfrica

a

b

s

t

r

a

c

t

Rapid,reliableandaccuratediagnosticmethodsprovideessentialsupporttoprogrammesthatmonitor andcontrolfoot-and-mouthdisease(FMD).Whilepan-specificmoleculartestsforFMDvirus(FMDV) detectionarewellestablishedandwidelyusedinendemicandFMD-freecountries,current serotyp-ingmethodsmainlyrelyeitheronantigendetectionELISAsornucleotidesequencingapproaches.This reportdescribesthedevelopmentofapanelofserotype-specificreal-timeRT-PCRassays(rRT-PCR) tai-loredtodetectFMDVlineagescurrentlycirculatinginEastAfrica.Theseassaystargetsequenceswithin theVP1-codingregionthatsharehighintra-lineageidentity,butdonotcross-reactwithFMDviruses fromotherserotypesthatcirculateintheregion.Theseserotype-specificassaysoperatewiththesame thermalprofileasthepan-diagnostictestsmakingitpossibletoruntheminparalleltoproduceCT

val-uescomparabletothepan-diagnostictestdetectingthe3D-codingregion.Theseassayswereevaluated alongsidetheestablishedpan-specificmoleculartestusingfieldsamplesandvirusisolatescollected fromTanzania,KenyaandEthiopiathathadbeenpreviouslycharacterisedbynucleotidesequencing. Samples(n=71)representingserotypeA(topotypeAFRICA,lineageG-I),serotypeO(topotypesEA-2and EA-4),serotypeSAT1(topotypeI(NWZ))andserotypeSAT2(topotypeIV)werecorrectlyidentifiedwith theserRT-PCRassays.Furthermore,FMDVRNAfromsamplesthatdidnotcontaininfectiousviruscould stillbeserotypedusingtheseassays.Theseserotype-specificreal-timeRT-PCRassayscandetectand characteriseFMDVscurrentlycirculatinginEastAfricaandhenceimprovediseasecontrolinthisregion. ©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Foot-and-mouthdisease(FMD)isahighlycontagiousviral dis-easeaffectingbothwildanddomesticatedcloven-hoovedanimals. AlthoughFMDgenerallyhasalowmortalityrate,itaffectsalarge numberofanimalsofmultiplespeciesleadingtolossorreduction inlivestockproductioninendemicregionsinAsiaandAfrica.Itis estimatedthatcollectivelythree-quartersoftheworld’slivestock

∗Correspondingauthor.

E-mailaddress:[email protected](K.Bachanek-Bankowska).

populationisconcentrated inFMDendemicareas(Knight-Jones andRushton,2013)leadingtoanenormoussourceofinefficiency andwasteinfoodproductionespeciallyinthecontextofagrowing demandforlivestockproducts.Inaddition,outbreaksofthedisease canspreadintocountriesnormallyfreeofFMDposingsignificant coststolivestockindustries.

FMD is caused by FMD virus (FMDV), belonging to genus

Aphthovirus within the family Picornaviridae (Knowles et al., 2012).Thenon-envelopedvirusparticleenclosesasingle-stranded positive-senseRNAgenomeofapproximately8.3kilobases.Seven immunologicallydistinctserotypesofthevirusexist:O,A,C,SAT (SouthernAfrican Territories)1, SAT2,SAT 3and Asia 1;each

http://dx.doi.org/10.1016/j.jviromet.2016.08.002

0166-0934/©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.

containingmultiplevariants(topotypes)oftenrestrictedto spe-cificgeographicallocations.GlobaldistributionofFMDVserotypes isuneven.Currently,serotypesOandAhavethewidestdistribution andarereportedinmostendemicviruspools.

Thesurfaceexposed capsidprotein,VP1, playsanimportant roleinantigenicandphylogeneticcharacterisationofFMDVasit containsmajorimmunogenicsites,includingaminoacidresidues withintheG-HloopandtheC-terminus(Baranowskietal.,2000; Jacksonetal.,2002).TheVP1-codingnucleotidesequencevaries between FMDV serotypes. Further classification of viruses into topotypesandlineagesisalsopossiblebasedonphylogenetic anal-ysisofthesequenceoftheVP1-codingregion(KnowlesandSamuel, 2003;SamuelandKnowles,2001).Thegeneticrelationshipofthe VP1nucleotidesequencebetweendifferentstrainsiscommonly appliedfortracingtheoriginandmovementofoutbreakviruses (Abdul-Hamid etal.,2011;DiNardoetal.,2014;Knowlesetal., 2014).

TheestablishedmethodsforFMDcontrol,suchasvaccination and movement restrictions (Sumption et al., 2012), are under-pinned by rapidand accuratediagnosisof clinical cases ofthe disease. Currently, pan-specific real-time reverse transcription polymerasechain reaction(rRT-PCR)assaysforFMDVdetection havebeendescribed(Callahanetal.,2002;Moniwaetal.,2007;Reid etal.,2002,2009).TheseamplifyhighlyconservedRNAsequences withinthe5-untranslatedregion(UTR)or theRNApolymerase (3D-coding region), respectively. As these genome regions are highlyconservedforallsevenserotypes,FMDVcanbedetected withhighdiagnostic sensitivity and specificity. However,rapid identificationof theserotype ofan outbreak virusis necessary forvaccineselection,especiallyinendemiccountries.Thisis nor-mallyachievedbyantigendetectionELISAand/orVP1sequencing (FerrisandDawson,1988;Knowlesetal.,inpress).Withboth meth-odsoftenrequiringvirusisolationandpropagationforincreased sensitivity,thetimeforserotypeidentificationandthereforefor implementationofeffectivevaccinationcanbeprolonged.

AstheVP1codingregionnucleotidesequencevariesaccording tothe serotypeof FMDV, typing canbe achieved by serotype-specificdetectionoftheVP1 codingregion,ideally,withoutthe needforviruspropagation.ConventionalRT-PCRassaystargeting theVP1codingregionhavebeendescribed(Alexandersenetal., 2000;CallensanddeClercq,1997;VangrysperreanddeClercq, 1996)butduetothehighgeneticdiversitywithinthetargetregion thesehaverelativelypoorsensitivityandspecificity(Reidetal., 2001).Amicroarray-basedsystemfortypingFMDVhasalsobeen described(Baxietal.,2006)butthiscanbelengthyandcostlyand alsorequiresuseofspecialisedequipment.Sequencedata analy-sisoftheVP1 codingregionindicatesthat,duetotheextensive intra-serotypevariationoftheVP1codingnucleotidesequences, developmentofreliableserotypespecificRT-PCRassaysmightbe difficulttoachieveonaglobalbasis(Reidetal.,1999).

AsFMDVcirculationoftenoccursinregionalreservoirs(Paton etal.,2009), topotypesand lineagesof FMDVspecificto differ-entgeographicalareashaveevolved.Thesecanbeidentifiedand characterisedbasedonphylogenetic analysisoftheVP1 coding sequences. Exploiting the fact that genetic variability between strainswithinlineagesisreducedincomparisontothenucleotide variabilitywithinserotypes,RT-PCRassaystailoredtoFMDV lin-eagesandthereforegeographicareascanbedeveloped.Thisidea hasbeenappliedinconventionalRT-PCRsystemstodistinguish betweenstrainsofFMDVbelongingtodifferentserotypes circu-latinginIndia(Giridharanetal.,2005).Thissystemwasfurther developedandappliedtoserotypevirusescirculatinginthe Mid-dleEast(Reidetal.,2014),WestEurasia(JamalandBelsham,2015) aswellasforthedetectionoftheSAT2serotype(topotypeVII) inEgypt(Ahmedetal.,2012)andtheserotypeOvirusesinNorth AfricabelongingtoInd-2001dlineage(Knowlesetal.,2014).

Table1

ListofprimersandprobesforFMDVtype-specificassaysinEastAfrica.

OLIGONAME NUCLEOTIDESEQUENCE(5→3)

FMDV/A/FP GCCACRACCATCCACGA FMDV/A/RP GAAGGGCCCAGGGTTGGACTC FMDV/A/P FAM-CTCGTGCGMATGAARCGGGC-BHQ1 FMDV/O/FP CCTCCTTCAAYTACGGTG FMDV/O/RP GCCACAATCTTYTGTTTGTG FMDV/O/P FAM-CCCTCTTCATGCGGTARAGCAG-BHQ1 FMDV/SAT1/FP CTYGACCGGTTCACYCTG FMDV/SAT1/RP CCGAGAAGTAGTACGTRGC FMDV/SAT1/P FAM-CAGGAYTGCGCCCACCA-BHQ1 FMDV/SAT2/FP CRATCCGCGGTGAYCG FMDV/SAT2/RP CGCTTCATYCTGTAGTARACGTC FMDV/SAT2/P FAM-TTTGGACAYGTGACCGCCG-BHQ1

Thenamesoftheprimersandprobesindicatethetype-specificityoftheassays.FP, RPorPstandfor“forwardprimer”,“reverseprimer”or“probe”respectively;R=A orG,Y=CorTandM=AorC.

ThisstudydescribesthedevelopmentofasetoftypingrRT-PCR

assaystailoredtodetectFMDVvirusescurrentlycirculatinginEast

Africa,withaparticularfocusonTanzania,KenyaandUganda.

2. Materialsandmethods 2.1. Primersandprobedesign

The design of primersand probes wasbased on nucleotide

sequencesencodingVP1originatingfromEastAfricaobtainedfrom

GenBank(http://www.ncbi.nlm.nih.gov/).

TheVP1codingregionsequenceswerealignedusingBioEdit (Hall,1999)andanalysedcollectivelyandseparatelyforserotypes A,O,SAT1andSAT2.Followingtheanalyses,uniqueregionsofthe VP1 codingsequencewereidentifiedastargetsforprimersand probesforeach serotype,in accordancewithTaqMan specifica-tions.However,toachievesatisfactoryresultsinthetypeAassay, amodified NK72primerofbroadspecificitythat annealstothe nucleotidesequence atthejunctionof2Aand2Bcoding region (Knowlesetal.,in press),wasusedasthereverseprimer.Each oligonucleotide(oligo)setwasevaluatedinsilicotoensure diagnos-ticspecificitybetweenthedifferentlineageswithintheserotypes withoutacompromiseoncross-reactivitybetweenserotypes.

AlloligonucleotidesweresynthesisedbySigma-Aldrich(USA) andallprobeswerelabelledwithBHQ-1(BlackHoleQuencher-1) andFAMattheir3and5termini,respectively.

At least two candidate oligo sets (primers and probe) were designedforeachoftheassaysandevaluatedinmultiplepossible combinationsfordetectionofthehomologousserotype.Thebest performingoligowasselectedforeach oftherespectiveassays. Table1liststhesequencesofthebestperformingoligosets.

2.2. Virusisolates

A panelof FMDV clinicalsamples(n=61) and virusisolates (n=10)wasselectedfromthesamplerepositoryheldattheWorld ReferenceLaboratoryforFMD(WRLFMD)(ThePirbrightInstitute, UK)and contemporary diagnosticsubmissions from EastAfrica (Fig.1).TheseincludedFMDVsamplespreviouslycharacterisedas serotypeA,topotypeAFRICA,lineageG-I;serotypeO,topotype EA-2andEA-4;serotypeSAT1,topotypeI(NWZ)andserotypeSAT2, topotypeIV.Viruswasisolatedfromthesesamplesandtestedwith antigenELISA(Ag-ELISA)toestablishtheFMDVserotype(Ferris andDawson,1988).SamplesthatoriginatedfromEastAfrica,from whichFMDVRNAwasdetectedbyrRT-PCR(Callahanetal.,2002), butneithervirusisolationnorAg-ELISAwerepositive(TAN/9/2013,

TAN/16/2013,TAN/18/2013,TAN/23/2013andTAN/28/2013)were

88 100 100 100 100 78 78 84 100 99 95 100 100 100 76 93 94 100 100 100 100 91 10071 0.01 100 84 87 0.01 0.02 0.005

Serotype O

Serotype A

Serotype SAT1

Serotype SAT2

Sample name origin 3D assay type O type A type SAT1 type SAT2 KEN/15/2011 clinical 31.34 30.95 No Ct No Ct No Ct

KEN/16/2011 clinical 25.02 27.61 No Ct No Ct No Ct

KEN/137/2010* clinical 23.83 37.90 23.18 No Ct No Ct

KEN/151/2010 clinical 14.46 23.51 No Ct No Ct No Ct

TAN/39/2012 clinical 13.50 16.51 No Ct No Ct No Ct

KEN/145/2010 clinical 15.56 17.94 No Ct No Ct No Ct

KEN/146/2010 clinical 12.37 13.86 No Ct No Ct No Ct

TAN/16/2008 clinical 18.47 21.08 No Ct No Ct No Ct

KEN/10/2009 clinical 20.41 34.97 No Ct No Ct No Ct

KEN/154/2010 clinical 19.90 21.14 No Ct No Ct No Ct

TAN/5/2009 clinical 16.23 15.75 No Ct No Ct No Ct

KEN/11/2011 clinical 25.18 27.45 No Ct No Ct No Ct KEN/1/2011 clinical 15.39 16.34 No Ct No Ct No Ct KEN/148/2010 clinical 15.46 16.88 No Ct No Ct No Ct KEN/152/2010 clinical 13.70 14.45 37.99 No Ct No Ct KEN/100/2010 T/C 21.60 22.35 No Ct No Ct No Ct ETH/04/2013 T/C 15.64 20.83 No Ct No Ct No Ct ETH/8/2013 T/C 27.45 28.65 No Ct No Ct No Ct ETH/13/2013 T/C 25.45 24.15 No Ct No Ct No Ct ETH/14/2013 T/C 25.25 23.95 42.40 No Ct No Ct

TAN/7/2013 clinical 14.56 No Ct 17.51 No Ct No Ct

TAN/5/2013 clinical 10.48 No Ct 13.88 No Ct No Ct

TAN/6/2013 clinical 11.60 No Ct 11.87 No Ct No Ct

TAN/14/2013 clinical 14.90 No Ct 17.43 No Ct No Ct

TAN/15/2013 clinical 11.07 No Ct 13.43 No Ct No Ct

TAN/1/2013 T/C 13.24 No Ct 14.78 No Ct No Ct

TAN/61/2012 T/C 18.25 No Ct 18.87 No Ct No Ct

TAN/71/2012 T/C 15.74 No Ct 15.99 No Ct No Ct

TAN/42/2009 clinical 18.45 No Ct 18.86 No Ct No Ct

TAN/45/2009 clinical 16.03 No Ct 15.97 No Ct No Ct

KEN/28/2008 clinical 18.37 No Ct 18.67 No Ct No Ct

TAN/26/2013 clinical 13.99 No Ct 18.10 No Ct No Ct

KEN/22/2009 clinical 23.23 No Ct 21.80 No Ct No Ct

KEN/5/2012 T/C 16.05 No Ct 16.94 No Ct No Ct

KEN/6/2012 T/C 14.15 No Ct 14.42 No Ct No Ct

TAN/20/2013 clinical 12.98 No Ct 34.70 16.85 No Ct

TAN/21/2013 clinical 13.43 No Ct 38.23 17.85 No Ct

TAN/22/2013 clinical 15.43 No Ct No Ct 18.99 No Ct

TAN/25/2013 clinical 12.50 No Ct No Ct 16.23 No Ct

TAN/27/2013 clinical 7.49 No Ct No Ct 20.05 No Ct

TAN/24/2013 clinical 11.88 No Ct No Ct 15.88 No Ct

TAN/49/2012 clinical 12.44 No Ct No Ct 13.77 36.33 TAN/50/2012 clinical 12.63 No Ct No Ct 13.53 No Ct

KEN/16/2009 clinical 15.12 28.67 No Ct 16.16 No Ct

KEN/26/2008 clinical 13.84 No Ct No Ct 15.39 No Ct

KEN/9/2009 clinical 15.95 No Ct No Ct 17.47 No Ct

KEN/139/2010 clinical 21.07 No Ct No Ct 17.73 No Ct

KEN/71/2010 clinical 18.71 No Ct No Ct 21.17 No Ct

KEN/72/2010 clinical 14.17 No Ct No Ct 16.16 No Ct

KEN/140/2010 clinical 19.97 No Ct No Ct 21.66 No Ct

KEN/1/2010 clinical 22.83 No Ct No Ct 23.76 No Ct

KEN/121/2009 clinical 21.76 36.88 No Ct 23.39 No Ct

KEN/12/2009 clinical 16.44 No Ct No Ct 18.31 No Ct

KEN/123/2009 clinical 31.65 No Ct No Ct 19.83 No Ct

TAN/7/2011 clinical 19.85 No Ct No Ct No Ct 22.07 KEN/12/2011 clinical 15.32 No Ct No Ct No Ct 17.89 TAN/6/2011 clinical 20.14 No Ct No Ct No Ct 21.90 TAN/3/2011 clinical 18.92 No Ct No Ct No Ct 19.11 KEN/21/2011 clinical 13.27 35.59 No Ct No Ct 15.68 TAN/64/2012 clinical 17.56 No Ct No Ct No Ct 20.07 TAN/14/2012 clinical 14.06 No Ct No Ct No Ct 15.17 TAN/16/2012 clinical 17.96 No Ct No Ct No Ct 18.32 TAN/19/2012 clinical 15.95 No Ct No Ct No Ct 15.81 KEN/2/2007 clinical 21.04 No Ct No Ct No Ct 25.40 KEN/11/2007 clinical 17.70 No Ct No Ct No Ct 19.91 KEN/2/2008 clinical 15.07 No Ct No Ct No Ct 16.23

Fig.1.DetailsoftheFMDVpositivesamplesbyvirusisolationusedinthisstudy.PhylogeneticcomparisonsarebasedontheanalysisofVP1codingsequence.T/Cstandsfor

viruspropagatedintissueculture.

*SampleKEN/137/2010sequencedatawasobtainedfromviruspropagatedintissueculture(BTy2)(serotypeO)whilerRT-PCRresults(serotypeA)wereobtaineddirectly

fromclinicalsamples.

Additionally27fieldsamplescollectedinTanzaniaweretested intheTanzaniaVeterinaryLaboratoryAgency(TVLA)toconfirm thediagnosticperformanceofthedevelopedassaysinanAfrican setting.

2.3. RNAsamplepreparation

Nucleicacidwasextractedeitherusinganautomated extrac-tionplatform,MagNAPure(Roche)aspreviouslydescribed(Reid etal.,2003)ormanuallyusingaQIAampViralRNAMiniKit(Qiagen) accordingtothemanufacturer’sinstructions.Eachoftheisolates

washandledwithcaretoavoidanycross-contamination.Theviral RNAwasstoredat₋70◦Cuntilusedintheexperiments.

2.4. RT-PCR

Therespectiveprimersandprobesetsweredesignedto per-formunderthesameprotocolandamplificationconditionsasthose describedearlierforthepan-FMDVassays(Shawetal.,2007)with theviewthatpan-specificandtailoredassayscouldbeperformedin parallelonthesamereactionplate.FMDVsampleswerealsotested usingapan-specificassaydetectingthe3D-codingregion(Callahan etal.,2002)tovalidatetheresults.Allreactionswereperformedin

duplicate,setupmanuallyandamplifiedinaStratageneMx3005P thermalcycler.

Theamplification efficiencyof each of therespectivetyping assayswasestimatedonthebasisofa10-folddilutionofa represen-tativesampleforeachserotype(serotypeO:ETH/4/2013;serotype A:TAN/45/2009;serotypeSAT1:KEN/136/2010;serotypeSAT2: KEN/21/2011).Theefficiencywascalculatedbythefollowing for-mula:E%=10−1/slope×100.

2.5. Sequencing

TheVP1sequencesweregeneratedusingthestandardmethod andprimers(Knowlesetal.,inpress)by3730DNAAnalyzer(ABI), accordingtothemanufacturer’sinstructionsandassembledusing SeqManPro13Software(DNAStarInc.,USA).Sequencedatawere alignedusingCLUSTALWv1.83software(Thompsonetal.,1994) andphylogenetictreesconstructedbyNeighbor-Joiningmethod. Theevolutionarydistances werecomputedusingtheKimura 2-parametermethodinMEGAversion6.06software(1000bootstrap replicates)(Tamuraetal.,2013).

3. Results

3.1. Diagnosticsensitivityandspecificityoftype-specificrRT-PCR assays

A panel of 68 RNA samples representing FMD viruses of

serotypesO,A, SAT1and SAT2circulating inKenya, Tanzania andEthiopiacollectedbetween2007and2013wastestedusing thetype-specificrRT-PCRassays.ResultsforduplicateRNA sam-ples weredirectly compared tothose ofthe 3D rRT-PCRassay (Callahanetal.,2002).AllFMDVRNAsamplesweredetectedby theassaytargetingthe3D-codingregionandtheserotypeofthese positivesampleswasdeterminedusingthecorresponding type-specificrRT-PCRassay,identifyingvirustypewithcomparableCT valuesinmostcases(Fig.1).MeanCTvaluesforthe3Dassayand serotype-specificassays(O,A,SAT1andSAT2)werecalculatedfor theisolatestestedshowingthat,onaverage,isolatesweredetected earlierwiththepan-specific3Dassaywithareductionof2.9,1.1, 1.7and 1·7 CT values, respectively.Thevirustype identifiedby thetype-specificreal-timeRT-PCRassaywasconfirmedfromVP1 nucleotidesequencedata(datanotshown).

InnineRNAsamples,apositivesignalwasproducedwithtwo differentserotype-specificassays.Insevenofthese,theserotype of the virus was correctly identified by the assay which pro-ducedaCTvaluesimilartotheCTvalueofthe3Dtargetedassay, whilethesecondassayproducedaCTvalue>12.44higherinthe panelofsamplestested.However,insampleKEN/137/2010,which

was characterised by VP1 sequencing and Ag-ELISA (WRLFMD

report: http://www.wrlfmd.org/fmd

genotyping/2011/WRLFMD-2011-00008%20O%20Kenya%202010-2011.pdf)asserotypeO,RNA ofFMDVserotypesAandOwasdetected.InthetypeA-specific rRT-PCRassaystheCTvalue(23.18)wascomparabletothe3D pan-specificassay(23.83),whiletheserotypeOsignalwasweaker(CT valueof37.90).TheAtype-specificrRT-PCRassayampliconwas sequenced,confirmingthepresenceoftheserotypeARNAinthis

sample.The amplicon sequencewascompared tootherknown

contemporaryserotypeAsequencesfromthegeographical area showingthatthisvirusinKEN/137/2010hasanovelsequencenot previouslyhandledatthePirbrightLaboratory(datanotshown).

Inadditiontosamplesthathadbeenpreviouslycharacterised byAg-ELISAand/orVP1sequencing,thevirusserotypewas

iden-tifiedinfivesamplesinwhichtheFMDVgenomewasdetected

withthe pan-specific assay targeting the 3D regionbut which werenegativebyvirusisolationmethods.FMDVtypeA-specific

assayproduced a positivesignalin three ofthesamplestested (TAN/9/2013,TAN/16/2013andTAN/18/2013)whileSAT1virus templatewasdetectedwiththeSAT1-specificassayintwoother samples(TAN/23/2013andTAN/28/2013).TheCTvaluesineach casewerecomparabletotheCTvalueofthe3Dtargetingassay.

3.2. Analyticalsensitivity

The comparative analytical sensitivity of the type-specific assayswasevaluatedbasedonten-folddilutionseriesof homol-ogousviralRNAperformedinduplicatedindirectcomparisonto thepan-specific assay.DilutionsofhomologousviralRNA were detectedatsimilarCT valuesforeachofthetype-specificassays incomparisontothepan-specificassay.However,theAandSAT 2-specificassaysdetectedsevenserialdilutionoftheRNAtemplate, whiletypeO-andSAT1-specificassaysyieldedpositiveresultswith sixdilutions,onefewerthanthepan-specificassay(Fig.2).

The efficiency of the respective type-specific assays varied

between 92.9 and 101.3% and was comparable to that of the

assaydetectingthe3Dcodingregion(efficiencybetween89.2and 100.2%)(Fig.2),confirmingsimilarrobustnessofthetype-specific assaytothatofthe3Dspecificassay.

3.3. EvaluationofassaysinEastAfrica

Theperformanceofthetype-specificassayswasfurther eval-uatedalongsidethepan-specific3Dassay(Callahanetal.,2002) intheTanzaniaVeterinaryLaboratoryAgencywhereanadditional panelof27clinicalsamplescollectedinTanzaniabetween2008 and2013,twoFMDVpositivecontrolsandanegativecontrolwere tested.Allsamples,apartfromthenegativecontrol,weredetected withthepan-specificassaytargetingthe3DregionwithCTvalues rangingfrom10.19to37.17.Thetype-specificCTvalues ranged from9.72to31.89withallbutthenegativecontrolandtwo sam-ples(#29and#179)whichproducedaweak3Dsignal(CT37.17and 34.32,respectively)(Table2).Thetype-specificsignalgaveaclear indicationoftheserotypeforthemajorityofsamples,however,a positivesignalwasrecordedinsample#374withtwotype-specific assays–AandSAT1,possiblyindicatingamixinfection.Ahigh CTvalue(31.90–39.02)wasrecordedwitha numberofsamples indicatingdetectionofalowlevelofviralRNA.

4. Discussion

Theavailabilityofrapid,reliableandaccuratediagnostic meth-odsisimportantforeffectivecontrolandsurveillancestrategiesfor FMD.MethodsforthedetectionofFMDVsuchasvirusisolation, conventionalandrRT-PCRarewidelyusedwhiletherepertoireof serotypingmethods(e.g.ELISA)ismorelimited(OIE,2012).The serotypingantigenELISAoftenreliesoninitialvirusisolationand propagationandrequires accesstostandard biologicalreagents (FerrisandDawson,1988).Thesefactorsmayextendtheperiodfor implementationofcontrolstrategiesespeciallyinendemic coun-triesandmakesurveillancestrategieslabour-andcost-intensive.

TheFMDVgenomeregionencodingthecapsidproteins, par-ticularlytheVP1 codingsequence,variesaccordingtoserotype. However,the highlevel ofintra-serotype nucleotidevariability makesitdifficulttodesignbroadlyreactingmolecularteststhat candetectallrepresentativesofasingleFMDVserotypethatare circulating in differentgeographical regions (Reid et al., 2014). Instead,anumberofmolecularassaystailoredtoaspecificlineage foundinmorerestrictedgeographicallocationshavebeen success-fullydeveloped (Ahmedet al.,2012;Jamal andBelsham,2015; Knowlesetal.,2014;Reidetal.,2014).Thisstudyalsoemployed this approachand describesthedesign andvalidation ofFMDV type-specificassaystailoredfortheFMDvirusesfromEastAfrica.

FMDV-SAT2

y

= 3.

3825x

+ 12.6

24

R² = 0.

9946

Eff=97.6%

3D

y = 3.3611x

+ 12.4

73

R² = 0.

9991

Eff=98.4%

0

5

10

15

20

25

30

35

40

0

2

4

6

8

FMDV-SA

T2

3D

FMDV-O

y = 3.3154x + 12.023

R² = 0.

9989

Eff=100.3%

3D

y = 3.3168x + 10.579

R² = 0.9999

Eff=100.2%

0

5

10

15

20

25

30

35

40

0

2

4

6

8

FMDV-O

3D

FMDV-SAT1

y = 3.2917x + 11.721

R² = 0.

9991

Eff=101.3%

3D

y = 3.3225x + 11.094

R² = 0.9999

Eff=100%

0

5

10

15

20

25

30

35

40

0

2

4

6

8

FMDV-SA

T1

3D

FMDV-A

y = 3.5054x + 14.041

R² = 0.

9992

Eff=92.9%

3D

y

= 3.

6118x

+ 14.5

09

R² = 0.

9985

Eff=89.2%

0

5

10

15

20

25

30

35

40

45

0

2

4

6

8

FMDV-A

3D

Ct va

lue

Ct va

lue

Number of se

ria

l diluons

Number of

se

ria

l diluons

Fig.2. ComparisonofdetectionofserialdilutedviralRNAinpan-specific(3D-specific)andindividualtype-specificassays.

FMDVcirculationoftenoccurswithinregionalreservoirswhere strainsspecifictotheregionevolve(Patonetal.,2009).Thus,mainly fourserotypesofFMDV(O,A,SAT1andSAT2)havebeendescribed tocirculateinEastAfrica.Withineachserotype,arestricted num-beroftopotypes/lineageshavebeenidentified,namely:topotype EA-2andEA-4inFMDVO,topotypeAFRICA,lineageG-IinFMDV A,topotypeI(NWZ)inFMDVSAT1andtopotypeIVinFMDVSAT2 (Kasambulaetal.,2012;Kasangaetal.,2015;Namatovuetal.,2015; Wekesaetal.,2014,2015a,b).Inthisstudy,asetoffour serotype-specificTaqMantechnologybasedassayswasdesigned,tailored todetectthevariousFMDVserotypescurrentlycirculatinginEast Africa.Thiswasachievedbytargetingtheserotype-specificVP1 codingsequence inmostcases.However,thereverseprimer in theFMDV-Aspecificassaywasdesigned toannealtothemore conserved2A/2Bcodingregionofthegenome,duetohigh het-erogeneitywithintheVP1codingregionoftheanalysedstrains.

Asintended, the assays were shown to specificallyidentify thehomologousvirusesinallassays withtheCT valuessimilar tothoseofthepan-specific testdetectingthe3Dcodingregion, identifyingtheserotypeofthesample.Inadditiontostudies under-takeninEurope(atThePirbrightInstitute),validationexperiments werealsoconductedattheTanzaniaVeterinaryLaboratoryAgency showingthatrapidandreliableidentificationofthevirustypecan beachievedinanendemicsetting.Insomesamples,however,a positivesignalwasrecordedinmore thanoneofthe serotype-specificRT-PCRassays.Thiscouldbeindicativeofamixedinfection, alaboratorycontaminationorpoorspecificityof thedeveloped tests.However,insilicoanalysesoftherespectiveoligo-template

annealingregionsindicatedhighspecificityoftheseassays.In addi-tion,theassay’sspecificitywasinvestigatedbysequencingofthe ampliconsandthepresenceofVP1templatesasidentifiedbythe developedrRT-PCRassayswasconfirmedineachcase.

Theanalysisoftheresultsobtainedfromtestingdifferent pas-sagesoftheKEN/137/2010samplegiveinsightintotheveracity of the assays developed. FMDV from this clinical sample was isolated and passagedtwice in BTy cells and theserotype was identifiedasFMDV-OinAg-ELISAbytheWRLFMDlaboratory(The PirbrightInstitute).TheVP1 sequenceofthesameserotypewas alsoobtainedonthevirusisolateconfirmingtheresultsof sero-logical typing.However,when RNAextracted directlyfromthe clinicalmaterialwastested withthedeveloped typingrRT-PCR assays,thepresenceoftwoserotypeswasidentified.The FMDV-Ospecificassayproducedalatesignal(highCTvalue)(indicating alowleveloftheserotypeOgeneticmaterial)andtheFMDV-A specificassaygeneratedalowerCTvaluecomparabletothe pan-specific assay,indicating that thepredominant serotypein the initialsamplewasFMDV-A.TherRT-PCRampliconofthe

FMDV-A specificassay wassubsequently sequencedand compared to

FMDV-Asequencesavailable intheWRLFMDdatabaseshowing

itasanovelsequence.Theseresultsleadtoaconclusionthatthe FMDV-Ocomponentoftheviruspresentintheclinicalsamplesmay havebecomedominantduringsubsequentcellpassages outcom-petingthepredominantAviruswhichwaspresentintheclinical sample.Alternatively,laboratorycontaminationcouldhaveplayed arole.

Table2

ValidationoftheserotypespecificassaysinlaboratorysettingsinTanzania.

PC—positivecontrol;NC—negativecontrol.

ThesedataalsoshowthattherRT-PCRassayscanbeusedon

specimenswherevirusisolationisnotavailableornotpossible.This

featuregivestheabilitytoobtainepidemiologicalinformationeven

fromsamplesnotsuitableforserotypingbyAg-ELISA.Theability

toestablishthetypeofthevirusbyselectivegenomeamplification

makesthissetoftype-specificassayssuitableforfurther

applica-tionssuchasadaptationformobilediagnosticplatforms(Howson

etal.,2015;Madietal.,2012)and/orvalidationforusein combina-tionwithnucleicacidrecoveredfromlateral-flowdevices(Fowler etal.,2014).Furthermore,thetype-specificassaysoperatewiththe samethermalprofileasthepan-diagnostictestsmakingitpossible tomultiplexdiagnosticandserotypingtestsfurtherenhancingthe potentialfortheseassays.

ThesefindingshighlightthevalidityoftheEastAfrican serotyp-ingRT-PCRassayinawiderangeofapplicationsincludingtesting for mixed infectionor lab contaminationof viral RNAsamples designedforfurtherdownstreamapplicationssuchasproduction ofdiagnosticsera.FMDVisarapidlyevolvingvirusandthe devel-opedassays targetthemostvariableregionofitsgenome.It is therefore possiblethat, with time, new lineageswill evolve or strainsarediscoveredwithsignificantchangeswithintheoligo annealingsites,prohibitingnormalfunctionoftheseassays.Itis thereforerecommendedthatsuchassaysarenotusedaloneasa

frontlinediagnostictestbutareusedincombinationwith pan-specificdiagnosticapproaches.

Acknowledgments

Thisworkwassupportedbyaresearchgrantfromthe

Euro-pean Commission for the Control of Foot-and-Mouth Disease

(EuFMD) fund for applied research (FAR) and Wellcome Trust

(grantsWT087546MAandWT104017MA).Theauthorswouldlike

toacknowledgethemembersoftheWorldReferenceLaboratory forFMD(WRLFMD,ThePirbrightInstitute)andTanzania Veteri-nary Laboratory Agency (TVLA) for supporting characterisation data.ThefieldteamsinTanzania,KenyaandEthiopiaarethanked forcollectingthesamples,someofwhichwereprovidedviathe CombatingInfectiousDiseasesofLivestockforInternational Devel-opment(CIDLID)initiativefundedbyBiotechnologyandBiological SciencesResearchCouncil,theDepartmentforInternational Devel-opmentandtheScottishGovernment(BB/H009302/1).Thework oftheWRLFMDissupportedwithfundingprovidedtotheEuFMD fromtheEuropeanUnion.Theviewsexpressedhereincaninno waybetakentoreflecttheofficialopinionoftheEuropeanUnion. ThePirbrightInstitutealsoreceivesgrant-aidedsupportfromthe

Biotechnology and Biological Sciences Research Council of the UnitedKingdom(BBS/E/I/00001713).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.jviromet.2016. 08.002.

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