Duplex nested-PCR for detection of small ruminant lentiviruses

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

Duplex

nested-PCR

for

detection

of

small

ruminant

lentiviruses

Rebeca

C. Marinho

a,∗

_,

_Gabrielle

_R.

_Martins

a

_,

_Kelma

_C.

_Souza

b

_,

_Ana

_Lídia

_M.

_Sousa

a

_,

Sabrina

Tainah

C. Silva

a

_,

_Juliana

_A.

_Nobre

a

_,

_Maria

_F.S.

_Teixeira

a

a_Universidade_Estadual_do_Ceará,_Programa_de_{Pós-Graduac¸ão}_em_Ciências_{Veterinárias,}_Laboratório_de_Virologia,_Fortaleza,_CE,_Brazil b_Embrapa_Ovinos_e_Caprinos,_Sobral,_CE,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9May2017 Accepted27April2018 Availableonline13August2018 AssociateEditor:MauricioNogueira

Keywords: Standardization Multiplex Viruses CAEV MVV

a

b

s

t

r

a

c

t

Smallruminantlentiviruses(SRLV)havehighgeneticvariabilitywhichresultsindifferent viralstrainsaroundtheworld.Thiscreateachallengetodesignsensibleprimersfor molecu-lardiagnosisindifferentregions.Thisworkproposesaprotocolofduplexnested-PCRforthe precisediagnosisofSRLV.Thetechniquewasdesignedandtestedwiththecontrolstrains CAEVCoandMVV1514.Then,fieldstrainsweresubmittedtothesameprotocolofduplex nested-PCR.BloodsamplesofsheepandgoatsweretestedwithAGIDandnestedPCRwith specificprimersforpol,gagandLTR.TheAGIDresultsshowedlowdetectioncapacityof positiveanimals,whilethenestedPCRdemonstratedagreatercapacityofvirusdetection. ResultsdemonstratedthatLTR-PCRwasmoreefficientindetectingpositivesheepsamples, whereasgag-PCRallowedagooddetectionofsamplesofpositivegoatsandpositivesheep. Inaddition,pol-PCRwasmoreefficientwithgoatsamplesthanforsheep.Duplexnested PCRperformedwithstandardvirussamplesandfieldstrainsdemonstratedthatthe tech-niqueismoreefficientforthedetectionofmultiplepro-viralDNAsequences.Thisstudy demonstratedasuccessfulduplexnestedPCRassayallowingamoreaccuratediagnosisof SRLV.

Introduction

Smallruminantlentiviruses(SRLV)arepathogensthataffect sheepandgoatscausingpersistentandprogressiveinfections. These agents are represented by two phylogenetic groups

∗ _{Corresponding}_author.

E-mail:beca.marinho@hotmail.com(R.C.Marinho).

characterizedbytheMaedi-Visnavirus(MVV)andthecaprine arthritis encephalitisvirus (CAEV).1–3 _Phylogenetic _analysis dividesthesevirusesintoﬁvemajorgroupsclassiﬁedasA-E. TheMVVandCAEVprototypesofgroupsAandB,respectively, arewidelydistributedthroughouttheworld,whileC-Egroups aregeographicallyrestricted.4,5

Theselentivirusesarecloselyrelatedandwereconsidered species-speciﬁcinfectingsheepandgoats,respectively, caus-ingprogressivelungdisease,encephalomyelitis,mastitisand arthritis.6 _However, _molecular _and _{epidemiological} _studies https://doi.org/10.1016/j.bjm.2018.04.013

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conductedinthelastdecadesindicatethattheseagentsare capableofinfectingbothgoatsandsheep,7_{transgressing}_the barrierbetweenspeciesconsistentlyandeasily.4,8,9

Thesevirusespresenthighgeneticvariabilityrelatedtothe viralreplicationprocess,resultingintheformationofseveral viralstrainsindifferentgeographicregions.2_Due_to_the het-erogeneityoftheSRLVgenomeitisdifﬁculttousethesame setofprimersindifferentgeographicregions.10

The serological diagnosis of infected animals by the Agarose Gel Immunodiffusion (AGID) technique is widely used and accepted due to its low cost and practicality. 11 Inaddition,thistechniqueisrecommendedbyOIE.12 How-ever, some animals may present low antibody titers, late seroconversionorintermittentreactionsofseropositivityand seronegativity.13,14

Numerous studies have successfully demonstrated the useofthePCRtechniqueinthedetectionofpro-viralDNA fromSRLV15–18_due_to_the_high_sensitivity_and_speciﬁcity_this techniquepresents.19_A_multiplex_PCR_is_a_variant_of_the con-ventionalPCRinwhichtwoormoretargetsequencescanbe ampliﬁedincludingmorethanonepairofprimersinthesame reaction.20

Basedontheseadvancesofmoleculardiagnosis,thisstudy aimedtodevelopaduplexnestedPCRprotocolforaccurate diagnosisofsmallruminantlentiviruses,aswellastoverify theefﬁciencyofthemethodindiagnosingsheepandgoatﬁeld samples.

Material

and

methods

ThisprojectwasapprovedbythelocalEthicsCommitteefor theUseofAnimalsfromtheStateUniversityofCeará,protocol number0784899-15.

Samples

Fortestingduplexnested-PCRtechnique,standardsamplesof theCAEV-Cork21_and_MVV-K151422_strains_were_used_and_this virussampleswereampliﬁedincellcultureandstoredfrozen forlateruse.

These samples were provided byDr. Roberto Soaresde Castro (Federal Rural University of Pernambuco – UFRPE) and originated from the Institut National de la Recherche AgronomiqueLyon-France.

Todemonstratetheefficiencyofthetechniquein detect-ingSRLV,sampleswerecollectedfromanimalshousedintwo farmsofnortheasternmesoregionfrom Parástateand two farmsinthemetropolitanregionofFortaleza,Ceará–Brazil. HerdsofSantaInêsbreedandundefinedbreedwereusedin thisstudy.Similarly,samplesofgoatswereobtainedfrom dif-ferentpropertiesinmetropolitanregionofFortaleza,Ceará– Brazil.HerdsofSaanen,Alpina,Murcianabreedandcrossbred animalswerecollected.Allanimalswereagedfrom1to2years andthefindingsofclinicalsymptomatologyweredescribedin Table2.

Bloodsamplesof20goatsand15sheepwerecollectedby venipunctureofthejugularveinusingvacutainer® tubeswith EDTAtoperformmoleculartestsandtubeswithout anticoag-ulantforagarosegelimmunodiffusion(AGID)assay.

Agarosegelimmunodiffusionassay(AGID)

Before performingduplexnPCRinﬁeldstrains, allsamples were tested by AGID for screening animals and identify-ingpositivesandnegatives.AGIDtestwasperformedusing the national commercial kit developedbyBiovetech®. The above-mentionedAGIDkitisproducedusingsupernatant con-centrateantigenfromMVVand CAEVinfectedcellcultures and with positiveserum harvested fromnaturally infected animal.Thistestisindicatedfordetectionofanti-p28 anti-bodiesfromgoatsinfectedwithCAEVandsheepinfectedwith MVV.

DNAextraction

For DNA extraction from whole blood leukocytes,samples weresubmittedtotheprotocoldescribedbyCaldasetal.23_. Thesampleswerecentrifugedat1500gfor10min.The leuko-cytelayerwascollectedandtransferredto1.5␮Lmicrotubes containing 500␮LTE(10mMTris, 1mM). Thematerialwas centrifugedinmicrofugeat2700gfor5mintoobtainthe pel-let, then the supernatantwas discardedand the operation repeated.ThepelletwasresuspendedinKbuffer(10mMTris HClpH8.0,50mMKCl,2mMMgCl2,0.5%Tween20,100␮g/mL

proteinase K=0.1␮g/␮L).Thesampleswere vortexed, incu-batedat56◦Cfor45minandthenboiledat96◦Cfor10min. Thematerialwasstoredat4◦Cfor24hforabetter homoge-nizationofsampleandthenat−20◦_C_until_duplex_nPCR_was

performed. Primers

ForduplexnPCR,apairofprimerswasusedforeachregion (gag,pol,LTR),whicharelocatedinmoreconservedregions (Table1).

Nested-PCR

Before performing duplex nPCR in field samples, all sam-ples were tested by nested-PCR (nPCR) for screening the animalsandidentifyingpositivesandnegatives.The detec-tion of pro-viralDNA was performed with nPCRfollowing the methodology described by Barloughet al.26 _with mod-ifications. Modificationswere made tothe methodology as proposedbyAndriolietal.27_in_which_KCl,_gelatin_and tetram-ethylammoniumchloridewereremovedfromthePCRmix.In addition,modificationsweremadetotheamplificationcycles withadecrease inthenumberfrom45to35cyclesandan increaseintheannealingtemperaturefrom50◦Cto63◦C.

Thiswas performed toamplifya fragmentofthe CAEV andMVVpro-viralDNAbasedonthegag,polandLTRregions adjustingtheofprimerconcentration.IndividualnPCRwas performedforeachsetofprimers,andallsamplesweretested forthegag,polandLTRprimerspriortoperformingtheduplex nPCR.ThenPCRwasperformedwithgagprimerswere consid-eredasstandardtodiscriminatepositiveornegativesamples forthemoleculartests.Thischoicewasmadeonthebasisof ﬁndingsfromtheliteratureconﬁrmingthatthegagregionis themostconservedwithintheSRLVgenome.37,41–43

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Table1–Primersusedforduplexnested-PCRstandardization.

Primer Sequence Round Ampliconsizeexpected

insecondround

Reference

GEX5 5-CAAGCAGCAGGAGGGAGAAGCTG-3 First

187bp

Saltarellietal.24

GEX3 5-TCCTACCCCCATAATTTGATCCAC-3 First Saltarellietal.24

GIN5 5_{-GTTCCAGCAACTGCAAACAGTAGCAATG-3} _Second _Saltarelli_et_al.24

GIN3 5_{-ACCTTTCTGCTTCTTCATTTAATTTCCC-3} _Second _Saltarelli_et_al.24

LTREXEFW 5-ACTGTCAGGRCAGAGAACARATGCC-3 First

203bp

Ryanetal.25

LTREXERV 5-CTCTCTTACCTTACTTCAGG-3 First Ryanetal.25

LTRINIFW 5-AAGTCATGTAKCAGCTGATGCTT-3 Second Ryanetal.25

LTRINIRV 5-TTGCACGGAATTAGTAACG-3 Second Ryanetal.25

POLEXP28 5-CATGAAGAGGGGACAAATCAGCA-3 First

1175bp

Shahetal.4

POLEXP33 5CTTCCCAVAGTACCTGDGTTGGTC-3 First Shahetal.4

POLINP29 5-GGTGCCTGGACATAAAGGGATTC-3 Second Shahetal.4

POLINP35 5-GCCACTCTCCTGRATGTCCTCT-3 Second Shahetal.4

The reactions was performed in a thermal cycler (Eppendorf®)inaﬁnalvolumeof50␮Landthesolutionmix consistedof10mM TrisHCl buffer (pH8.3);1.5mM MgCl2;

100␮Mofeach dNTP;2U/␮LtaqDNApolymerase recombi-nant(Invitrogen®);3␮LoftestsampleandDNAse-freewater tocompleteaﬁnalvolume.Differentconcentrationsofthegag, polandLTRprimerspreparedforeachreactionweretested withtheconcentrationsof20pM,10pM,7.5pMand5pM.

Theamplificationconditionswereasfollows:initial dena-turingat94◦Cforfivemin,followedby35cycles:denaturation at94◦Cforonemin,annealing63◦Cforonemin,extension at72◦Cfor45s,followedbyanadditionalextensionphaseat 72◦Cforsevenmin.Analiquotof1.0␮LofthisPCRproductwas transferreddirectlytoasecondreaction.Thefirstandsecond roundsofthePCRwereperformedunderthesameprevious conditions.Allreactionswereperformedunderthesametime andtemperatureconditions.

PCRampliﬁedfragmentswereanalyzedbyhorizontalcube electrophoresis and the bands were visualized under the ultravioletlighttransilluminator(VilberLourmat– Transillu-minatorUV/whitelight)andphotodocumented(Logic200PRO Serie).Thesizesoftheampliﬁedfragmentswerecomparedto a100bpDNAladder(Promega)andtothepositivecontrol.

Duplexnested-PCRusingstandardSRLVsamples

AftertheserologicaltestandtheindividualnPCRperformed witheach set ofprimers, the duplex nPCR wasperformed withthesameprimerconcentrationsoftheprevioustest.The followingconcentrationswereused20pM,10pM,7.5pMand 5pMforgag,polandLTRprimers.

Theﬁrststepofstandardizationinvolvedperformingthe duplexnPCRusingtheprimersforthegagandpolgenesand thesecondstepinvolvedatestusingtheprimersfortheLTR andpolregion.

ThecombinationgagandLTRwasnotevaluated.Primers designedforthiscombination didnotgeneratesatisfactory resultsinthetestsperformed,sincetheampliconsgenerated havevery closesizes,making it impossibletodifferentiate them.

The reactions were performed in a thermal cycler (Eppendorf®)inaﬁnalvolumeof50␮Landthesolutionmix consistedof10mMTrisHClbuffer(pH8.3);2U/␮LtaqDNA

polymerase;3␮LoftestsampleandDNAse-freewaterto com-plete aﬁnalvolume. Theprimerconcentrationswere used accordingtotheresultsobtainedfortheconventionalnPCR previouslyperformed.ToadjusttheduplexnPCRtechnique, differentconcentrationsofdNTPsandmagnesiumwerealso tested.Ampliﬁcationconditionsfollowedthesame protocol aspreviouslydescribedforthenPCR.

PCRamplifiedfragmentswereanalyzedbyhorizontalcube electrophoresis.Differentconcentrationsofagarosegelwere tested to visualize PCR products, in which the following were tested1%,1.3%,1.5%and 2%.Theconcentrationthat allowedthebestvisualizationofthedifferentbandsformed intheduplexnPCRwasverified.Thebandswerevisualizedin ultravioletlighttransilluminator(VilberLourmat– Transillu-minatorUV/whitelight)andphotodocumented(Logic200PRO Serie).Thesizesoftheamplifiedfragmentswerecomparedto 100bpDNAladder(Promega)andtothepositivecontrol.

Evaluationoftechniqueperformanceinﬁeldsamples Afterobtainingthebestconcentrationofreagentsforduplex nPCRandevaluatingeffectivenessindetectingthestandard virussamples,thedevelopedtechniquewasusedtotestthe samplesobtainedfromanimalsintheﬁeld.

Fifteensheepandtwentygoatsamplesweretestedwith duplexnPCRusingthepolandgagprimersetsandthepoland LTRprimersetsasdescribed fortheexperimentperformed withthestandardvirussamples.

DNAsequencingandsequenceanalysis

PositivesamplesobtainedfromGoats6,7and8andSheep4, 5and6weresentforsequencing.

Thepartialdirectsequencingofthegaggenerelativeto theampliﬁcationproductsobtainedinthesecondroundof nested-PCRwasperformed.Wereusedtheprimersinternals GIN5andGIN3.

Thesampleswerepuriﬁedon1%agarosegelusing com-mercial kit (BigDye Terminator v3.1 CycleSequencing) and sequencedonABI3500platform(GeneticAnalyzer).Thegene sequenceswerecomparedwithMEGA7analysissoftware,and BasicLocalAlignmentSearchTool–Blast.Allpositionswith

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ambiguouscodesoralignmentgapswereexcludedfromthe analyses.

TheSRLVstrainswerecomparedtoK1514-MVV,28_CAEV,24 7-29Ont,29 _{Br/UFRGS-2/V27,}30 _21G-MG/Br,31 _39QPI/Br,31 19F-MG/Br31 _and _BR/CNPC-G432 _with _GenBank _accession no_. _M60609, _M33677, _KC155690.1, _AJ305039.1, _KF861573.1,

KF861553.1,EU300979.1andKF861559.1respectively.

ThroughtheanalysisbyBLASTN2.8.0toolitwaspossible todeterminethesimilarityofthestrainsfoundwiththeother strainsdepositedintheGenBank.33,34

Results

AGIDandnestedPCR

AllﬁeldsamplesweretestedwithAGIDandnested-PCR tech-niques,inwhichtheprimersforthegaggenewereusedand consideredthediagnosticcontrolstandardsforcomparison withthedevelopedduplexnested-PCRtechnique.Inatotal of20goatsevaluatedbyAGID,onlytwoanimals(10%)were positive,whilenineanimalshad inconclusive.Inconclusive resultsconsistingin inadequateformation ofthe expected precipitationline.

SamesamplesweretestedwithnPCRwithgagprimers,in which17goats(85%)testedpositive.Onlythreeanimalsthat werenegativeinAGIDtestcontinuedtodemonstratenegative resultswithnPCRperformedwithgagprimers.

Inatotalof15sheepevaluatedbyAGID,threepresented positiveresultsand12werenegative.InthenPCRresultsusing

gag primers,9(60%)sheepwere positiveand 6sheepwith negativeresultsinAGIDwerealsonegativeinnPCR.

As for the technique performed with the pol primers, results demonstrated that primers were also efficient for detectingpro-viralDNAfromgoatsamples,butnotasefficient asprimersforgagregion.Amongthe17animalspositivein nPCRperformedwithgag,15animals(75%)werealsopositive forthepolprimers,showinganagreementof88%.However, thenPCRresultsofsheepsamplesusingpolprimers demon-stratedthatthesewerenotasefficientindetectingthepositive animals.Fromthetotaloffifteentestedanimals,only3were positiveforthetechniqueusingthepolprimers(Table2).

ThenPCRperformedwiththeLTRprimersdemonstrated that these primers were not efﬁcient for the detection of pro-viralDNA ingoat samplesofthis study,sincenoneof thetestedanimalspresentedapositiveresult.Amongthe15 sheepsamplestestedforthissetofprimers,10werepositive. Onesamplethathadbeenconsiderednegativeforthepoland

gagprimersshowedpositivityfortheLTRprimers.

ThebestprimerconcentrationsfornPCRinthisstudywere 10pMforgagprimersand20pMforLTR.Forpolprimersthe bestconcentrationwas7.5pMperreaction.Forthistest,there wasnoneedtomodifytheconcentrationofanyotherreagent oradjustthethermalcycler.

Duplexnested-PCRstandardizationusingstandardSRLV samples

For standardization of the duplex nPCR technique, tests were elaborated with standard samples ofCAEV-Cork and

POL 1175 bp LTR 203 bp GAG 187bp

Fig.1–Duplexnested-PCR.1.3%agarosegelstainedwith ethidiumbromide,presentingproductsofamplificationof thepro-viralDNAtargetfragmentsofsmallruminant lentivirusessamples.Productsareequivalentto1175bpfor amplificationwithPOLprimers,203bpforamplification withprimersLTRand187bpforamplificationwithprimers GAG.MM=molecularmarker(DNAladdermarker100pb), C−=negativeControl,CA=CAEVstandardsample, MV=standardMVVsample.

POL 1175pb

GAG 187pb MM C - G1 Sheep3 G3 G4 G5 G16 G11 C+

Fig.2–Duplexnested-PCR.Agarosegel(1.3%)stainedwith ethidiumbromide,presentingproductsfromthe

ampliﬁcationofpro-viralDNAtargetfragmentsfromsmall ruminantlentivirussamples.Productsequivalentto 1175bpforampliﬁcationwithpolprimersand.

MVV-K1514 viruses. This step was performed in order to demonstratethattheprimerspreparedandusedfor perform-ingthetechniqueareeffectiveindetectingtheSRLV.

Standardization of the duplex nPCR technique demon-stratedthattheoptimalconcentrationsforthereactionusing the gagand polprimerswas5pMforpoland10pMforthe

gag,whichgeneratedtwosatisfactorybandsbothallowinga conclusiveandreliablediagnosis.

For the protocol of duplex nPCR performed with the primers pol and LTR, the best primer concentrations were 7.5pMfortheprimerpoland20pMfortheLTRprimersfor bothﬁrstandsecondrounds.Inthisstep,therewasabetter resultofduplexnPCRwithanincreaseofdNTPconcentration usedto200␮Mofeach(Fig.1).

Toperformtheagarosegelelectrophoresis,different con-centrations of agarose were tested. Among all the values tested,the1.3%concentrationallowedthebestvisualization ofthebands.Thisconcentrationwasadoptedasthestandard fortheelectrophoresisofsamplesobtainedbyduplexnPCR. Evaluationtechniqueperformanceinﬁeldsamples

TheresultsobtainedwiththeﬁrstduplexnPCRtestwithgag

andpolprimersusinggoatandsheepsamplesshowedthat

polprimerswereefﬁcientfordetectionoflargenumberofthe positivesamplesofgoats,butwasnotasefﬁcientasthegag

primers.Fromatotalof17samplespositiveforgag,15showed positivityforpolandcontinuedtodemonstratethisexpected result with the duplex technique (Fig. 2). In the evaluated sheepsamples,onlythreewerepositiveforthepolprimers, demonstratingthattheduplextechniqueperformedwithgag

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Table2–DescriptionoftheclinicalﬁndingsandtheresultsobtainedintheAGIDandNested-PCRtests.

Samples Clinicalsymptoms AGID gag pol LTR

CAEVCo – + + + +

MVVK1514 – + + + +

Goat1 Asymptomatic Inconclusive + + −

Goat2 Asymptomatic Negative + + −

Goat3 Arthritis

Productivity decrease

Inconclusive + + −

Goat4 Arthritis Inconclusive + + −

Goat8 Arthritis

Productivity decrease

Inconclusive + + −

Goat10 Asymptomatic Positive + + −

Goat14 Asymptomatic Positive + + −

Goat15 Arthritis Negative + − −

Goat16 Asymptomatic Negative − − −

Goat17 Muscularatrophy

Arthritis

Negative + + −

Goat19 Asymptomatic Negative + − −

Sheep1 Asymptomatic Negative − − −

Sheep3 Asymptomatic Negative + − +

Sheep4 Neurological symptomatology, weightloss, pneumoniaand prostration Positive + + +

Sheep5 Asymptomatic Positive + + +

Sheep6 Asymptomatic Positive + + +

Sheep7 Asymptomatic Negative − − +

Sheep8 Asymptomatic Negative + − −

Sheep11 Asymptomatic Negative − − +

samples.Allsamplesthatwerepositiveinthetestsof individ-ualnPCRforeachsetofprimersalsodemonstratedpositivity whentestedwithduplexnestedPCR.

ThesecondstepofstandardizationusingtheLTRandpol

primers in field samples demonstrated that these primers areidealforduplexnested-PCR,consideringthatthesizeof thebandsformedisverydifferent,allowingagood visualiza-tionoftheresultswhenanalyzedontheagarosegel(Fig.3). In addition, results demonstrated that LTR primers were moreefficientatdetectingsheepsamples, failingtodetect insamples from goats.The resultsdemonstrated that pol primersweremorespecificforSRLVpresentingoatsandLTR primersforSRLVobtainedfromsheepsamples.Theseresults alsoshowed thattheduplexnested-PCRtechniqueismore

sensitive to generate morereliable results fordetection of lentivirusinsheepandgoats.

DNAsequencingandsequenceanalysis

Theanalysesrevealedthatsequencesfromsheepandgoats describedherearerelatedtogenotypeB1(CAEV-Co)and geno-typeA (MVVK514) ofSRLV,respectively. Inaddition, some geneticrelationshipwasobservedwithotherBrazilianstrains described and the samples of sheepshowed relation with Canadianstrain(7-29Ont).

A phylogenetic tree (Fig. 4) was obtained using the NJ method with the gag nucleotide sequences, and this tree showed the relationship between the strains detected by

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MM C- G 7 G8 S7 S10 S15

POL 1175 bp

LTR 203 bp

Fig.3–Duplexnested-PCR.Agarosegel(1.3%)stainedwith ethidiumbromidepresentingproductsoftheampliﬁcation ofthepro-viralDNAtargetfragmentsofsmallruminant lentivirussamples.Bandsequivalentto1175pbfor

ampliﬁcationwithPOLprimersand203pbforampliﬁcation withprimersLTRwereobserved.MM=molecularmarker (DNAladdermarker100pb),C−=negativecontrol.Positive samplesforpol–Goat7andGoat8.PositivesamplesforLTR –Sheep7,10and15. 0.0100 Goat 6 Goat 8 Goat 7 Sheep 4 Sheep 5 Sheep 6 CAEV Cork MVV K1514 7-29Ont Br/UFRGS-2/V27 21G-MG/Br 39QPI/Br BR/CNPC-G4 19F-MG/Br

Fig.4–Molecularphylogeneticanalysisbyevolutionary relationshipsoftaxausingtheNeighbor-Joiningmethod.

duplexnPCRandtheotherknownSRLVstrains.The phylo-genetictreewasdeterminedusingtheneighborjoining(NJ) method35 _implemented _in _MEGA, _the _Tamura-Nei _gamma distance.36

Whencomparingthesequencesobtainedwiththesamples depositedintheGenBankitwaspossibletoverifythedegree ofidentitybetweenthestrains.InTable3itispossibletoverify theconvergenceandidentitybetweentheanalyzedstrains.

Afteranalysisofthepartialsequencesofthegaggenefrom ovineand caprine samplesthesampleswere submitted to the GenBankand are availablefor access withthe follow-ingaccessnumbers:MH251628(Goat6),MH251627(Goat7), MH251626(Goat8),MH251631(Sheep4),MH251630(Sheep5) andMH251629(Sheep6).

Afterthealignmentofthesamplesunderstudywiththe standardsamples,itispossibletoidentifythesiteswhere dif-ferencesinpro-viralDNAoccur.Thesedifferencescanbeseen inFig.5.

Discussion

Severalstudieshaveshownthatthemoleculardiagnosisof lentiviruses are increasingly sensitive,37–39 _being _a _suitable techniqueformonitoringanimals,particularlythoseofhigh zootechnical value. In this study, the developed technique provedtobeefﬁcientforthesimultaneousdetectionofsheep andgoatsamples,allowingafasterandmoresensitive diagno-sisoflentivirusescomparedtoAGID.Consideringthatduplex nPCRallowsthesimultaneousdiagnosisofdifferentregions oftheviralgenomeinasingleassay,itispossiblebymeans ofasingleassaytoperformamoreaccuratediagnosisofthe affectedanimals.Inaddition,costsoflaboratorymaterialand timearereducedwhencomparedtotheconventionalnPCR technique.

TheAGIDtechniqueisrecommendedbyOIE12_and_is_widely used for the serological diagnosis of animals infected by SRLV.However,individualvariationsinimmuneresponseare observedandsomeanimalsmaypresentlowantibodytiters, late seroconversion. Inthis study, amongthe 35 evaluated animals,comprisedbysheepandgoats,onlyﬁveanimals pos-itiveforSRLVinfectionintheAGID.Nevertheless,theproviral DNAofSRLVwasdetectedin27animalswiththenested-PCR. Theseﬁndingsdemonstratethatthemoleculartechniquewas superiorindetection capacitywhencomparedtothe AGID technique.

Theresultsobtainedcorroboratethestudycarriedoutin breedingherds,inwhichanimalsknowntobepositivehad negativeresultsforAGIDandpositivefornPCR.40_In_this con-text,severalotherstudieshavesuccessfullydemonstratedthe useofPCRtechniquetodetectpro-viralDNAofSRLV,15–18,37,39 consideringthatthistechniquepresentshighsensitivity col-laboratingforanearlydiagnosis.19

Barquero et al.10 _consider _that _there _is _no _“gold stan-dard” test for the diagnosis of lentiviruses and with the advancesinmolecularbiologyseveralPCRprotocolshavebeen developed.Therefore,itisdifﬁculttousethesameprimers in different geographic regions due to the SRLV genome heterogeneity.

In most studies involving CAEV41,42 _and _MVV37,43_, _this detection is performedusing primers designedfor the gag

gene, since it has conserved sequences in different SRLV samples.Howeverotherregionsaredescribedthatstillhave somedegreeofconservationandcanbeusedtoimprovethe sensitivityofthePCRtechnique,suchaspolandLTR, allow-ing detection of a wide spectrum of CAEV and MVV ﬁeld strains.10,44–46

TheresultsofDNAsequenceanalysisdemonstratedthat thesamplesobtainedfromgoatsareclosetotheCAEVgroup, while the sheep samples are similar to those of MVV. In addition, throughthe analysisperformedbythe alignment ofthesequencedsamples,itwaspossibletoverifythe dif-ferences thatoccurintheviralgenome,demonstratingthe heterogenicityofthesamples.Reinforcingtheneedforamore sensitiveandspeciﬁctesttodetectalargernumberof sam-ples.

With the molecular tests, this study veriﬁed that the primersdesignedforthegagandpolregionspresentedhigh detection capacityforthecirculating strainsinthestudied

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Table3–Similarityoftheﬁeldstrainswiththestrainsusedinthemultiplesequencingandconstructionofthe phylogenetictree.

Goat6 Goat7 Goat8

Identity CAEV-Co39Q-PI/BrBR-CNPC-G4KF861559.1 95%97%95%95% 97%97%97%96% 95%97%95%95%

Sheep4 Sheep5 Sheep6

Identity MVV-K15147-29OntBr/UFRGS-2/V2721G-MG/Br 93%94%94%91% 93%94%94%91% 93%94%94%91% Goat 1 ---TGTTCCAGCAACTGCAAACAGTAGCAATGCAGCATGGCCT Goat 2 ---GTTCCAGCAACTGCAAACAGTAGCAATGCAACATGGCCT Goat 3 - - - G T T C C A G C A A C T G C A A A C A G T A G C A A T G C A A C A T G G C C T Sheep 1 - - - T G T T C C A G C A A C T G C A A A C A G T A G C A A T G C A G C A T G G A C T Sheep 2 ---TGTTCCAGCAACTGCAAACAGTAGCAATGCAGCATGGACT Sheep 3 ---TTCCAGCAACTGCAAACAGTAGCAATGCAGCATGGACT AJ305039.1 Maedi-Visna virus proviral partial gag gene for Gag CA protein isolate MVV Br/UFRGS-2/V27 T T C A G T A G T T T T C C A A C A A T T G C A A A C A G T G G C A A T G C A A C A T G G A C T EU300979.1 Caprine arthritis-encephalitis virus isolate BR/CNPC-G4 capsid protein p28 (gag) gene par TTCTGTAATGTTCCAGCAACTGCAAACAGTAGCAATGCAGCATGGCCT KC155690.1 Visna/maedi virus isolate 7-29Ont gag protein (gag) gene partial cds CTCAGTAGTCTTCCAGCAGCTGCAAAATGTAGCAATGCAGCATGGACT KF861553.1 Small ruminant lentivirus strain 39Q-PI/Br gag protein gene partial cds CGCATCAATGTTCCAGCAACTGCAAACAGTAGCAATGCAGCATGGCCT KF861559.1 Small ruminant lentivirus strain 19F-MG/Br gag protein gene partial cds C T C T G T A A T G T T C C A G C A A C T G C A A A C A G T A G C A A T G C A G C A T G G C C T KF861573.1 Visna/maedi virus strain 21G-MG/Br gag protein gene partial cds G T C A G T A G T C T T C C A A C A A C T G C A A A C A G T A G C A A T G C A G C A T G G C C T M33677.1 Caprine arthritis encephalitis virus complete proviral genome TTCTGTAATGTTCCAGCAACTGCAAACAGTAGCAATGCAGCATGGCCT M60609.1 Visna virus Icelandic strain 1514 complete genome GTCAGTAGTCTTCCAGCAACTGCAAACAGTGGCAATGCAGCATGGACT

Goat 1 CG-TGTCCGAGGATTTTGAAAGGCAGTTGGCATATTATGCTACTACCT Goat 2 CG-TGCTCGAGGATTTTGAAAGGCAGTTAGCATATTATGCTACTACCT Goat 3 CGCTGTCCGAGGATTTTGAAAGGCAGTTAGCATATTATGCTACTACCT Sheep 1 GG-TGTCCGAGGATTTTGAGAGGCAACTGGCTTATTATGCCACTACAT Sheep 2 GG-TGTCCGAGGATTTTGAGAGGCAACTGGCTTATTATGCCACTACAT Sheep 3 GG-TGTCCGAGGATTTTGAGAGGCAACTGGCTTATTATGCCACTACAT AJ305039.1 Maedi-Visna virus proviral partial gag gene for Gag CA protein isolate MVV Br/UFRGS-2/V27TG-TGTCCGAGGATTTTGAGAGGCAAATGGCTTATTATGCCACTACAT EU300979.1 Caprine arthritis-encephalitis virus isolate BR/CNPC-G4 capsid protein p28 (gag) gene par CG-TGTCTGAGGACTTTGAAAGGCAGTTGGCATATTATGCTACTACCT KC155690.1 Visna/maedi virus isolate 7-29Ont gag protein (gag) gene partial cds TG-TGTCCGAGGATTTTGAGAGGCAAGTGGCATATTATGCCACCACAT KF861553.1 Small ruminant lentivirus strain 39Q-PI/Br gag protein gene partial cds CG-TGTCCGAGGATTTTGAGAGGCAGTTAGCATATTATGCTACTACCT KF861559.1 Small ruminant lentivirus strain 19F-MG/Br gag protein gene partial cds CG-TGTCAGAGGACTTTGAAAGGCAGTTGGCATATTATGCTACTACCT KF861573.1 Visna/maedi virus strain 21G-MG/Br gag protein gene partial cds CG-TGTCCGAGGATTTTGAAAGACAGCTAGCATATTATGCCACTACCT M33677.1 Caprine arthritis encephalitis virus complete proviral genome CG-TGTCTGAGGACTTTGAAAGGCAGTTGGCATATTATGCTACTACCT M60609.1 Visna virus Icelandic strain 1514 complete genome TG-TGTCCGAGGATTTTGAGAGGCAATTGGCATATTATGCTACTACCT

Goat 1 GGACAAGTAAAGACATATTAGAGGTATTGGCCATGATGCCTGGCAATA Goat 2 GGACAAGTAAAGATATCTTAGAAGTATTGGCCATGATGCCTGGAAATA Goat 3 GGACAAGTAAAGATATATTAGAAGTATTGGCCATGATGCCTGGCAATA Sheep 1 GGACAAGTAAAGATATATTAGAAGTATTGGCCATGATGCCTGGGAACA Sheep 2 GGACAAGTAAAGATATATTAGAAGTATTGGCCATGATGCCTGGGAACA Sheep 3 GGACAAGTAAAGATATATTAGAAGTATTGGCCATGATGCCTGGGAACA AJ305039.1 Maedi-Visna virus proviral partial gag gene for Gag CA protein isolate MVV Br/UFRGS-2/V27GGACAAGTAAAGATATATTAGAGGTATTAGTCATGATGCCTGGGAACA EU300979.1 Caprine arthritis-encephalitis virus isolate BR/CNPC-G4 capsid protein p28 (gag) gene par GGACAAGTAAAGACATACTAGAAGTATTGGCCATGATGCCTGGAAATA KC155690.1 Visna/maedi virus isolate 7-29Ont gag protein (gag) gene partial cds GGACAAGTAAGGATATATTAGAAGTGTTGGCCATGATGCCTGGGAACA KF861553.1 Small ruminant lentivirus strain 39Q-PI/Br gag protein gene partial cds GGACAAGTAAAGACATCTTAGAAGTATTGGCCATGATGCCTGGAAATA KF861559.1 Small ruminant lentivirus strain 19F-MG/Br gag protein gene partial cds GGACAAGTAAAGATATATTAGAGGTATTGGCCATGATGCCAGGAAATA KF861573.1 Visna/maedi virus strain 21G-MG/Br gag protein gene partial cds GGACAAGTAAAGATATATTAGAGGTACTGGCTATGATGCCTGGGAATA M33677.1 Caprine arthritis encephalitis virus complete proviral genome GGACAAGTAAAGACATACTAGAAGTATTGGCCATGATGCCTGGAAATA M60609.1 Visna virus Icelandic strain 1514 complete genome GGACTAGTAAAGATATATTAGAAGTATTGGCTATGATGCCTGGGAATA

Goat 1 GAGCTCAAAAAGAGTTAATTCAAGGGAAATTAAATGAAGAAGCAGAAA Goat 2 GAGCTCAAAAAGAGTTAATTCAAGGGAAATTAAATGAAGAAGCAGAAA Goat 3 GAGCTCAAAAAGAGTTAATTCAAGGGAAATTAAATGAAGAAGCAGAAA Sheep 1 G A G C A C A A A A A G A G T T A A T A C A A G G G A A A T T A A A T G A A G A A G C A G A A A Sheep 2 GAGCACAAAAAGAGTTAATACAAGGGAAATTAAATGAAGAAGCAGAAA Sheep 3 GAGCACAAAAAGAGTTAATACAAGGGAAATTAAATGAAGAAGCAGAAA AJ305039.1 Maedi-Visna virus proviral partial gag gene for Gag CA protein isolate MVV Br/UFRGS-2/V27GAGCACAAAAAGAGTTGATACAGGGGAAATTAAATGAAGAAGCAGAAA EU300979.1 Caprine arthritis-encephalitis virus isolate BR/CNPC-G4 capsid protein p28 (gag) gene par GAGCTCAAAAGGAGTTAATTCAAGGGAAATTAAATGAAGAAGCAGAAA KC155690.1 Visna/maedi virus isolate 7-29Ont gag protein (gag) gene partial cds GAGCACAGAAAGAATTAATACAAGGGAAATTAAATGAAGAAGCAGAAA KF861553.1 Small ruminant lentivirus strain 39Q-PI/Br gag protein gene partial cds GAGCTCAAAAAGAGTTAATTCAAGGGAAATTAAATGAAGAAGCAGAAA KF861559.1 Small ruminant lentivirus strain 19F-MG/Br gag protein gene partial cds GGGCTCAAAAAGAATTAATTCAAGGGAAATTAAATGAAGAAGCAGAAA KF861573.1 Visna/maedi virus strain 21G-MG/Br gag protein gene partial cds GAGCTCAAAAAGAATTAATACAAGGAAAGTTAAATGAAGAAGCAGAAA M33677.1 Caprine arthritis encephalitis virus complete proviral genome GAGCTCAAAAGGAGTTAATTCAAGGGAAATTAAATGAAGAAGCAGAAA M60609.1 Visna virus Icelandic strain 1514 complete genome GAGCACAGAAGGAATTAATACAAGGAAAATTAAATGAAGAAGCAGAAA

Goat 1 GGTAA--- -Goat 2 GGT--- -Goat 3 GGTAA--- -Sheep 1 GGT--- -Sheep 2 GGT--- -Sheep 3 GGT--- -AJ305039.1 Maedi-Visna virus proviral partial gag gene for Gag CA protein isolate MVV Br/UFRGS-2/V27GGTGGGTAAGGCAAAACCCACCGGGTCA---AAATGTCCTTACA EU300979.1 Caprine arthritis-encephalitis virus isolate BR/CNPC-G4 capsid protein p28 (gag) gene par G G T G G A G A A G G A A T A A T C C A C C A C C T C C A G C A G G A G G A G G A T T A ? A C A KC155690.1 Visna/maedi virus isolate 7-29Ont gag protein (gag) gene partial cds GGTGGGTGAGACAAAATCCACCAGGGCC---AAATGTCCTAACA KF861553.1 Small ruminant lentivirus strain 39Q-PI/Br gag protein gene partial cds GGTGGAGAAGAAACAATCCACCACCTCCCCAAGGAGGAGGATTAA-CA KF861559.1 Small ruminant lentivirus strain 19F-MG/Br gag protein gene partial cds GGTGGAGAAGAAATAATCCCCCACCTCCAGCAGGAGGAGGGTTAA-CA KF861573.1 Visna/maedi virus strain 21G-MG/Br gag protein gene partial cds GATGGGTAAGGCAGAATCCACCGGGTCC---GAATGTCCTTACA M33677.1 Caprine arthritis encephalitis virus complete proviral genome GGTGGAGAAGGAATAATCCACCACCTCCAGCAGGAGGAGGATTAA-CA M60609.1 Visna virus Icelandic strain 1514 complete genome GGTGGGTAAGACAAAATCCACCCGGGCC---GAATGTCCTCACG

Fig.5–AlignmentofnucleotidesequencesfromthepartialgagregionofSRLVstrains.Themarkingsidentifytheregions

wheretherearedifferences.

regionwithsamplesfromgoats.However,theprimersofthe gagregionaremoreefﬁcient.Gregoryetal.,45_used_pol_primers todetectDNApro-viralofCAEVinsamplesfromlung, mam-mary gland, brain and synovial ﬂuid, obtaining promising results.

Forsheepsamplesinthisstudy,gagandLTRprimerswere efﬁcientindetectingpositiveanimals. Severalstudies have described the use ofPCR with primers LTR obtained good results.15,47_However,_other_researchers_have_shown_that gag-PCRwasmoresensitivethanLTR-PCRfordetectionofSRLVin sheepandgoats.16,43,48

Inourstudy,asimilardetectionwasobservedbetweenthe detectionvaluesofpositivesheepsamplesusinggagandLTR primers.However,onesamplewaspositiveforLTRandwas negativeforgag.Thisdemonstratesthattheprimersforthe LTRregionaremorespeciﬁcforviraldetectionofsheep.This ﬁndingcorroborateswiththeresultsobtainedbyGlariaetal.46 inwhichdifferenttissuesamplesevaluatedbygag-PCRand LTR-PCRwerepositiveforLTRprimersandnegativeforgag primers.

Similar resultswere obtainedbyMarinhoet al.38 _which reportedthatthedetectionofsheeplentiviruseswasmuch

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moresensitivewhenperformingnested-PCRwithLTRprimers thanwhenusinggagprimers.

MVVinfectionwasinvestigatedinthesamesamplesusing twoPCRtestsdirectedtothe LTRandpolsequences ofthe MVVgene.LTR-PCRwasmoresensitiveandallowedtodetect infectionearlierthantheothertests.49 _Similar_results_were observedinthepresentstudyinwhichthesamplesofsheep werebetterdetectedwithLTR-PCRthanwhenthesame tech-niquewasdoneusingpolprimers.

From thesedata, wecanconclude thatnested-PCR per-formedwithprimersfromthepolregionwasnotefﬁcientfor thedetectionofsheepsamplesinthisstudy.However,when usedforthedetectionofgoatsamples,itdemonstratedhigh detectioncapacity.Thenested-PCRperformedwiththegag

primerswasabletodetectsomesheepsamples,butnotallof them,whichcouldleadtoafalsenegativeresultwhenthePCR isperformedonlywiththegagprimers.Concerningprimers forthe LTRregion, thesehavebeen shown tobethe most efﬁcientforthedetectionofsheepsamples.

Considering the importance of diagnosing etiological agentsinvolvedindiseases toavoidthespread among ani-mals,itisnecessarytoestablishasystemofrapidandhighly speciﬁcdiagnosisforthesepathogens.Amethodthatis sen-sitive,speciﬁcandcapableofdetectingallthevariantsofthe virusevenifpresentinlowconcentrationarefundamental prerequisitesforthesuccessofthecontrolanderadicationof lentiviruses.3

Inthissense,duplexnested-PCRtechniqueforthe diag-nosisoflentivirusesofsmallruminantsallowedforamore accuratediagnosis,consideringthatitisanampliﬁcation reac-tiondesignedtodetectmultipletargetsequencesinthesame sample.Thispromotesthedetectionoflentivirusesbothin sheepandgoats,eveninthecaseofcross-infection.Through phylogenetic analysis, it canbeconcluded thatthe duplex nested-PCRtechniquewasabletodetectCAEVandMVV sam-plesinasingleassay.

Inveterinarymedicine,multiplexPCRhasbeendeveloped andappliedforthesimultaneousdetectionofdifferent dis-eases inpigs,50 _cattle,51 _birds,52 _dogs53 _and _equines54 _in _a singleassay.

As well as SRLV, equine herpesviruses are considered geneticallyandantigenicallyrelated,whichpromotesa difﬁ-cultyindifferentiatingthesepathogensthroughconventional serological tests.55 _A _multiplex _nested-PCR _technique _was developed and successfully applied for the simultaneous detectionofequineherpesvirusesindifferentclinical sam-ples.Thesameresultcouldbeobtainedinthisstudywiththe applicationofaduplexnested-PCRtechnique.

Thecritical factors ofPCR reaction are the selection of primers, theconcentration ofmagnesium chlorideand the annealingtemperature.56_It_was_not_necessary_to_change_the magnesiumconcentrationortheannealingtemperaturefor performingthetechnique.However,fortheduplexnested-PCR usingpolandLTRprimers,itwasnecessaryadjustmentsinthe mixtooptimizethereaction.ThesealterationsofthePCRmix forperformingtheduplextechniquewerealsonecessaryin thestudybySilvaetal.57

Theresultsobtainedallowedustoconcludethattheduplex nested-PCRisanimportanttoolforamoreaccurate,sensitive andspeciﬁcdiagnosisofthedifferentstrainsofSRLVthatare

circulatinginBrazil,allowingadecreaseoffalsenegativesand increasingthecapacityofviraldetection.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

ThisworkwassupportedbytheNationalCouncilof Scien-tiﬁcandTechnologicalDevelopment(CNPq),grantnumbers 487425/2012-0 and 308995/2013-9;Coordinationof Improve-ment of Higher Level Personnel (CAPES) for AUXPE-PROEX 533/2014grantnumbers23038.003104/2014-58andCearense FoundationinSupportofScientiﬁcandTechnological Devel-opment (FUNCAP), which provideda PhD scholarship.The authors are thankful tothe Post GraduateProgram in Vet-erinaryScience(PPGCV)oftheStateUniversityofCearáfor supporting the implementation of the experiments; to the LaboratoryofParasiticDiseases,particularlytoClaudiaMaria LealBevilaqua,PhD,forunconditionalsupportinperforming themoleculartests.Theauthorsalsoexpresstheirgratitude toownersofthefarmsthatallowedtheuseoftheiranimals forthisstudy.

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