Bovine papillomavirus isolation by ultracentrifugation

(1)

ContentslistsavailableatScienceDirect

Journal

of

Virological

Methods

jou rn al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / j v i r o m e t

Short

communication

Bovine

papillomavirus

isolation

by

ultracentrifugation

R.P.

Araldi

a,b

,

D.N.S.

Giovanni

b,c

,

T.C.

Melo

a,d

,

N. Diniz

a

,

J. Mazzuchelli-de-Souza

a,b

,

T.A.

Sant’Ana

a,b

,

R.F.

Carvalho

a

,

W. Bec¸

ak

a,e

,

R.C.

Stocco

a,b,∗

a_Laboratório_de_Genética,_Instituto_Butantan,_Av._Vital_Brasil,_1500,_São_Paulo_05503-900,_SP,_Brazil

b_Programa_de_{Pós-graduac¸}_ão_{Interunidades}_em_{Biotecnologia,}_Universidade_de_São_Paulo,_Av._Prof._Lineu_Prestes,_2415,_Ed._ICB_III,_Cidade_{Universitária,}_São

Paulo05508-900,SP,Brazil

c_Laboratório_de_{Parasitologia,}_Instituto_Butantan,_Av._Vital_Brasil,_1500,_São_Paulo_05503-900,_SP,_Brazil

d_Programa_de_{Pós-graduac¸ão}_em_Biologia_Estrutural_e_Funcional,_Universidade_Federal_de_São_Paulo,_Ed._Leitão_da_Cunha,_R._Botucatu,_740,_São_Paulo

04023-900,SP,Brazil

e_Departamento_de_Biologia,_Universidade_Federal_da_Integrac¸_ão_{Latino-Americana,}_Av._Silvio_Américo_Sasdelli,_1842,_Vila_A,_Ed._Comercial_Lorivo,_Foz_do

Iguac¸ú85866-000,PR,Brazil

Articlehistory: Received25April2014

Receivedinrevisedform21July2014 Accepted25July2014

Availableonline4August2014

Keywords:

Bovinepapillomavirus Diagnosis

Virusisolation Ultracentrifugation

a

b

s

t

r

a

c

t

Thebovinepapillomavirus(BPV)istheetiologicalagentofbovinepapillomatosis,whichcausessignificant economiclossestolivestock,characterizedbythepresenceofpapillomasthatregressspontaneouslyor persistandprogresstomalignancy.Currently,thereare13typesofBPVsdescribedintheliteratureaswell as32putativenewtypes.ThisstudyaimedtoisolateviralparticlesofBPVfromskinpapillomas,using anovelviralisolationmethod.Thevirustypeswerepreviouslyidentifiedwithnewprimersdesigned. 77cutaneouspapillomasamplesof27animals,Simmentalbreed,weresurgicallyremoved.TheDNA wasextractedandsubjectedtoPCRusingDelta-EpsilonandXiprimers.Thebandswerepurifiedand sequenced.ThesequenceswereanalyzedusingsoftwareandcomparedtotheGenBankdatabase,by BLASTtool.TheviraltypingshowedaprevalenceofBPV-2in81.81%ofsamples.Itwasalsodetected thepresenceoftheputativenewvirustypeBR/UEL2inonesample.Virusisolationwasperformedby ultracentrifugationinasingledensityofcesiumchloride.Themethodofvirusisolationislesslaborious thanthosepreviouslydescribed,allowingtheisolationofcompletevirusparticlesofBPV-2.

Papillomavirusesareoncogenicviruseswithdouble-stranded circular DNAgenome, notcoiled, approximately8kblong with a55–60nmdiameter.Thegroupisincludedinthe Papillomaviri-dae family,which displaystropism forsquamous epithelialand mucoustissues.Thesevirusesaffectvertebrates,includinghuman (Albertietal.,2010),associatedwithbenignandmalignant epithe-liallesions(StoccodosSantoset al.,1998; Ropertoetal.,2008;

Carvalhoetal.,2013).

According to the International Committee on Virus Taxon-omy(http://ictvonline.org/virusTaxonomy.asp),papillomaviruses

∗Correspondingauthorat:InstitutoButantan,LaboratóriodeGenética,Av.Vital Brasil,1500Butantã,SãoPaulo05503-900,SP,Brazil.Tel.:+551126279701;fax: +551126279701.

E-mailaddresses:rodrigo.araldi@butantan.gov.br(R.P.Araldi),

dalton.silva@butantan.gov.br(D.N.S.Giovanni),thatianacmelo@yahoo.com.br (T.C.Melo),nara.diniz@gmail.com(N.Diniz),jackmazzuchelli@yahoo.com.br (J.Mazzuchelli-de-Souza),thalita.a.santana@hotmail.com(T.A.Sant’Ana), rodsfc@butantan.gov.br(R.F.Carvalho),wbecak@pq.cnpq.br(W.Bec¸ak), rita.stocco@butantan.gov.br(R.C.Stocco).

areclassifiedinto30generainaccordancewiththediversityof nucleotidesequencesoftheL1OpenReadingFrame(ORF).Over 10%differencesbetweentwoDNAsequencesoftheL1ORFdefine anewvirustype,whiledifferencesbetween2and10%defineanew viralsubtype(DeVilliersetal.,2004).

Specificallyinbovines,currently,thereare13typesofBovine Papillomaviruses(BPV)describedintheliterature,althoughthis numbermaybegreaterthan20(Lunardietal.,2013).BPVtypes areclassifiedintothreegenera,basedonhomologytothegenomic regionsoftheL1ORF,themostconservedsequence(DeVilliers etal.,2004;DeVilliers,2013):Deltapapillomavirus(BPV-1,2and 13),Epsilonpapillomavirus(BPV-5and8)Xipapillomavirus(BPV-3, 4,6,9,10,11and12)andBPV-7thatremainsnotincludedinany genre(Araldietal.,2013).

TheBPVicosahedralcapsidhas360copiesoftheL1proteinof 55kDa,organizedin72capsomersandapproximately12copiesof theL2proteinwith39kDa(Campo,1997;Góesetal.,2008;Meinke andMeinke,1981)havingamolecularweightof5.2×106Da(Orth

etal.,1977).TheBPVgenomeisdividedintothreeregions:early, late and long control, separated by two polyadenylation sites

(2)

120 R.P.Araldietal./JournalofVirologicalMethods208(2014)119–124

(Zheng and Baker, 2006) and the viral DNA is associated with histone-likeproteins(Letoetal.,2011).Theearlycontrolregion (ECR)occupies50%oftheviralgenomeandencodestheproteins E1,E2,E4,E5,E6andE7.Thelatecontrolregion(LCR)occupies40% ofthegenomeandencodestheL1andL2proteins.Thenot cod-ingregion(NCR)occupies10%ofthegenome,withapproximately 850bpand,althoughitisnotcoding,itcontainstheoriginof repli-cation(ori)andthebindingsitesformultipletranscriptionfactors, importantfortheregulationofRNApolymeraseII(Hatamaetal., 2008;ZhengandBaker,2006).

TheBovinepapillomavirus(BPV)istheetiologicagentofbovine papillomatosis,aninfectiousdisease,clinicallycharacterizedbythe presenceofhyperproliferativelesions(papillomas),causing signif-icanteconomiclossestothelivestock(Carvalhoetal.,2003,2013). Theinfectioncanresultinobstructionoftheteat,hinderingthe hygiene,mastitis,bleeding ofteatsand difficultyinplacingthe mechanicalmilking.Otherthanthat,itisassociatedwitheconomic depreciationofanimalleather(Catroxoetal.,2013).Thevirusalso cancausebladderandgastrointestinalcancerinbovinesandits presencehasbeendetectedinperipheralblood(StoccodosSantos etal.,1998;Freitasetal.,2003;Araldietal.,2013;Camposetal., 2013).TheBPVcanalsocausesarcoidsinequines(Martensetal., 2000;Wobeseretal.,2012).Althoughconsideredspecie-specific, BPV-1and2canaffectyaks(Bametal.,2013),tapirs(Kidneyand Berrocal,2008),zebras,giraffes,antelopes(VanDyketal.,2011); buffaloes(Pangtyetal.,2010)andhorses(Chambersetal.,2003)

TheBPVinfectionbeginsinamicro-tissuelesion,whichexposes thepeptydoglicansofheparansulfate(Hartletal.,2011;McBride etal.,2012).TheBPVbindstoheparansulfate incytoplasmatic membrane of the epithelium cell from the basal layer. In this processthevirusparticlesareinternalizedthroughendocytosis, mediatedbyclathrin(Hartletal.,2011;McBrideetal.,2012).

Aftercellinfection,BPVreproductivecycleoccursthrough:(1) theformationoflow-copyviralgenome,(2)themaintenanceof replicationand(3)amplificationofdifferentiatedvegetativecells (McBrideetal.,2012).So,thereproductiveviruscycledependsof epithelialcelldifferentiation,whichjustifiestheabsenceofa sys-temforinvitroreplication(Shafti-keramatetal.,2003;DeVilliers, 2013).

Consideringthehugesizeofthenationalcattleherdandthe significanteconomiclossesduetoBPVrelateddiseases,the devel-opmentofvaccinestrategiesisveryrelevant(Mazzuchelli-Souza etal.,2013).

Purificationofvirionsisessentialtoobtaininformationabout viruschemical,physical,biochemicalandbiologicalpropertiesand toreachtheproductionofantiserum(AliandRoossinck,2007).

The greatest difficulties in vaccine strategies against papil-lomavirus (PV) is the viral isolation of a particular viral type (Kirnbauer et al., 1996), since the co-infection is frequently observed(Lindseyetal.,2009;Araldietal.,2013).However,asfaras weconcerned,thereisnoinvitroPVreplicationsystemdescribed (Kirnbaueretal.,1996),andPVisnotcultivable(Favreetal.,1974). ThereareseveralmethodsforBPVisolationdescribedinthe lit-erature,allbasedonultracentrifugationincesiumchloride(CsCl) gradients(Bujard,1967;Larsen etal.,1987;Löhrdr etal.,2005) orsucrosegradients(Favre,1975;Liuetal.,1997).However,the publishedmethodstodatearelaboriousandinvolvecomplicated procedurestoobtainsufficientamountsofvirionsafter ultracen-trifugation(Bujard,1967;Wangetal.,2007).

This study shows a novel method for the isolation of BPV virionsinvolvingultracentrifugation,whichallowsisolateviral par-ticlesfrommonoviralcutaneouspapillomassamples.Themethod proposedinthisworkcanbeusedforisolationofother Papillo-maviruses,includingtheHumanpapillomavirus(HPV).

77sampleswere collectedof cutaneouspapillomasfrom 27 adult bovines (Bos taurus) at an experimental farm. Surgical

procedureswereperformedbyaveterinarian,usingaseptic meth-ods,localanesthesia(2%lidocaine),incisionwithsterilebistouries, and sutured with synthetic 3.0 mononylon. The protocol was approved bythe EthicCommitteeon AnimalUse. Thesamples weretransportedincoolersat4◦_C_and_a_fragment_of_{approximately} 25mgofeachsampleweredestinedtomoleculartype identifica-tion.

The DNA extraction was performed using the PureLinkTM

GenomicDNAkit(Invitrogen,Carlsbad,USA)accordingtothe man-ufacturerinstruction,using25mgoftissue.TheextractedDNAwas quantifiedinspectrophotometerBioPhotometerPlus(Eppendorf, Hamburg,Germany).ItwasalsodeterminedtheratioA260/A280 andA260/A230toevaluatetheDNAextractionquality.

TheDNAqualitywasverifiedthroughPCR,usingtheprimerfor bovine␤-globin (forward 5′_{-AACCTCTTTGTTCACAAAGAG-3}′ _and reverse 5′_{-CAGATGCTTAACCCACTGAGC-3}′_), _according _to _Yaguiu

etal.(2008),using200ng/␮lofDNAtemplate.Reactionswere per-formedinthethermocyclerVeriti96WellThermalCycler(Applied Biosystems,Carlsbad,USA)andsubjectedtothefollowingcycles: aninitialstepof3minat94◦_C,₃₅_cycles_with₅₀_s_at₉₄◦_C (dena-turation),60sat60◦_C_(annealing)_and₆₀_s_at₇₂◦_C_(extension)_and afinalstepof5minat72◦_C.

Theampliconswereanalyzedin2%agarosegelinTAEbuffer, stainedwith2.0␮lofGelRedTM_(Biotium,_Hayward,_USA)/100_ml

of agarose solution. The electrophoretic run was performed at 100V, 400mAfor120min,withthe100bpDNAladdermarker (Invitrogen,Carlsbad,USA).Thegelwasvisualizedunder transillu-minatorMiniBISPro(DNRBio-ImagingSystems,Jerusalem,Israel) andimageswerecapturedusingthesoftwareGelCapture7.1(DNR Bio-ImagingSystems,Jerusalem,Israel).

The viral identification was performed using a pair of degenerated primers Delta-Epsilon (forward 5′ -CCAGAYT-AYYTMAAAATGGC-3′ _and_reverse₅′ -ATAAMKGCTAGCTTATATTC-3′₎_and_Xi_(forward₅′_{-TWYAATAGDCCVTTTTGGAT-3}′ _and_reverse 5′_{-TTMCGCCTACGCTTTGGCGC-3}′_), _originally _designed _based _on homologyofsequencesofBPVs,allowingtodetectBPVsofgenera Delta, Epsilonpapillomavirus (Delta-Epsilon) and Xipapillomavirus (Xi).TheseprimersamplifytheL1ORF,resultinginproductswith 430bpand 600bp,respectively.These primerpairs werefirstly designed by Maeda et al. (2007), that named them as subAup (Delta-Epsilon)andsubAdw(Xi)andsubsequentlymodifiedbyus. InordertoemploythenintheidentificationofBPVinaccordance toviralgenres,theninthbaseofthesubAdwforwardprimer(G) wassubstitutedbyD,andthetwelfthbaseofthis sameprimer (C),byV.ThesechancesresultintheXiprimer.Thus,changesin theoriginalPCRcycleweredonetooptimizetheseprimerstoBPV genresidentification.

Totestthespecificity ofbothprimers todetectBPV accord-ingtothegenera,theywerefirstlytestedinclonedgenomesof BPV-1(AB626705),BPV-2(M20219.1),BPV-3(AF486184.1),BPV-4 (X05817.1),BPV-5(NC004195.1)andBPV-6(AB845589.1).

PCRparameters:reactionswereperformedinatotalvolumeof 50.0␮l,using:200ng/␮lofDNAtemplate.Reactionsweredoneon thethermocyclerVeriti96WellThermalCycler(Applied Biosys-tems,Carlsbad, USA), andsubjected tothefollowing cycles:an initialstepof10minat94◦_C,₃₅_cycles_with₆₀_s_at₉₄◦_C (dena-turation),60sat52◦_C _(annealing),₆₀_s_at₇₂◦_C _(extension)_and 10minat72◦_C_(final_step)._The_amplicons_were_analyzed_in pre-viouslydescribedconditions,withpositiveandnegativecontrols. Aspositivecontrol,BPV2viralgenomeforDelta-Epsilonprimerand BPV-3forXiprimer,previouslyclonedinbacterialvectorpAT153 inEscherichiacoliD5H␣strainwereselected.

TheampliconbandswerepurifiedusingthePureLinkTM_Quick

(3)

Fig.1. Electrophoresisgel:430bpampliconsobtainedwithprimerpairDelta-EpsilonthatisabletoamplifyBPVsgenraDeltapapillomavirus(BPV-1and2)and Epsilonpapil-lomavirus(BPV-5)and600bpampliconsobtainedwithprimerpairXiindetectBPVsofgenreXipapillomavirus(BPV-3,4and6).

3730DNAAnalyzerTM_(Applied_Biosystems,_Carlsbad,_USA),_using

theBigDye®_Terminator_v3.1_(Applied_Biosystems,_Carlsbad,_EUA),

employing2.5␮loftherespectiveprimerat5pM.

ThequalityoftheDNAsequenceswerecheckedandoverlapping fragmentsweremountedusingthebioinformaticssoftwareBioEdit version7.0.9.0(IbisTherapeutics,Carlsbad,USA)andthesequences werecomparedusingtheNCBIdatabase,throughtheBLASTtool (http://blast.ncbi.nlm.gov).

AfterthemolecularidentificationofBPVtypes,40gofpapilloma sampleswithonlyoneviraltypeweremechanicallymaceratedfor twominuteswith320mloflysesbuffer(1MNaCl,0.02MPBSpH 8.0).Theproductwastransferredtocellulosenitratetubes and wascentrifugedfor 30minat9000×gin SorvallOTD-75 Ultra-centrifuge(DuPont,Wilmington,USA),usingtheAH-629(36ml) swingingrotor,at4◦_C_for_a_first_clearance._The_supernatant, con-tainingthevirusparticles,wascollectedandstored.Theresulting pelletsweresuspendedin320mloflysesbufferandcentrifuged again under the above conditions for the enrichment of virus extraction.Thesupernatantswerecombinedandaddedof0.25% ofUltraPureTM_SDS_(Invitrogen,_Carlsbad,_USA)_and_0.01%_trypsin

1:250(Sigma–Aldrich,St.Louis,USA)/Milli-Qautoclavedwater.The finalvolumewasincubatedalong2hat37◦_C_in_a_shaker,_under rotationof175×g.Afterincubation,thematerialwascentrifuged for1hat100,000×ginaSorvallAH-629(36ml)swingingrotor at4◦_C, _discarding_the_supernatant._The_pellet¸_consisting_mainly ofcollagenfibers,wassuspendedin2mg/mlcollagenasesolution inlysesbuffer,resultinginafinalvolumeof5ml.Thesuspension wasincubatedforfourhoursat37◦_C_in_a_shaker,_under_rotation_of 175g.

Thesuspensionwascentrifugedfor1hat100,000×ginSorvall AH-650swingingrotorincellulosenitratetubesof5ml,discarding thesupernatant.Thepelletwassuspendedin400␮lsuspension buffer(0.05MNaCl,0.01MEDTA,0.05MPBSpH7.4),whichwas transferredtocellulosenitratetubes of5ml,containing4mlof asolutionofCsCl(densityof1.3g/ml)insuspensionbuffer.The tubeswerecentrifugedalong24hat136,000×ginSorvallAH-650 (36ml)swingingrotor.Afterultracentrifugation,theBPVvirions werecollectedbyaspirationandtransferredtocryogenictubesthat hadtheirvolumecompletedto2mlofPBS(137mMNaCl,2.7mM KCl,10mMNa2HPO4,2mMKH2PO4,pH7.4)andstoredat−80◦C. Analiquotof500␮lofisolatedvirionswasdestinedto elec-tronic transmission microscopy, being fixed in glutaraldehyde bufferat2%,dehydratedinethanol,submittedtonegativestaining

andincludedinepoxyresin.Thematerialwasanalyzedin elec-tronictransmissionmicroscopyLeo906E(CarlsZeiss,Oberkochen, Germany)andtheimageswerecapturedbyMegaViewIII cam-eraand usingthesoftwareITEMversion E23082007(Olympus SoftImagingSolutions,Hamburg,Germany),employingavoltage of80kVandaconstantcurrentof1Aandatotalmagnificationof 60,000–120,000×.

TheDelta-EpsilonandXiprimersshowedspecificityforthe dif-ferentBPVsgenera,accordingtoFig.1.Usingtheseprimers,itwas possibletoidentifythepresenceofBPV-2in81.82% ofsamples (63/77),beingthemostprevalentvirustype.Itwasdetectedthe presence ofBPV-5 in 11.68% ofsamples(9/77), BPV-9in 3.89% (3/77).TheresultsalsopointoutthepresenceofBPV-3andthe putativevirustypeBR/UEL-2inonesampleeach,beingthefirst descriptionofthisputativevirustypeinSãoPauloState,Brazil.It wasalsoobservedtheco-infectionformorethanonevirustypein fiveanimals,representing18.51%ofanalyzedanimals.

Usingthenovelmethodologyproposedinthis presentwork, itwassuccessfullyachievedtheisolationofcompleteBPV-2 viri-ons.Althoughthemoleculartypifyinghadshowedthepresenceof fourdifferenttypesofBPV(BPV-2,3,5and9)andaputativenew type(BR/UEL-2),weonlyisolatedBPV-2,themostfrequenttype observed.

Toconfirmtheisolatedvirus,thematerialwassubmittedto electronictransmissionmicroscopyinamaximummagnification of120,000×,revealingtheefficacyofthenovelmethodology, indi-catingthepresenceofhighamountofBPV-2,with55nmdiameter particles,asexpected(Fig.2).

TheisolatedparticlesofBPV-2werestockedat−80◦C.Thenovel methodologyproposedcanbeappliedwithequalsuccessfor isola-tionofanotherBPVstypesaswellasfortheHumanPapillomavirus (HPV),sincethemethodologyallowsisolatingthevirusin accor-dancewiththemolecularweight.

A high frequency of bovine cutaneous papillomatosis was observedinthestudiedpopulation,especiallyamongyoung ani-malsagedbetweentwoandthreeyears.Similarresultsinanimals oftheSimmentalbreedwereobservedbyTurketal.(2005).The highincidenceofpapillomatosisisrelatedtothedensificationof animals,sinceconfinedpopulationsaremoresusceptibleto out-breaksofthedisease.

(4)

Fig.2.IsolatedBPV2particlesfromcutaneouspapillomasusingtheproposedmethodologyinfourdifferentmagnifications:16,000×(A),36,000×(B),75,000×(C)and 120,000×(D).

Thisamountmaybeevenhigherbecausethevirusinfectioncanbe asymptomatic(Araldietal.,2013),leadingtotheneedof prophy-lacticand/ortherapeuticvaccines.

Theviraltypingbysequencingrequirestheuseofprimersthat allowefficientidentificationofviralsequences.Accordingly,the primersetDelta-EpsilonandXiwerehighlyefficientinidentifying sequencesofBPVinaccordancewiththevirusgenera.The Delta-Epsilonprimer wasalreadydescribed withthenameof subAup (Maedaetal.,2007).Howeverthecyclingchangesalloweditsuse todetectBPVsofDeltaandEpsilonpapillomavirusgenres.Inrelation totheXiprimer,thiswasthefirsttimethatitwasusedandallowed thesuccessfulidentificationofXipapillomavirus.

Viraltypingwasperformedbysequencing,revealingthe pres-ence of BPV-2 in 81.81% (63/77) of samples, being the most prevalentvirustype in thestudiedpopulation,followed bythe BPV-5,observedin11.68%(9/77),BPV-9,detectedin3.89%(3/77) andBPV-3,observedinonlyonesample.Itwasalsodetectedthe presenceofsupposednewviraltypeBR/UEL-2inonesample.

TheBR/UEL-2wasfirstlyidentified inStateof Paraná,being observedinskinpapillomaoftheaxillaregion(Clausetal.,2009). Theviruspresentsanidentitymatrixof78%withtheBPV-4, allow-ingitsclassificationasaXipapillomavirus(Clausetal.,2009).

WedetectedtheBR/UEL-2inacutaneouspapillomainthe tho-racicregionofafemaleanimal.ThisfindingpointsouttotheBPV distributionand disseminationfordifferentareas,justifyingthe needforinvestmentinlargerepidemiologicalstudiesto under-standthedifferentviraltypescirculatinginBrazilandworldwide.

Currently,differentvirustypesandputativenewtypesofBPVhave beenidentifiedinBrazil(Lindseyetal.,2009;Meloetal.,2014;

Araldietal.,2014).

Itwasobservedco-infectioninfiveanimals.Althoughfew stud-ieshavereportedtheco-infection,similarresultswereobservedby

Lindseyetal.(2009),Araldietal.(2013)andCarvalhoetal.(2013). Theinfectionwithmorethanonevirustypedemandsinvestments invaccineswithabroadspectrumofcoverage,particularlyrelated tothemostprevalentBPVstypes(NichollsandStanley,2000).

Since thedevelopmentof ultracentrifugationtechnique, this hasfrequentlybeenusedforvirusisolationofPapillomavirus viri-onswithgradientofcesiumchloride(CsCl),plusacushionof40% sucrose(Bujard,1967;Favre,1975;Bakeret al.,1991;Vanslyke etal.,1993;Zhouetal.,1995).

Theultracentrifugationpromotesacompressionofsolutions, resultingina continuousincreaseofdensity ofsolutionsinthe directionofcentrifugalforce(Meselsonetal.,1957).This condi-tionallowstheisolationof BPVvirions, witha buoyantdensity of1.34g/mlofCsClandemptyparticles(composedonlyby cap-sidswithoutgeneticmaterial)withabuoyantdensityof1.29g/ml (LancasterandOlson,1982).

(5)

Theuseofthis novelmethod,resultsofadaptationof those describedpreviouslyintheliterature,allowedtheisolationof BPV-2satisfactorily. Wedidnotobtainenoughsamples,interms of weight,of othertypes identifiedin this study(BPV-3, 5,9 and BR/UEL2),astheisolationtechniquerequiresatleast40gof papil-lomainfectedwithonlyonevirustype.

Theresultsofelectronictransmissionmicroscopyrevealedthe efficacyof thenovelmethodologytoBPVisolation,indicating a highnumberofviralparticles,withapproximately55nm,which correspondstothediameterofBPV(Fig.2).

Themethodpresentedinthisworkshowedtobelesslaborious thanthatreportedinpreviousstudiesthatemployedtheuseofCsCl gradientplusacushionofsucrose,whilethatprovedeffectivein theisolationofBPVvirions.Anotheradvantageobtainedfromthe useofthismethodwastoobtainasingleband,comprisingwhole virusBPVparticles,withoutformingasecondbandcomposedof DNAwithoutviralcapsids.Theformationofthissecondband rep-resentedoneofthedifficultiesreportedintheliterature,because itreducesthenumberofvirionsisolated(Favre,1975).

Otherchangewasthereplacementofthesarcosil,anionic sur-factantderived fromsarcosine bysodiumdodecylsulfate(SDS). Thereplacementwasdoneinviewoftheirsimilarpropertiesand theirrelativelyclosemolecularweights,being293.38g/molofthe sarcosiland288.38g/moloftheSDS.Thus,theSDSisverycommon reagentanditsuseallowedtheBPVisolation.

Themethodproposedinthispresentworkcanbeusedwith equalsuccessforisolationofanotherpapillomaviruses,including theHPV.Underthisview,theisolationofspecifictypesofBPVis criticaltoimmunechallenge,allowingthevalidationofvaccine productsthroughinvivoassaysandtheguaranteeofthe immuno-genicefficacyofcandidateproductstoentranceintoamarket.

Acknowledgements

TheauthorsthanktoDr.GérardOrthfortheacademicsupport, the Ministério de Ciência, Tecnologia e Inovação (MCTI), Con-selhoNacionaldeDesenvolvimentoCientíficoeTecnológico(CNPq) (#554816/2006-7and#402539/2011-7),FundaçãoButantanand Coordenação de Aperfeiçoamentode Pessoal de Nível Superior (CAPES)forfinancialsupport.CarolinadaPazSabinoforher edi-torialsupport.

References

Alberti,A.,Pirino,S.,Pintore,F.,Addis,M.F.,Chessa,B.,Cacciotto,C.,Cubeddu,T., Anfossi,A.,Benenati,G.,Coradduzza,E.,Lecis,R.,Antuofermo,E.,Carcangiu,L., Pittau,M.,2010.Ovisariespapillomavirus3:aprototypeofanovelgenusin thefamilypapillomaviridaeassociatedwithovinesquamouscellcarcinoma. Virology407,352–359.

Ali,A.,Roossinck,M.J.,2007.Rapidandefficientpurificationofcowpeachlorotic mottlevirusbysucrosecushionultracentrifugation.J. Virol.Methods 141, 84–86.

Araldi,R.P.,Carvalho,R.F.,Melo,T.C.,Diniz,N.S.P.,Ana,T.A.S.,2014.Bovine papillo-mavirusinbeefcattle:firstdescriptionofBPV-12andputativetypeBAPV8in Brazil.Genet.Mol.Res.13,5644–5653.

Araldi,R.P.,Melo,T.C.,Diniz,N.,Carvalho,R.F.,Bec¸ak,W.,Stocco,R.C.,2013.Bovine papillomavirusclastogeniceffectanalyzedincometassay.BiomedRes.Int.2013, 1–7.

Baker,T.S.,Newcomb,W.W.,Olson,N.H.,Cowsert,L.M.,Olson,C.,Brown,J.C., 1991.Structuresofbovineandhumanpapillomaviruses.Analysisby cryoelec-tronmicroscopyandthree-dimensionalimagereconstruction.Biophys.J.60, 1445–1456.

Bam,J.,Kumar,P.,Leishangthem,G.D.,Saikia,A.,Somvanshi,R.,2013.Spontaneous cutaneouspapillomatosisinyaksanddetectionandquantificationofbovine papillomavirus-1and-2.Transbound.Emerg.Dis.60,475–480.

Bujard,H.,1967.Studiesoncirculardeoxyribonucleicacid.J.Virol.1,1135–1138. Campo,M.,1997.Vaccinationagainstpapillomavirusincattle.Clin.Dermatol.15,

275–283.

Campos,S.R.C.,Melo,T.C.,Assaf,S.,Araldi,R.P.,Mazzuchelli-de-Souza,J.,Sircili, M.P.,Carvalho,R.F.,Roperto,F.,Bec¸ak,W.,Stocco,R.C.,2013.Chromosome aberrationsincellsinfectedwithbovinepapillomavirus:comparingcutaneous

papilloma,esophaguspapilloma,andurinarybladderlesioncells.ISRNOncol. 2013,910849.

Carvalho,R.F.,Sakata,S.T.,Giovanni,D.N.S.,Mori,E.,Brandão,P.E.,Richtzenhain, L.J.,Pozzi,C.R.,Arcaro,J.R.P.,Miranda,M.S.,Mazzuchelli-de-Souza,J.,Melo,T.C., Comenale,G.,Assaf,S.L.M.R.,Bec¸ak,W.,Stocco,R.C.,2013.Bovinepapillomavirus inBrazil:detectionofcoinfectionofunusualtypesbyaPCR-RFLPmethod. BiomedRes.Int.2013,270898.

Carvalho,C.,DeFreitas,A.C.,DeBrunner,O.,DePindamonhangaba,F.,2003.Bovine papillomavirustype2inreproductivetractandgametesofslaughteredbovine females.Braz.J.Microbiol.34,82–84.

Catroxo,M.,Martins,A.,Petrella,S.,Souza,F.,Nastari,B.,2013.Ultrastructuralstudy ofbovinepapillomavirusduringoutbreaksinBrazil.Int.J.Morphol.31,777–784. Chambers,G.,Ellsmore,V.,O’Brien,P.,Reid,S.,Love,S.,Campo,M.,Nasir,L.,2003. Associationofbovinepapillomaviruswiththeequinesarcoid.J.Gen.Virol.84, 1055–1062.

Claus,M.P.,Lunardi,M.,Alfieri,A.F.,Sartori,D.,Fungaro,H.P.,Alfieri,A.A.P.,Lunardi, A.C.M.,Alfieri,M.,Sartori,A.F.,Fungaro,D.M.H.P.,2009.Identificationofthe recentlydescribednewtypeofbovinepapillomavirus(BPV-8)inaBrazilian beefcattleherd1.Pesqui.Vet.Bras.29,25–28.

DeVilliers,E.-M.,2013.Cross-roadsintheclassificationofpapillomaviruses. Virol-ogy445,2–10.

DeVilliers,E.-M.,Fauquet,C.,Broker,T.R.,Bernard,H.-U.,zurHausen,H.,2004. Classificationofpapillomaviruses.Virology324,17–27.

Favre,M.,1975.Structuralpolypeptidesofrabbit,bovine, andhuman papillo-maviruses.J.Virol.15,1239–1247.

Favre,M.,Breitburd,F.,Croissanr,O.,Orth,G.,1974.Hemagglutinatingactivityof bovinepapillomavirus.Virology578,572–578.

Freitas,A.C.,DeCarvalho,C.,DeBrunner,O.,Birgel-Junior,E.H.,Maria,A.,Paiva, M.,Benesi,F.J.,Gregory,L.,Bec¸ak,W.,DeCassia,R.,2003.ViralDNAsequences inperipheralbloodandverticaltransmissionofthevirus:adiscussionabout BPV-1.Braz.J.Microbiol.34,76–78.

Góes,L.G.B.,deFreitas,A.C.,Ferraz,O.P.,Rieger,T.T.,DosSantos,J.F.,Pereira,A., Bec¸ak,W.,Lindsey,C.J.,deCassiaStocco,R.,2008.Bovinepapillomavirustype 4L1genetransfectioninadrosophilaS2cellexpressionsystem:absenceofL1 proteinexpression.Braz.J.Microbiol.39,1–4.

Hartl,B.,Hainisch,E.K.,Shafti-Keramat,S.,Kirnbauer,R.,Corteggio,A.,Borzacchiello, G.,Tober,R.,Kainzbauer,C.,Pratscher,B.,Brandt,S.,2011.Inoculationofyoung horseswithbovinepapillomavirustype1virionsleadstoearlyinfectionof PBMCspriortopseudo-sarcoidformation.J.Gen.Virol.92,2437–2445. Hatama,S.,Nobumoto,K.,Kanno,T.,2008.Genomicandphylogeneticanalysisoftwo

novelbovinepapillomaviruses,BPV-9andBPV-10.J.Gen.Virol.89,158–163. Karger,A.,Bettin,B.,Granzow,H.,Mettenleiter,T.C.,1998.Simpleandrapid

purifica-tionofalphaherpesvirusesbychromatographyonacationexchangemembrane. J.Virol.Methods70,219–224.

Kidney,B.A.,Berrocal,A.,2008.Sarcoidsintwocaptivetapirs(Tapirusbairdii): clin-ical,pathologicalandmolecularstudy.Vet.Dermatol.19,380–384.

Kirnbauer,R.,Chandrachud,L.M.,O’Neil,W.,Wagner,E.,Grindlay,G.,Armstrong, A.,McGrarvie,G.,Schiller,J.,Lowy,D.,Campo,M.,1996.Virus-likeparticlesof bovinepapillomavirustype4inprophylacticandtherapeuticimmunization. Virology44,37–44.

Lancaster,W.D., Olson,C.,1982. Animal papillomaviruses.Microbiol. Rev. 46, 191–207.

Larsen,P.M.,Storgaard,L.,Fey,S.J.,1987.Proteinspresentinbovinepapillomavirus particles.J.Virol.61,3596–3601.

Leto,M.,Santos-Júnior,G.,Porro,A.,Tomimori,J.,2011.Humanpapillomavirus infec-tion:etiopathogenesis,molecularbiologyandclinicalmanifestations.An.Bras. Dermat.86,306–317.

Lindsey,C.L.,Almeida,M.E.,Vicari,C.F.,Carvalho,C.,Yaguiu,a,Freitas,aC.,Bec¸ak, W.,Stocco,R.C.,2009.BovinepapillomavirusDNAinmilk,blood,urine,semen, andspermatozoaofbovinepapillomavirus-infectedanimals.Genet.Mol.Res.8, 310–318.

Liu,W.J.,Gissmann,L.,Sun,X.Y.,Kanjanahaluethai,A.,Müller,M.,Doorbar,J.,Zhou,J., 1997.SequenceclosetotheN-terminusofL2proteinisdisplayedonthesurface ofbovinepapillomavirustype1virions.Virology227,474–483.

Löhrdr,C.,Juan-sallésd,C.,Rosas-Rosas,A.,García,A.,Garnerd,M.,Teifke,J.,2005. Sarcoidsincaptivezebras(Equusburchellii):associationwithbovine papillo-mavirustype1infection.J.ZooWildl.Med.36,74–81.

Lunardi,M.,deAlcântara,B.K.,Otonel,R.A.A.,Rodrigues,W.B.,Alfieri,A.F.,Alfieri, A.A.,2013.Bovinepapillomavirustype13DNAinequinesarcoids.J.Clin. Micro-biol.51,2167–2171.

Maeda,Y.,Shibahara,T.,Wada,Y.,Kadota,K.,Kanno,T.,Uchida,I.,Hatama,S.,2007. Anoutbreakofteatpapillomatosisincattlecausedbybovinepapillomavirus (BPV)type6andunclassifiedBPVs.Vet.Microbiol.121,242–248.

Martens,A.,DeMoor,A.,Demeulemeester,J.,Ducatelle,R.,2000. Histopatholog-icalcharacteristicsoffiveclinicaltypesofequinesarcoid.Res.Vet.Sci.69, 295–300.

Mazzuchelli-de-Souza,J.,Carvalho,R.F.,Ruiz,R.M.,Melo,T.C.,Araldi,R.P.,Carvalho, E.,Thompson,C.E.,Sircili,M.P.,Bec¸ak,W.,Stocco,R.C.,2013.Expressionandin SilicoanalysisoftherecombinantbovinepapillomavirusE6proteinasamodel forviraloncoproteinsstudies.BiomedRes.Int.2013,421398.

McBride,A.A.,Sakakibara,N.,Stepp,W.H.,Jang,M.K.,2012.Hitchhikingonhost chro-matin:howpapillomavirusespersist.Biochim.Biophys.Acta1819,820–825. Meinke,W.,Meinke,G.C.,1981.Isolationandcharacterizationofthemajorcapsid

proteinofbovinepapillomavirustype1.J.Gen.Virol.52,15–24.

(6)

Phylogeneticclassificationandclinicalaspectsofanewputative Deltapapillo-mavirusassociatedwithskinlesionsincattle.Genet.Mol.Res.13,2458–2469. Meselson,M.,Stahl,F.,Vinograd,J.,1957.Equilibriumsedimentationof

macro-moleculesindensitygradients.Proc.Natl.Acad.Sci.U.S.A.43,581–588. Nicholls,P.K.,Stanley,M.A.,2000.Theimmunologyofanimalpapillomaviruses.Vet.

Immunol.Immunopathol.73,101–127.

Orth,G.,Favre,I.M.,Croissant,O.,1977.Characterizationofanewtypeofhuman papillomavirusthatcausesskinwartscharacterizationofanewtypeofhuman papillomavirusthatcausesskinwarts.J.Virol.24,108.

Pangty,K.,Singh,S.,Goswami,R.,Saikumar,G.,Somvanshi,R.,2010.Detectionof BPV-1and-2andquantificationofBPV-1byreal-timePCRincutaneouswarts incattleandbuffaloes.Transbound.Emerg.Dis.57,185–196.

Roperto,S.,Brun,R.,Paolini,F.,Urraro,C.,Russo,V.,Borzacchiello,G.,Pagnini,U., Raso,C.,Rizzo,C.,Roperto,F.,Venuti,A.,2008.Detectionofbovine papillo-mavirustype2intheperipheralbloodofcattlewithurinarybladdertumours: possiblebiologicalrole.J.Gen.Virol.89,3027–3033.

Shafti-keramat,S.,Handisurya, A.,Meneguzzi, G.,Slupetzky, K., Kirnbauer,R., Kriehuber,E.,2003.Differentheparansulfateproteoglycansserveascellular receptorsforhumanpapillomaviruses.J.Virol.77(24),13125–13135. StoccodosSantos,R.C.,Lindsey,C.J.,Ferraz,O.P.,Pinto,J.R.,Mirandola,R.S.,Benesi,

F.J.,Birgel,E.H.,Pereira,C.A.,Bec¸ak,W.,1998.Bovinepapillomavirus transmis-sionandchromosomalaberrations:anexperimentalmodel.J.Gen.Virol.79(Pt 9),2127–2135.

Turk,N., ˇZupanˇci ć, ˇZ.,Stareˇsina,V.,Kovaˇc,S.,Babi ć,T.,Kreszinger,M., Ćuri ć,S., Barbi ć,L.,Milas,Z.,2005.Severebovinepapillomatosis:detectionofbovine

papillomavirusintumourtissueandefficacyoftreatmentusingautogenous vaccineandparammunityinducer.Vet.Arh.75,391–397.

VanDyk,E.,Bosman,A.,vanWilpe,E.,Willians,J.,Bengis,R.,vanHeerden,J., Venter,E., 2011. Detection and characterisationofpapillomavirus inskin lesionsofgiraffeandsableantelopeinSouthAfrica.J.S.Afr.Vet.Assoc.82, 80–85.

Vanslyke,J.K.,Lee,P.,Wilson,E.M.,Hruby,D.E.,1993.Isolationandanalysisof vac-ciniavirusprevirions.VirusGenes7,311–324.

Wang,J.W.,Roden,R.B.S.,2013.L2,theminorcapsidproteinofpapillomavirus. Virology445,175–186.

Wang,R.,Wang,J.,Li,J.,Wang,Y.,Xie,Z.,An,L.,2007.Comparisonoftwogelfiltration chromatographicmethodsforthepurificationoflilysymptomlessvirus.J.Virol. Methods139,125–131.

Wobeser,B.K.,Hill,J.E.,Jackson,M.L.,Kidney,B.A.,Mayer,M.N.,Townsend,H.G.G., Allen,A.L.,2012.Localizationofbovinepapillomavirusinequinesarcoidsand inflammatoryskinconditionsofhorsesusinglasermicrodissectionandtwo formsofDNAamplification.J.Vet.Diagn.Invest.24,32–41.

Yaguiu,A.,Dagli,M.L.,BirgelJr.,E.H.,Reis,B.C.A.A.,2008.Simultaneouspresenceof bovinepapillomavirusandbovineleukemiavirusindifferentbovinetissues: insituhybridizationandcytogeneticanalysis.Genet.Mol.Res.7,487–497. Zheng,Z.M.,Baker,C.,2006.Papillomavirusgenomestructure,expression,and

post-transcriptionalregulation.Front.Biosci.11,2286–2302.