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RESEARCHOpenAccessHumanPapillomavirus(HPV)genotype18variantsinpatientswithclinicalmanifestationsofHPVrelatedinfectionsinBilbao,SpainSaraLArroyo1,MirenBasaras1*,ElixabeteArrese1,SilviaHernáez2,DanielAndía3,ValentínEsteban2,KoldoGarcia-Etxebarria4,BegoaMJugo4andRamónCisterna1,2AbstractBackground:Humanpapillomavirus(HPV)variantsdifferintheirbiologicalandchemicalproperties,andtherefore,maypresentdifferencesinpathogenicity.
MostauthorsclassifiedvariantsbasedonthephylogeneticanalysisofL1region.
Nevertheless,recombinationinHPVsamplesisbecomingausualfindingandthus,characterizinggeneticvariabilityinotherregionsshouldbeessential.
Objectives:WeaimedtocharacterizethegeneticvariabilityofHPV18in5genomicregions:E6,E7,E4,L1andtheUpstreamRegulatoryRegion(URR),workingwithbothsingleinfectionandmultipleHPVinfectionsamples.
Furthermore,weaimedtoassesstheprevalenceofHPV18variantsinourregionandlookforpossibleexistenceofrecombinationaswellasanalyzetherelationshipbetweenthesevariantsandthetypeoflesion.
Methods:From2007to2010,ClinicalMicrobiologyandInfectionControlDepartmentanalyzed44sampleswhichwerepositiveforHPV18.
GeneticvariabilitywasdeterminedinPCRproductsandvariantswereassignedtoEuropean,Asian-amerindianorAfricanlineage.
Recombinationandassociationofvariantswithdifferenttypesoflesionwasstudied.
Results:Geneticanalysisoftheregionsrevealedatotalof56nucleotidevariations.
European,AfricanandAsian-amerindianvariantswerefoundin25/44(56.
8%),10/44(22.
7%)and5/44(11.
4%)samples,respectively.
Wedetectedthepresenceofrecombinantvariantsin2/44(4.
5%)cases.
Samplestakenfromhigh-gradesquamousintraepitheliallesions(H-SIL)onlypresentedvariantswithspecific-africansubstitutions.
Conclusions:MultipleHPVinfection,non-europeanHPVvariantsprevalenceandexistenceofrecombinationareconsideredriskfactorsforHPVpersistenceandprogressionofintraepithelialabnormalities,andtherefore,shouldbetakenintoconsiderationinordertohelptodesignandoptimizediagnosticsprotocolsaswellasimproveepidemiologicstudies.
OurstudyisoneofthefewstudiesinSpainwhichanalysesthegeneticvariabilityofHPV18andweshowedtheimportanceofcharacterizingmorethanonegenomicregioninordertodetectrecombinationandclassifyHPVvariantsproperly.
Keywords:Humanpapillomavirusinfection,Genotype18,Variants,Recombination,Multipleinfection*Correspondence:miren.
basaras@ehu.
es1Immunology,MicrobiologyandParasitologyDepartment,UniversityofBasqueCountry,Leioa48940,SpainFulllistofauthorinformationisavailableattheendofthearticle2012Arroyoetal.
;licenseeBioMedCentralLtd.
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Arroyoetal.
VirologyJournal2012,9:258http://www.
virologyj.
com/content/9/1/258BackgroundBasedontheepidemiologicclassificationintermsoftheirrisktoinducecervicalcancer,humanpapilloma-viruses(HPV)canbedividedinto3groups:"high-risk"genotypesassociatedwithagreaterriskofdevelopingcancer,"low-risk"genotypesassociatedwithlowgradecellchangesorbenignepitheliumproliferationsinthegenitalarea,butnotwithcancer,and"probablehigh-risk"genotypesfromwhichthereisnotenoughdataabouttheirrelationshipwithcervicalcancertoclassifythem[1].
About15genotypesareclassifiedashigh-risktypes,andtwoofthem(16and18)causeover70%ofallcervicalcancercases[2,3].
Nucleotidevariabilityofthesegenotypeshasbeenlargelystudiedanddifferentmolecularvariantsweredescribed[4,5].
Thesevariantsdifferintheirbiologicalandchemicalproperties[6–8],andtherefore,maybecomeanimportantriskfactorincervicalcancerduetopossibledifferencesinpathogenicity.
Mostauthorsclassifiedvariantsbasedonthephylogene-ticanalysisofonegenomicregionnucleotidevariations[9].
Nevertheless,somepublicationshaveconfirmedthepresenceofrecombinationinHPVsamples[10,11].
ThiseventmayoccurduetoahomologousrecombinationortoarepeatedinfectionofthesameHPVgenotypebutdif-ferentvariantanditismoreoftenfoundsincecoinfectionswithmorethanoneHPVtypearebecomingausualfind-ing[12–14].
Therefore,itshouldbeessentialtodetermineHPVvariantsanalyzingdifferentgenomicregionsandmultipleinfections.
ThereareveryfewepidemiologicalnationalstudiesinSpainandallofthemrefertoHPV16whichisthemostinvestigatedHPVtypeworldwide.
However,thereisnopreviousnationalworkrelatedtoHPV18nucleotidevariability,whichisthesecondmostprevalentHPVgenotypefoundincervicalcancer.
Theaimofthepresentstudywas:i)tocharacterizethegeneticvariabilityofHPV18in5genomicregions:E6,E7,E4,L1andtheUpstreamRegulatoryRegion(URR),workingwithbothsingleinfectionandmultipleHPVinfectionsamples,ii)assesstheprevalenceofHPV18variantsinourregionandlookforexistenceofre-combination,andiii)analyzetherelationshipbetweenvariantsandtypesoflesion.
ResultsSamplescollectedSince2007to2010,atotalof1085positivesamplesforHPVwerereceivedandanalyzed.
HPV18wasdetectedin65samples(6%).
Forty-fourpatientsconsentedtohavetheirsamplesanalyzedandstudied,sothisstudywasbasedontheirsamples:10singleHPVinfections(22.
7%)and34multipleHPVinfections(77.
3%).
WewereabletoamplifyHPVDNAin43/44samplesforE6region,41/44forE7,35/44forE4,43/44forL1regionand44/44forURRregion.
AllPCRproductsweresequencedandsequencesfromeachregionweresubmittedtoGenBank.
NucleotidevariationsVariantdistributionwasdeterminedthroughE6,E7,E4,L1andURRsequences.
Geneticanalysisoftheregionsrevealedatotalof56nucleotidevariations(Figure1).
IntheE6geneninenucleotidevariationsweredetected.
SixofthemwerespecifictotheAfricanlineage:T317C(6/10Africanvariants),T251C(9/10),A548G,G266AandG374A(presentinallAfricaniso-lates)andC342T(5/10Africanvariants)whichleadtoanon-synonymousaminoacidalterationHis/Tyr.
Anon-synonymoussubstitutionT318C(Tyr/His)wasfoundtobespecifictotheEuropeanlineage(2/25Europeaniso-lates),whilethesynonymoussubstitutionC549Awasdetectedamongthethreedifferentbranches(35/43sequencedE6amplimers).
Inourstudy,C287GwasobservedinallHPV18isolates.
E7genegeneticvariabilityanalysisrevealedfivenucleotidessubstitutions.
ThreenucleotidevariationswerespecificfortheAfricanlineage:C665T(3/10Africanvariants),C593T(His/Tyr),C640CandT864G(Asn/Ser).
AllofthembutC665TandT864GwerepresentinallAfricanvariants.
Onesynonymoussubstitution(C751T)wasdetectedinbothEuropeanandAsian-amerindianisolates(26/34non-Africanvariants).
E4geneanalysispresentedmostnucleotidevariations(17substitutions)andalmosthalfofthem(8/17)leadtoaminoacidchanges.
AllAfricanvariantsshowed4non-synonymoussubstitutions(C3558AHis/Gln,C3578TSer/Leu,A3586CSer/ArgandT3593GIle/Ser),onesynonymousvariation(T3534C)andadeletionof6aminoacids(3627–3632).
Europeanvariantsshowed4specificnonsynonymoussubstitutionsG3482A(Ser/Asn,3/25isolates),T3563A(Leu/Gln,4/25),C3617T(Ser/Leu,4/25)andC3630G(His/Gln,allEuropeaniso-lates).
Twonon-synonymoussubstitutionswerealsodetectedintwoEuropeanisolates:T3492AandC3615T.
L1geneandURRsequenceanalysisdemonstratedthepresenceofsubstitutionsC6842GandT7592Cinallourisolates.
MostnucleotidevariationsfoundinourstudyhavebeenalreadydescribedinliteratureexceptforT318C,C665T,C3615T,C3617T,G6897A,G6993A,A7000T/C,T7001C,T7007GandT7765G.
Onlysubstitutionsinpositions318and3617leadtoaminoacidchanges(Tir/HisandSer/Leu,respectively).
T318Csubstitutionwaspresentin2EuropeanisolateswhereasC3617Tnucleo-tidechangewasnotspecifictoanylineageandwasdetectedin5samples.
Arroyoetal.
VirologyJournal2012,9:258Page2of8http://www.
virologyj.
com/content/9/1/258HPVvariantsInourstudy,thepredominantvariantfoundwastheEuropean(25/44samples)followedbytheAfrican(10/44)andtheAsian-amerindianvariants(5/44).
PhylogeneticanalysisoftheallregionsstudiedshowedthatEuropeanandAsian-amerindianlineagesformedcloselyrelatednodesaswellasamaximalnucleotidediver-sitybetweenAfricanandnon-Africanvariants(Figure2)(Additionalfile1).
Isolateswhichshowednucleotidediversityfromthethreebranches(European,Asian-amerindianandAfrican)-LSM3,LSCM,LSCE,LSMK1,LSCR,LSMH1,LSC5,LSM7andLSCB–wereanalyzedforpossiblerecombina-tion(Table1).
PhylogenetictreeswereconstructedforeachsequencedregionfromtheseisolatesandwefoundthatsamplesLSCR,LSMH1,LSC5andLSM7belongedtotheEuropeanbranchinallregionsandtherefore,wereclassifiedasEuropeans.
IsolateLSCBbelongedtotheEuropeanlineageinallregionsbutinE6(Asian-amerindian)duetothelackofonenucleotidesubstitution(C549A).
ThissamplewasalsoclassifiedasEuropean.
SamplesLSM3,LSCM,LSCE,LSMK1belongedtotheAfricanbranchinsomeregionsbutwereclassifiedasEuropeaninothers(Table1).
RDP[15],Maxchi[16]andChimaerawereusedforthedetectionofrecombinationinthese9samplesandonly2ofthemwerefoundtoberecombinant,onesingle(LSMK1)andonemultipleHPVinfection(LCE)(Table1).
LSCMandLSM3wereclassifiedasXvariants(unknown).
Variants,typeoflesionandinfectiontypeOutof44samples,32wereclassifiedbypathologistsasnormal(nolesionwasfound),9sampleswerediagnosedFigure1NucleotidesequencevariationsamongHPVisolates.
NumberingreferstothefirstnucleotideoftheHPV18referencegenome(accessionnumberNC001357).
EachrowindicatestheisolateidentificationandthePCRnucleotidesequencealignmentcomparedtothereference.
IsolatesEF202143-EF202155areHPV18knownvariantsequenceswhichbelongtoAsian-amerindianlineage,AfricanandEuropeanlineage.
Nucleotidepositionswhereasubstitutionleadstoachangeofaminoacidarehighlightedingray.
Inthefirstcolumn,samplesthatarehighlightedingraycorrespondtosingleHPVinfectionsamples,whereasnothighlightedsamplescorrespondtomultipleHPVinfections.
Dashesindicateabsenceofnucleotidesequencedata.
Recombinantvariantsareindicatedbyanasterisk.
Arroyoetal.
VirologyJournal2012,9:258Page3of8http://www.
virologyj.
com/content/9/1/258asL-SILwhilepresenceofH-SILwasdetectedin3specimens.
HighgradelesionsonlypresentedAfricanvariants(2/3isolates,66.
7%)andvariantsthatpresentedbothAfricanandEuropeansubstitutions(1/3isolates,33.
3%)whereasmostEuropeanandAsian-amerindianvariantsweredetectedinnegativecytologies(Table2).
Presenceoflesionsassociatedwithnon-Europeanva-riantswasfoundtobestatisticallysignificant(p=0.
01053).
Nevertheless,therewasnotastatisticallysignificant0.
2LSCLHPV18Qv16302LSMH1LSCPLSCWLSCS1HPV18Qv04924LSM6LSCT1LSCBLSCALSC5LSCM1HPV18Qv17955HPV18Qv02876HPV18Qv21751LSCHLSCXLSCSHPV18Qv15957LSCILSMG1LSCR1LSCKHPV18Qv15586LSCE1LSM4LSM7LSCFLSCH1LSCRLSCGHPV18Qv17052LSCJLSCDLSCJ1LSCP1LSM2LSCA1HPV18Qv03132LSC9HPV18Qv03814HPV18Qv17199LSMK1LSCI1LSCQ1LSM3LSCCLSCC1LSCYLSCU1LSCMHPV18Qv16306HPV18Qv21444LSMF1LSCELSM1LSML110.
8278110.
8414111111111110.
93931111111111110.
993410.
96530.
99340.
89321110.
618410.
9993110.
646710.
72431110.
8540.
526910.
6777110.
7244111110.
6049110.
52520.
58030.
6657111111110.
8302111Figure2PhylogenetictreeoftheHPV18isolates.
E6,E7,E4,L1andURRnucleotidesequencesofisolatesusingtheBayesianinferencemethodimplementedinMrBayes3.
1.
IsolatesEF202143-EF202155areincludedasHPV18referencevariantsequenceswhichbelongtoAsian-amerindianlineage,AfricanandEuropeanlineage.
Table1EvidenceforrecombinantsamplesSample\GeneE6E7E4L1URRPrograminferenceLSM3AfAfXXENorecombinationdetected.
LSCEAfAfEEEMaxchidetectedrecombination.
(LSCMisoneofthedonors)LSMK1AsAEAfEAfMaxchidetectedrecombination.
(LSCIandLSCR1arethedonors)LSCREEEEENorecombinationdetected.
LSMH1EEEEENorecombinationdetected.
LSC5EEEEENorecombinationdetected.
LSM7EEEEENorecombinationdetected.
LSCBAsAEEEENorecombinationdetected.
LSCMAfEAfAfENorecombinationdetected.
Isolatesanalyzedforpossiblerecombination.
PhylogenetictreeswereconstructedforeachsequencedregionandisolateswereclassifiedasAsian-amerindian(AsA),European(E)orAfrican(Af)variants.
RDP,MaxchiandChimaerawereusedforthedetectionofrecombination.
URR:Upstreamregulatoryregion,X:absenceofdata.
Arroyoetal.
VirologyJournal2012,9:258Page4of8http://www.
virologyj.
com/content/9/1/258associationbetweentypeofinfections(singlevsmultiple)andpresenceoflesions(p=0.
18078).
DiscussionTherearealmostnoepidemiologicstudiesaboutHPV18variantscarriedoutinSpainandeventhoughna-tionalHPVprevalenceislow,itcannotbeforgottenthatthisgenotypetogetherwithgenotype16cause70%ofcervicalcancercases.
ManyauthorsconfirmthatdistributionofHPVvariantsisrelatedtogeographicorracedistribution[6,17]andtherefore,SpainshouldexpectpredominanceofEuropeanvariant,followedbyAfricanandAsian-amerindianvariants.
Ourresultsshowconcordancewiththisstating:25Europeanvariants(56.
8%),10African(22.
7%)and5Asian-amerindianvariants(11.
4%).
HPV18hasbeenassociatedwithbothrecurrentlesionswithverybadclinicalprognosis[18]andbenignlesions[19].
Thisfactmayreflecttheoncogenicpotentialdiffer-enceamongvariants.
Hechtetal[20]identifiedaHPV18variantwithloweroncogenicpotentialduetoitsabsenceincervicalcancerbutpresencein40%ofintraepitheliallesions.
Villaetal[21]suggestedthatnon-EuropeanHPV18variantspersistedmorefrequentlyandweremoreassociatedwithpre-invasivelesions.
Sincethen,moststudiesconfirmthatdifferentvariantsofthesamegeno-typedifferintheirpathogeniccharacteristicsandthere-fore,nucleotidesubstitutionsmayplayanimportantrole.
OurstudyresultsshowconcordancewiththesestatementsasAfricanvariantsandvariantswheremostofspecific-africansubstitutionsweredetectedweretheonlytypeofvariantsdetectedinH-SIL.
Mostnucleotidechangesreportedinourstudyhavebeenpreviouslydescribedandsomeofthemareofpar-ticularimportance.
InURR,themutationA41GislocatedintheSp-1bindingsiteandisolateswiththisnucleotidevariationhaveshowntohaveanincreasedtranscriptionalactivity[22].
Variationsinpositions41and104modulateSp1andYY1activitiesandareassociatedtoahigherac-tivityoftheE6/E7promoter.
PatientswithT104Csubsti-tutionarelesslikelytopresenttumourrecurrence[23].
OthernucleotidechangeslikeT7651C,A7658CandC7726Talsolaywithintranscriptionfactorbindingsites.
SubstitutionsC287G,C6842GandT7592Cwerefoundinallourisolates.
VariationC6842Ghasbeenpreviouslyreportedaserrorintheoriginalsequence[24],andH.
Arias–Pulidoetal.
sequencedtheoriginalreferenceHPV18plasmid(providedbyE-MdeVilliers,DeutschesKrebs-forschungzentrum,Germany)andobservedthesubstitu-tionT7592C[25],soitisconsideredasasequencingerrorintheoriginalHPV18referencesequencereport.
Furthermore,ten"new"nucleotidevariationshavebeendetectedandtwoofthemwerenon-synonymousandleadtoaminoacidchanges(T318CandC3617T,Tir/HisandSer/Leu,respectively).
KnowledgeonHPVvariantsandtheirnucleotidevariabi-lityisessentialforthreemainreasons:i)nucleotidevaria-tionsmayinterferewiththeviraloncogenicpotential,ii)hostcellularimmuneresponsecanbedifferentwhenTable2Humanpapillomavirus18variantsvstypeoflesionVariants\TypeofLesionNegL-SILH-SILEuropean71.
0%3.
3%0%Asian-amerindian12.
9%1.
1%0%African12.
9%4.
4%100%Recombinant3.
2%1.
1%0%Neg:nolesion,L-SIL:low-gradesquamousintrapehiteliallesion,H-SIL:high-gradesquamousintraephiteliallesion.
Table3PolymerasechainreactioncharacteristicsforHumanpapillomavirus18Primersequence(50–30)AnnealingTemp/CyclesNucleotidesamplified*AmpliconsizeE6FAGTAACCGAAAACGGTCGGGA55°C/40cycles38-491454pbE6RGTTGTGAAATCGTCGTTTTTCAE7FTGAAAAACGACGATTTCACAAC55°C/40cycles470-931462pbE7RACCTTCTGGATCAGCCATTGE4FGTAAAGGAAGGGTACAACACG57°C/35cycles3309-3792484pbE4RCTGTCCAATGCCAGGTGGALCR1FTCGGTTGCCTTTGGCTTAT55°C/40cycles7465-7775311pbLCR1RAAGGGTAGACAGAATGTTGGACA55°C/40cycles7718-163303pbLCR2FGCTAATTGCATACTTGGCTTGLCR2RTCCGTGCACAGATCAGGTAGMY11GCACAGGGTCATAACAATGG55°C/40cycles6558-7012455pbMY09CGTCCAAGGGATATTGATCL1seqACAGTCTCCTGTACCTGGG*PositionnumberingreferstothefirstnucleotideoftheHPV18referencegenome(accessionnumberNC001357).
F:forwardprimer,R:reverseprimer.
Arroyoetal.
VirologyJournal2012,9:258Page5of8http://www.
virologyj.
com/content/9/1/258therearesubstitutionsintheaminoacidsontheviralcap-sidwhichmayberelevantforthevaccination,iii)HPVinfectionswithavariantmaynotgiveimmunologicalpro-tectionagainstasubsequentinfectionwithothervariantofthesamegenotype.
Nowadays,HPVvariantsrecombinationhasalreadybeendescribedanditismoreoftenfoundsincecoinfec-tionwithmorethanoneHPVtypeprevalenceisnotaunusualfinding[10].
Inourstudywedetected2recombinantvariants(4.
5%)whichmighthavebeenmissedorwrongclassifiedifonlyamplifyingonegenomicregion.
Furthermore,non-recombinantsamplesasLSCMorLSM3showedspecific-africansubstitutionsinsomeregions(forex-ampleE6)whereastheywouldbeclassifiedaseuropeanvariantswhenonlyanalyzingnucleotidevariationinURR.
Therefore,characterizingmorethanonegenomicregionmaybeessentialinordertodetectrecombinationandclassifyHPVvariantsproperly.
WeamplifiedURRandE6,E7andL1genesfromatleast93%ofsamples.
However,whencharacterizingE4region,wewereonlyabletoamplify35samples.
E4geneisgenerallydisruptedduringDNAintegrationintothehostgenomeandthisdisruptionmayexplaintheinabil-itytoamplifyE4geneinsomeofoursamples.
Inconclusion,dataandknowledgeongeographicHPVintratypicvariantsdistributionmighthelptoestab-lishadatabaseaboutthediversityandpathogenicityofdifferentHPVvariants,whichmayhelptodesignandoptimizediagnosticsprotocolsinordertoreducethedisease.
MethodsRecruitmentofparticipantsClinicalMicrobiologyandInfectionControlDepartmentatBasurtoUniversityHospital(BasqueCountry,NorthofSpain)analyzedsampleswhichwereremittedfromdifferentHospitalServices,especiallytheConsultationofSexuallyTransmittedDiseasesandtheDepartmentofObstetricsandGynecology,from2007to2010.
AllsampleswerecollectedfrompatientswithclinicalmanifestationsofHPVrelatedinfections.
Lesionswereclassifiedbypathologistsintothreecategories:negative(nolesionwasfound),low-gradesquamousintraepithe-liallesion(L-SIL)orhigh-gradesquamousintraepitheliallesion(H-SIL).
Moleculargenotypingwascarriedoutusing"LinearArrayHPVGenotypingTest"kit(RocheMolecularTable4GeneBankaccessionnumbersforthesequencedisolatesIsolate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able4GeneBankaccessionnumbersforthesequencedisolates(Continued)LSCT1JN416260JN416209XJN416311JN416363LSCU1JN416261JN416210JN416161JN416312JN416364Arroyoetal.
VirologyJournal2012,9:258Page6of8http://www.
virologyj.
com/content/9/1/258Diagnostics).
Inourstudy,weanalyzedpositivesamplesforHPVgenotype18(bothsingleinfectionsandmultipleHPVinfections)frompatientswhohadgivenwritten,informedconsent.
GenomicDNAextractionDNAextractionwasperformedbyQIAampDNAminiKit(Qiagen,Hilden,Germany),accordingtothemanu-facturersinstructions.
ExtractedDNAwaselutedwith200μlAEbufferandstoredat20°Cuntilamplification.
PCRamplificationandsequencingAmplificationofHPVE6,E7,E4genesandtheURRwasperformedusingtype-specificprimersdesignedaccordingtoHPV18genomeprototypesequence(GenBankacces-sionnumberNC001357).
TheURRwasamplifiedusing2primersets.
InordertoamplifyL1region,consensusHPVprimerswereused(Table3).
PCRwasperformedin30μlofreactionmixturecon-taining10*PCRbuffer,25mmol/LMgCl2,25mmol/Lofeachdeoxynucleoside,100pmol/Lofsenseandanti-senseprimer,5μloftemplateDNAand2,5UofTaqDNApolymerase(Qiagen).
Thethermalprogramstartedwithapre-heatof95°Cfor15min,followedby35–40cyclesofsuitableannealingtemperaturewhichdependedontheprimersandfinishedwithafinalextensionat72°Cfor10min(Table3).
PCRproductswereconfirmedbasedonspecificbandsofamplifiedDNApresenceinagarosegel(2%).
After-wards,amplimerswereautomaticallysequencedusingthe"BigDyeTerminatorCycleSequencingkit"(AppliedBio-systems)accordingtothemanufacturersinstructions.
ForE6,E7,E4andURRampliconsthesameforwardspecificprimersasthoseusedinamplificationwerechosenassequencingprimers.
IntheL1region,aspe-cificprimerwasusedinordertosequenceHPV18andnototherHPVtypespresentincasesofmultipleinfec-tion(Table3).
Nucleotidevariations,phylogeneticanalysis:variantsandrecombinationHPVsequenceswerealignedandcomparedtotheHPV18prototypesequencewhichbelongstotheAsian-amerindianlineage(accessionnumberNC001357),usingBioEditSequenceAlignmentEditorv7.
0.
4.
1andClustalW(http://www.
genome.
jp/tools/clustalw/).
Amplificationandsequencingofthesampleswererepeatedtoconfirmnucleotidevariationswhichwerepresentinlessthanthreeisolates.
SequenceswereassignedtoalineageonthebasisoftheirsimilaritytoHPV18knownvariantsequences[26]whichbelongtoAsian-amerindianlineage(GenBankaccessionnumbers:EF202143-EF202146),African(EF202152-EF202155)andEuropeanlineage(EF202147-EF202149,EF202151).
PhylogenetictreeswerebuiltusingtheBayesianinferencemethodimplementedinMrBayes3.
1[27]andthreemethods(allimplementedinRDP3[15])wereusedforthedetectionofrecombination(RDP[15],Maxchi[16]andChimaera)toanalyzeisolateswhichdidnotadjusttotheclusters.
LesionsAssociationoflesionsandvariantsorinfectiontype(singleHPVvsmultipleHPVinfection)wasanalyzed.
Fisherexacttestwasusedforstatisticallysignificantassociation.
GenBankaccessionnumbersThefollowingaretheGenBankaccessionnumbersforallthesequencesusedinthisanalysis.
XindicatesabsenceofnucleotidesequencedataTable4.
EthicalapprovalAllproceduresfollowedwereapprovedbytheappropri-ateEthicsCommiteerelatedtoourinstitutions(BasurtoUniversityHospitalandUniversityofBasqueCountry)andcompliedwiththeguidelinesandethicalstandardsforexperimentalinvestigationwithhumansubjectsofHelsinkiDeclarationof1975,asrevisedin2000.
Allstudyparticipantsprovidedwritten,informedconsent.
AdditionalfileAdditionalfile1:E4,E6,E7,L1,LCR_bay.
Phylogenetictreeswereconstructedforeachregionindividually.
AbbreviationsHPV:Humanpapillomavirus;H-SIL:High-gradesquamousintraepitheliallesion;L-SIL:Low-gradesquamousintraepitheliallesion;SIL:Squamousintraepitheliallesion;URR:Upstreamregulatoryregion.
CompetinginterestsAllauthorsdeclarenopotentialconflictsofinterest.
Authors'contributionsLSAcarriedoutthemolecularstudies,participatedinthesequencealignmentanddraftedthemanuscript.
MBandEAparticipatedinthedesignofthestudyanditscoordinationandhavebeeninvolvedindraftingthemanuscriptandrevisingitcriticallyforimportantintellectualcontent.
SH,DAandVEhavemadesubstantialcontributionstoacquisitionofsamplesanddataandhaverevisedthemanuscriptcritically.
KGEandBMJperformedphylogeneticandstatisticalanalysis.
RCconceivedthestudy,participatedinitsdesignandcoordinationandhasgivenfinalapprovaloftheversiontobepublished.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsAuthorswishtothanktheDepartmentofHealthfromtheBasqueGovernmentforsupportingthisproject[projectnumber2008111058].
Authordetails1Immunology,MicrobiologyandParasitologyDepartment,UniversityofBasqueCountry,Leioa48940,Spain.
2ClinicalMicrobiologyandInfectionControlDepartment,BasurtoUniversityHospital,Bilbao48013,Spain.
3ObstetricsandGynecologyDepartment,BasurtoUniversityHospital,Bilbao48013,Spain.
4Genetics,PhysicalAnthropologyandAnimalPhysiologyDepartment,UniversityofBasqueCountry,Leioa48940,Spain.
Arroyoetal.
VirologyJournal2012,9:258Page7of8http://www.
virologyj.
com/content/9/1/258Received:4June2012Accepted:30October2012Published:2November2012References1.
MuozN,CastellsagueX,deGonzalezAB,GissmannL:HPVintheetiologyofhumancancer.
Vaccine2006,24:1–10.
2.
SmithJS,LindsayL,HootsB,KeysJ,FranceschiS,WinerR,etal:Humanpapillomavirustypedistributionininvasivecervicalcancerandhigh-gradecervicallesions:ameta-analysisupdate.
IntJCancer2007,121:621–632.
3.
MuozN,BoschFX,CastellsaguéX,DíazM,deSanjoseS,HammoudaD,etal:Againstwhichhumanpapillomavirustypesshallwevaccinateandscreentheinternationalperspective.
IntJCancer2004,111:278–285.
4.
YamadaT,ManosMM,PetoJ,GreerCE,MunozN,BoschFX,etal:Humanpapillomavirustype16sequencevariationincervicalcancers:aworldwideperspective.
JVirol1997,71:2463–2472.
5.
OngCK,ChanSY,CampoMS,CampoMS,FujinagaK,Mavromara-NazosP,etal:Evolutionofhumanpapillomavirustype18:anancientphylogeneticrootinAfricaandintratypediversityreflectcoevolutionwithhumanethnicgroups.
JVirol1993,67:6424–6431.
6.
deAraujoSouzaPS,SicheroL,MaciaPC:HPVvariantsandHLApolymorphisms:theroleofvariabilityontheriskofcervicalcancer.
FutureOncol2009,5:359–370.
7.
SicheroL,FerreiraS,TrottierH,Duarte-FrancoE,FerenczyA,FrancoEL,etal:Highgradecervicallesionsarecausedpreferentiallybynon-europeanvariantsofHPVs16and18.
IntJCancer2007,120:1763–1768.
8.
López-SavedraA,González-MayaL,dePonceLeónS,García-CarrancaA,MoharA,LizanoM:FunctionalimplicationofsequencevariationinthelongcontrolregionandE2geneamonghumanpapillomavirustype18variants.
ArchVirol2009,154:747–754.
9.
deVilliersEM,FauquetC,BrokerTR,BernardHU,ZurHH:Classificationofpapillomaviruses.
Virology2004,324:17–27.
10.
AnguloM,Carvajal-RodríguezA:Evidenceofrecombinationwithinhumanalpha-papillomavirus.
VirolJ2007,4:33.
11.
JiangM,XiLF,EdelsteinZR,GallowayDA,OlsemGJ,LinWC,etal:Identificationofrecombinanthumanpapillomavirustype16variants.
Virology2009,394:8–11.
12.
MéndezF,MuozN,PossoH,MolanoM,MorenoV,vandenBruleAJ,etal:Cervicalcoinfectionwithhumanpapillomavirus(HPV)typesandpossibleimplicationsforthepreventionofcervicalcancerbyHPVvaccines.
JInfectDis2005,192:1158–1165.
13.
TinelliA,LeoG,Dell'EderaD,StorelliF,GalanteMM,GuidoM,etal:MolecularmethodsforacorrectdiagnosisofmultipleHPVinfectionsandclinicalimplicationsforvaccine.
IntJGynecolCancer2011,21:545–550.
14.
NielsonCM,HarrisRB,FloresR,AbrahamsenM,PapenfussMR,DunneEF,etal:Multiple-typehumanpapillomavirusinfectioninmaleanogenitalsites:prevalenceandassociatedfactors.
CancerEpidemiolBiomarkersPrev2009,18:1077–1083.
15.
MartinDP,LemeyP,LottM,MoultonV,PosadaD,LefeuvreP:RDP3:aflexibleandfastcomputerprogramforanalyzingrecombination.
Bioinformatics2010,26:2462–2463.
16.
SmithJ:Analyzingthemosaicstructureofgenes.
JMolEvol1992,34:126–129.
17.
XiLF,KiviatNB,HildesheimA,GallowayDA,WheelerCM,HoJ,etal:Humanpapillomavirustype16and18variants:race-relateddistributionandpersistence.
JNatlCancerInst2006,98:1045–1052.
18.
ImSS,WilczynskiSP,BurgeRA,MonkBJ:Earlystagecervicalcancerscontaininghumanpapillomavirustype18DNAhavemorenodalmetastasisanddeeperstromalinvasion.
ClinCancerRes2003,9:4145–4150.
19.
BurgerR,MonkBJ,KurosakiT,Anton-CulverH,VasileySA,BermanML,etal:Humanpapillomavirustype18:associationwithpoorprognosisinearlystagecancer.
JNatlCancerInst1996,88:1361–1368.
20.
HechtJ,KadishA,JiangG,BurkR:Geneticcharacterizationofthehumanpapillomavirus(HPV)18E2geneinclinicalspecimenssuggeststhepresenceofasubtypewithdecreasedoncogenicpotential.
IntJCancer1995,60:369–376.
21.
VillaLL,SicheroL,RahalP,CaballeroO,FerenczyA,RohanT,etal:Molecularvariantsofhumanpapillomavirustypes16and18preferentiallyassociatedwithcervicalneoplasma.
JGenVirol2000,81:2959–2968.
22.
RoseB,StegerG,DongXP,ThompsonC,CossartY,TattersallM,etal:PointmutationsinSP1motifsintheupstreamregulatoryregionofhumanpapillomavirustype18isolatesfromcervicalcancersincreasepromoteractivity.
JGenVirol1998,79:1659–1663.
23.
RoseB,ThompsonCH,ZhangJ,StoeterM,StephenA,PfisterH,etal:SequencevariationintheupstreamregulatoryregionofHPV18isolatesfromcervicalcancers.
GynecolOncol1997,66:282–289.
24.
MeissnerJ:SequencingerrorsinreferenceHPVclones.
InHumanpapillomaviruses.
Acompilationandanalysisofnucleicacidandaminoacidsequences.
3rdedition.
EditedbyMyersG,BakerC,MungerK,etal.
LosAlamos(NM):TheoreticalBiologyandBiophysics,LosAlamosNationalLaboratory;1997:110–123.
25.
Arias-PulidoH,PeytonCL,Torrez-MartinezN,AndersonDN,WheelerCM:Humanpapillomavirustype18variantlineagesinUnitedStatespopulationscharacterizedbysequenceanalysisofLCR-E6,E2,andL1regions.
Virology2005,338:22–34.
26.
ChenZ,DeSalleR,SchiffmnM,HerreroR,BurkRD:Evolutionarydynamicsofvariantgenomesofhumanpapillomavirustypes18,45and97.
JVirol2009,83:1443–1455.
27.
RonquistF,HuelsenbeckJP:MrBayes3:Bayesianphylogeneticinferenceundermixedmodels.
Bioinformatics2003,19:1572–1574.
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