HBoV bocavirus.docx

HBoV bocavirus.docx

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HBoVbocavirus

BocavirusEpisomeinInfectedHumanTissueContainsNon-IdenticalTermini

AmitKapoor,1*MadyHornig,1AravindAsokan,2BrentWilliams,1JoseA.Henriquez,1andW.IanLipkin1

1CenterforInfectionandImmunity,ColumbiaUniversity,NewYork,NewYork,UnitedStatesofAmerica

2GeneTherapyCenter,TheUniversityofNorthCarolinaatChapelHill,ChapelHill,NorthCarolina,UnitedStatesofAmerica

Jean-PierreVartanian,Editor

InstitutPasteur,France

*E-mail:

ak3117@columbia.edu

Conceivedanddesignedtheexperiments:

AKWIL.Performedtheexperiments:

AKJAHMHBW.Analyzedthedata:

AKJAHAA.Contributedreagents/materials/analysistools:

AKJAHAAMHBW.Wrotethepaper:

AKMHWIL.

ReceivedMarch21,2011;AcceptedMay26,2011.

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Abstract

Introduction

MaterialsandMethods

Results

Discussion

References

Abstract

Humanbocaviruses（HBoV）arehighlyprevalenthumaninfectionswhosepathogenicpotentialremainsunknown.Recentidentificationofthefirstnon-humanprimatebocavirus[1]incaptivegorillasraisedthepossibilityofthepersistentnatureofbocavirusinfection.Tocharacterizebocavirusinfectioninhumans,wetestedintestinalbiopsiesfrom22childrenwithgastrointestinaldiseaseforthepresenceofHBoVDNA.FourHBoV-positivetissuesampleswereanalyzedtodeterminewhetherviralDNAwaspresentinthelineargenomic,theepisomalclosedcircularorthehostgenome-integratedform.WhereasonetissuesamplepositiveforHBoV3containedtheepisomalform（HBoV3-E1）,nonehadthegenome-integratedform.ThecompletegenomesequenceofHBoV3-E1contains5319nucleotidesofwhich513representthenon-codingterminalsequence.ThesecondarystructureofHBoV3-E1terminisuggestsseveralconservedandvariablefeaturesamonghumanandanimalbocaviruses.OurobservationthatHBoVgenomeexistsashead-to-tailmonomerininfectedtissueeitherreflectsthelikelyevolutionofalternativereplicationmechanisminprimatebocavirusesoramechanismofviralpersistenceintheirhost.Moreover,weidentifiedtheHBoVgenomicterminalsequencesthatwillbehelpfulindevelopingreversegeneticsystemsforthesewidelyprevalentparvoviruses.

Significance

HBoVhavebeenfoundinhealthyhumancontrolsaswellasindividualswithrespiratoryorgastrointestinaldisease.OurfindingssuggestthatHBoVDNAcanexistasepisomesininfectedhumantissuesandthereforecanlikelyestablishpersistentinfectioninthehost.PreviouseffortstogrowHBoVincellcultureandtodevelopreversegeneticsystemshavebeenunsuccessful.CompletegenomicsequenceoftheHBoV3episomeanditsgenomicterminiwillimproveourunderstandingofHBoVreplicationmechanismanditspathogenesis.

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Abstract

Introduction

MaterialsandMethods

Results

Discussion

References

Introduction

Parvovirusesaresmall,non-envelopedicosahedralviruseswithsingle-strandedlinearDNAgenomesthatfrequentlyinfectanimalsthroughthefecal-oralroute[2].TheyaremembersoftheParvoviridaefamily,whichcomprisestwosub-families,DensovirinaeandParvovirinaethatinfectnon-vertebrateandvertebratehosts,respectively[2],[3].TheInternationalCommitteeonTaxonomyofViruses（ICTV）hasfurtherclassifiedthesub-familyParvovirinaeintofivegenera:

Dependovirus,Bocavirus,Erythrovirus,ParvovirusandAmdovirus.BocavirusesareuniqueamongparvovirusesbecausetheycontainathirdORFbetweenthenon-structuralandstructuralcodingregions[4],[5],[6].ThegenusBocavirusincludesthebovineparvoviruses（BPV）,minutevirusofcanines（MVC）[2],porcinebocaviruses[7],gorillabocavirus[1]and4speciesofhumanbocaviruses（HBoV1–4）[4],[8],[9],[10],[11].

Parvovirusesareuniqueastheyhavealinearsingle-strandedDNAgenomewithhairpinsequencesateachend.ThelengthoftheDNAgenomeisbetween4500and5500nucleotides[2],[3].Dependingontheirreplicationrequirements,parvoviruses（PV）canbeautonomousPVordependoviruses;thelatterincludesadeno-associatedviruses（AAV）andrequirespresenceofahelpervirusforsuccessfulreplication[3].Bocavirusesareconsideredautonomousparvoviruses.Thenon-codingornon-translatedregionsonthegenomicterminicontainpalindromicsequences,commonlyknownasinvertedterminalrepeats（ITR）,thatplayavitalroleinviralreplication[12].Sincetheirdiscoveryusingnucleicacidamplification-basedtechniques[4],[8],[9],[11],theterminalregionsofallprimatebocavirusspeciesremainunsequenced,limitingthescopeofstudiesonviralreplicationandthedevelopmentofreversegeneticsystems.HerewereportthecompletegenomeoftheHBoVepisomefoundininfectedhumanintestinaltissueandthesecondarystructureofitstermini.

HBoV1infection,widelyconsideredacute,hasbeenlinkedwithmildtoseverelowerrespiratorytractinfectionsinchildren,frequentlyinassociationwithotherviralinfections[8],[13],[14],[15],[16],[17],[18],[19],[20].HBoV1hasalsobeendetectedatlowfrequencyinstoolsamples,althoughitsassociationwithentericdiseaseisweakerthanwithrespiratorydisease[9],[21],[22],[23],[24],[25].HBoV2wasfirstidentifiedinstoolsamplesofPakistanichildren[11].LowerfrequenciesofHBoV2weredetectedinthestoolsofScottishadultsandchildren[11].HBoV3andHBoV2wererecentlyfoundinstoolsamplesfromAustralianchildrenwithdiarrhea[9],andHBoV4wasfirstidentifiedinstoolsamplesofchildrenfromNigeriaandTunisia[26].WhetheranyofthefourHBoVspeciescauseshumandiseaseisstillundetermined[3],[27],[28].

Duringasurveyofdivergentbocavirusesinanimals,thefirstdistinctspeciesofnon-humanprimatebocavirus,GBoV1,wasidentifiedinstoolsamplesofgorillasfromacaptivecolony[1].PhylogeneticanalysissuggeststhatGBoV1ismostgeneticallyrelatedtoHBoV1.GBoV1anditsrelatedvariantwerealsodetectedinstoolsamplesofwildanimalsinCameroon,confirmingtheirnon-humanprimateoriginandworldwidedistribution[29].ThepresenceofGBoV1instoolsamplesofanimalslivingincaptivityforseveralyearssuggeststhatbocavirusinfectionmaybepersistentintheseanimals.Inhumans,HBoVinfectionispredominantlyacute;therefore,moststudiesdonetodeterminetheassociationbetweenHBoVanddiseasecomparedtheprevalenceofviralDNAinsamplesfromsymptomaticpatientsandhealthycontrols[9],[14],[17],[21],[22],[23],[30],[31],[32],[33],[34].HerewereportthefirstdetectionofepisomalHBoV3inhumanintestinaltissue.Theseresultsraisethepossibilitythatbocaviruses,likeotherwell-characterizedparvoviruses,maycausepersistentinfectionoftheirhosts[12],[35].

OtherSections▼

Abstract

Introduction

MaterialsandMethods

Results

Discussion

References

MaterialsandMethods

Samplesandsource

Patientbiopsieswerecollectedaspartofastudytoassessthefrequencyofmeaslesvirustranscriptsinileaofchildrenwithautisticdisorderandgastrointestinalsymptoms（AUT-GI）andchildrenwithgastrointestinalsymptomswithoutanyapparentneurologicdisorder（CON-GI）[36].TheInstitutionalReviewBoards（IRB）ofColumbiaUniversityMedicalCenter（CUMC）andPartnersspecificallyreviewedandapprovedthedesignofthepreviousstudyunderwhichthesesampleswereobtainedafterwrittenconsentofparentsandguardians,inadditiontochildassent,asappropriate.Aftertheconclusionofthepreviousstudy,allresidualsampleswerede-identifiedandstored,andthereafterweredeemedexemptfromadditionalreviewbytheIRB.AllproceduresemployedinthecurrentstudywereapprovedbytheCUMCIRBunderahumansubjectsprotocoldesignedfortheinvestigationofproperlyde-identifiedsamples,whereintheneedforanyadditionalconsenthasbeenspecificallywaived.Allsampleswereanalyzedanonymously.

Sampleprocessingandpan-bocavirusPCRassay

DNAwasextractedfromilealbiopsymaterialfrom22patients（15AUT-GIand7CON-GIpatients）withTRIzol（Invitrogen）,usingthemanufacturer'sprotocolsforsequentialextractionofRNAandDNA.DNAconcentrationandintegrityweredeterminedusingaNanodropND-1000Spectrophotometer（NanodropTechnologies）andaBioanalyzer（AgilentTechnologies）.Sampleswerestoredat−80°C.PCRprimerspanBOV-F1（5′-TAATGCAYCARGAYTGGGTIGANCC-3′）andpanBOV-R1（5′-GTACAGTCRTAYTCRTTRAARCACCA-3′）wereusedforthefirstroundofhemi-nestedPCR;primerspanBOV-F2（5′-GCAYCARGAYTGGGTIGANCCWGC–3′）andpanBOV-R1（sameasfirstround）wereusedforthesecondroundofsemi-nestedPCR.ForthefirstroundofnestedPCR,5µlofeachspecimen'sDNAweremixedwith5.2µl10×polymerasereactionbuffer（Qiagen）,3µlofMgCl2,1.25µleachdNTP（10mM）,50pmoleachofforward（bothpanBOV-F1）andreverseprimer（panBOV-R1）,0.65µlHotStartTaqDNApolymerase（Qiagen）and32µlnucleasefreewater,inatotalreactionvolumeof50µl.ThePCRreactionwasperformedusingthefollowingprotocol:

initialdenaturationat95°Cfor7min,followedby6cyclesat95°Cfor40sec,60°Cfor45secand68°Cfor30sec,followedby35cyclesat95°Cfor30sec,57°Cfor30secand68°Cfor30sec,andafinalextensionat72°Cfor10min.DuringthefirstroundofPCR,thefirst6cyclesweredoneatahighannealingtemperaturetoincreasestringency,andtheremainingcycleswereperformedatalowertemperaturetofacilitateprimerhybridizationbytoleratingsomenucleotidemismatches.Weadded0.5µlofthePCRproductfromthefirstroundtothereactionmixtureforthesecondroundofPCR.Weusedthesamecyclingconditionsforthesecondround,withanannealingtemperatureof64°Cforthefirst6cyclesand58°Cfortheremaining35cycles.Followingelectrophoresis,productswereimagedon