HBoV bocavirusWord格式文档下载.docx
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HBoV bocavirusWord格式文档下载.docx
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InstitutPasteur,France
*E-mail:
ak3117@columbia.edu
Conceivedanddesignedtheexperiments:
AKWIL.Performedtheexperiments:
AKJAHMHBW.Analyzedthedata:
AKJAHAA.Contributedreagents/materials/analysistools:
AKJAHAAMHBW.Wrotethepaper:
AKMHWIL.
ReceivedMarch21,2011;
AcceptedMay26,2011.
OtherSections▼
Abstract
Introduction
MaterialsandMethods
Results
Discussion
References
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.
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].
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,5µ
lofeachspecimen'
sDNAweremixedwith5.2µ
l10×
polymerasereactionbuffer(Qiagen),3µ
lofMgCl2,1.25µ
leachdNTP(10mM),50pmoleachofforward(bothpanBOV-F1)andreverseprimer(panBOV-R1),0.65µ
lHotStartTaqDNApolymerase(Qiagen)and32µ
lnucleasefreewater,inatotalreactionvolumeof50µ
l.ThePCRreactionwasperformedusingthefollowingprotocol:
initialdenaturationat95°
Cfor7min,followedby6cyclesat95°
Cfor40sec,60°
Cfor45secand68°
Cfor30sec,followedby35cyclesat95°
Cfor30sec,57°
Cfor30secand68°
Cfor30sec,andafinalextensionat72°
Cfor10min.DuringthefirstroundofPCR,thefirst6cyclesweredoneatahighannealingtemperaturetoincreasestringency,andtheremainingcycleswereperformedatalowertemperaturetofacilitateprimerhybridizationbytoleratingsomenucleotidemismatches.Weadded0.5µ
lofthePCRproductfromthefirstroundtothereactionmixtureforthesecondroundofPCR.Weusedthesamecyclingconditionsforthesecondround,withanannealingtemperatureof64°
Cforthefirst6cyclesand58°
Cfortheremaining35cycles.Followingelectrophoresis,productswereimagedon
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