feces玄幻小说排行榜2012前十名

1Anovelrecombinantlineage'scontributiontotheoutbreakofcoxsackievirusA6-associatedhand,footandmouthdiseaseinShanghai,China,2012-2013XiaoboFeng1,*,WencaiGuan2,*,YifengGuo1,*,HuijuYu3,*,XiaolingZhang2,RuhongCheng1,ZhenWang1,ZhenZhang1,JiaZhang1,HuaguoLi1,YinZhuang1,HuiZhang1,ZhiyongLu1,MingLi1,HongYu1,YixiaoBao3,YunwenHu2&ZhirongYao1Sincelate2012,coxsackievirusA6(CVA6)hasgraduallybecomethepredominantpathogenresponsibleforhand-foot-mouthdisease(HFMD)inseveralprovincesofChina.
Atotalof626patientsdiagnosedwithHFMDinShanghai,ChinafromJanuary2012toSeptember2013wereenrolledinthisstudy.
Ofthese,292CVA6infectedcasesweresubjectedtoclinicalanalyses.
Whole-genomesequencing,recombinationandphylogeneticanalyseswerealsoperformed.
ArecombinantCVA6monophyleticlineagewasfoundduringanoutbreakofCVA6-associatedHFMDsinShanghai,ChinainNovember2012,andaccountedfor21.
9%(64/292)oftheCVA6strainsduringthestudyperiod.
Recombinationanalysesshowedthatthe2CgeneofthenovelCVA6viruswasprobablyderivedfromacoxsackievirusA4(CVA4)straincirculatinginthepopulation.
ClinicalobservationshowedthatthisrecombinantCVA6virusledtoamoregeneralizedrashthandidthenon-recombinantCVA6virus.
ThisnewlyemergedCVA6lineagewasassociatedwithaconsiderableproportionofHFMDcasesfrom2012to2013inShanghai,andposesapotentialthreattopublichealth.
Theclinicalmanifestationsofhand-foot-monthdisease(HFMD)includemildtosevereskinrash,pul-monaryedema,circulatorydisorders,menigoencephalitis,asepticencephalitis,andevendeath.
LargeoutbreaksintheAsia-Pacificregionhavebeendescribedsince1997,anditisacommonepidemicdiseasearoundtheworld1,2.
Duringthelastfewyears,large-scaleoutbreaksofHFMDhaveoccurredinChina3–6,andhavebecomeasignificantpublichealthissue.
FromJanuary2012toSeptember2013,atotalof3,686,764casesofHFMDofwhich792werefatalwerereportedinmainlandChinaaccordingtotheChineseCenterforDiseaseControlandPrevention(http://www.
chinacdc.
cn/tjsj/fdcrbbg/).
ThecausativeagentsofHFMDarehumanenteroviruses(EVs)belongingtogenusEnterovirus,familyPicornaviridae.
EnterovirusA(EV-A)specieswasthemainpathogenforHFMD,followedbyEnterovirusB(EV-B).
EV-A(formerlynamedHumanenterovirusA)consistsof25serotypes:coxsackievirusA2(CV-A2),CV-A3,CV-A4,CV-A5,CV-A6,CV-A7,CV-A8,CV-A10,CV-A12,CV-A14,CV-A16,enter-ovirusA71(EV-A71),EV-A76,EV-A89,EV-A90,EV-A91,EV-A92,EV-A114,EV-A119,EV-A120,1DepartmentofDermatology,XinhuaHospitalAffiliatedtoShanghaiJiaotongUniversitySchoolofMedicine,Shanghai,China.
2PathogenDiagnosisandBiosafetyDepartment,ShanghaiPublicHealthClinicalCenter,KeyLabofMedicalMolecularVirology,FudanUniversity,Shanghai,China.
3DepartmentofPediatrics,XinhuaHospitalAffiliatedtoShanghaiJiaotongUniversitySchoolofMedicine,Shanghai,China.
*Theseauthorscontributedequallytothiswork.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoY.
H.
(email:ywhu0117@126.
com)orZ.
Y.
(email:dermatology.
yao@sohu.
com)received:21October2014accepted:02June2015Published:30June2015OPEN2EV-A121andthesimianenterovirusesSV19,SV43,SV46andbaboonenterovirusA13(BA13)(www.
picornaviridae.
com).
ThegenomeofEViscomposedofasingleopenreadingframeencodingapoly-proteinwhichiscleavedbyviralproteasesintothematureviralcapsidproteinsP1(VP4,VP2,VP3,andVP1)andnon-structuralproteinsP2(2Ato2C)andP3(3Ato3D)7.
Enterovirusesgenerategeneticdiversityandevolvethroughnucleotidemutationsorgeneticrecombination.
Epidemiologicstudieshaveindicatedthatdistinctenterovirusescouldrecombinewitheachother5,8,9.
Inter-typicrecombinantEVswerereportedtocausesevereorfatalcasesofHFMD10,11;however,therewasnoclearevidencethatinter-typicrecombinantEVshadpreviouslycontributetooutbreaksofHFMDs5,12.
Historically,EV71andCVA16werereportedtobethemajoretiologicalagentsofHFMDworld-wide7.
Since2008,CVA6hasemergedgraduallyandcausedatypicallygeneralizedandsometimessevereexanthemainHFMDcasesaroundtheworld13,14.
In2008,anoutbreakofHFMDcausedbyCVA6wasfirstreportedinFinlandandsimilaroutbreakswerethenreportedworldwide13,15–24.
Sincelate2012,CVA6hasgraduallybecomethepredominantpathogenresponsibleforHFMDinseveralprovincesofChina21,25–27.
BecausethenumberofHFMDcaseswithgeneralizedskinlesionshasincreasedinShanghai,ChinasinceNov2012,weperformedmolecularidentificationofenterovirusesfromHFMDcases,whole-genomesequencing,recombinationandphylogeneticanalysis,aswellasadeterminationofclinicalrelevance.
MethodsPatients,specimensanddatacollection.
FromJanuary2012toSeptember2013,atotalof626patientswerediagnosedwithHFMDbytheXinhuaHospitalaffiliatedtoShanghaiJiaotongUniversitySchoolofMedicineaccordingtodiagnosticcriteriadefinedbytheNationalHealthandFamilyPlanningCommissionofthePeople'sRepublicofChina(http://www.
nhfpc.
gov.
cn/yzygj/s3593g/201306/6d-935c0f43cd4a1fb46f8f71acf8e245.
shtml),and1251clinicalspecimens(626throatswabs,71stools,28vesicleswabs,526sera)werecollectedfromthesepatients.
Thedistributionofthepatientscoveredall18municipaldistrictsandonecountyinShanghai.
ThisstudywasapprovedbytheEthicsCommitteeofXinhuaHospital,andtheprocedureswerecarriedoutinaccordancewithapprovedguidelines.
Informedconsentwasobtainedfromthesubjects'parentsorguardians.
Patientswhometthefollowing3criteriawereselectedforclinicalcorrelationanalysis:1)under14yearsofage,2)skinlesionscompatiblewithtypicalHFMDoratypicalHFMD,3)CVA6identifiedbyvirologicalmethods.
TheskinlesionsoftypicalHFMDarecommonlymanifestassmallvesicles,papu-lovesicularlesionsormacularrashesonthepalms,soles,buttocksandoralmucosa.
AtypicalHFMDismanifestaslargevesiclesorbullae,maculopapularrashesortarget-likelesionspresentingonanysiteofthebodyincludingthetrunk,limbsorfacialareas,aswellassymptomsofacuteviralinfectionsuchasfever,coughordiarrhea.
Patientswhometanyofthefollowingconditionswereexcludedintheanalyses:1)lesionslocalizedontheoralmucosa,particularlythesoftpalateortonsillarpillars,withoutinvolvementofanyothersitesinthebody,whichwerediagnosedasherpangina;2)immune-compromisedsubjectswithahistoryofsystemicillnesswhoreceivedchemotherapyorimmunosuppressivetreatment;3)nonCVA6virusesdetectedbyvirologicalanalysis.
Demographicdata,clinicalmanifestations,andlaboratoryfindingswererecorded.
Amongtheclin-icalmanifestations,fever,timingofskinlesions,distributionofskinlesionsincludingthegeneralizedform,andsystemicsymptomswereevaluated.
Accordingtothenumberofanatomicalsitesinvolved(9sites:hands,feet,buttocks,oralmucosa,upperlimbs,lowerlimbs,anteriortrunk,posteriortrunkandface/neck),thegeneralizedformofHFMDwasdefinedasskinlesionsatfiveormoreofthesesites28.
Laboratoryfindingsincludingcompletebloodcount,biochemistryprofile,cardiacenzymes,chestX-ray,electrocardiogram(EKG),electroencephalogram(EEG)andMRIofthebrainwereperformed.
Serotypicidentificationofenteroviruses.
Clinicalspecimensincludingthroatswabs,vesicularfluids,fecesandserawerecollectedfromHFMDpatientsforlaboratoryexaminations.
ViralRNAwasextracteddirectlyfromclinicalspecimensusingaQIAampViralRNAMiniKit(Qiagen,SantaClara,CA)andstoredat80°C.
TheidentificationofEVandserotypingofEV71andCVA16fromsampleswereperformedbyareal-timereversetranscriptionpolymerasechainreaction(RT-PCR)aspreviouslydescribed29,30.
TofurtheridentifytheEVserotypesotherthanEV71andCVA16,semi-nestedRT-PCRandsequencingwereconductedaspreviouslydescribed31.
SerotypewasdeterminedbycomparisonoftheviralsequenceswithcorrespondingsequencesoftheEVprototypestrainsusingblastnonline(http://blast.
ncbi.
nlm.
nih.
gov/Blast.
cgi).
Completegenomeamplificationandsequencing.
Completegenomeamplificationandsequenc-ingwereperformedusingviralRNAextracteddirectlyfromclinicalspecimenscollectedduringpre-outbreakandoutbreakofCVA6-associatedHFMDs.
FourteenCVA6s,includingpre-outbreakstrainsof1827/CVA6/SH/CHN/2011,1232/CVA6/SH/CHN/2010,3913/CVA6/SH/CHN/2011and4368/CVA6/SH/CHN/2012,outbreakstrainsof5039/CVA6/SH/CHN/2013,PF3/CVA6/SH/CHN/2013,5056/CVA6/SH/CHN/2013,4592/CVA6/SH/CHN/2012,5068/CVA6/SH/CHN/2013,4645/CVA6/SH/CHN/2012,PF19/CVA6/SH/CHN/2013,5047/CVA6/SH/CHN/2013,PF001/CVA6/SH/CHN/2013and5084/CVA6/SH/CHN/2013,togetherwithtworecentstrainsofCVA4(701/CVA4/SH/CHN/2010and1047/CVA4/SH/CHN/2010)circulatingintheShanghaiareawererandomlyselectedandsubjectedtocomplete3genomeamplificationusingprimerslistedinSupplementalTable1.
Overlappingfragmentscoveringtheviralgenomewereamplifiedusingaone-stepRT-PCRkit(TaKaRa,Dalian,China),andprimersweredesignedbasedmainlyontherecentlyemergingCVA6andCVA4genomesequencespublishedinpreviousstudies32,33.
Thenon-structuralproteinsintheP2andP3regionsofpartialCVA6strainsweredeterminedbyprimerwalkingstrategyinwhichadditionalprimersweredesignedonthebasisoftheinitialPCRproductsforfurtheramplification.
One-stepRT-PCRwasperformedwithafinalvolumeof50μLcontaining5μLgenomicRNA,25μL2*onestepRT-PCRbuffer,1μLTaKaRaExTaqHS,1μLPrimeScriptRTEnzymeMix,1μLofeachprimer(10μM),and16μLRNaseFreedH2O.
RT-PCRwasperformedinanEppendorfMastercyclerat42°Cfor15min,95°Cfor1min,followedby40cyclesof95°Cfor20s,56°Cfor30s,and72°Cfor1.
2minwithafinalextensionstepat72°Cfor10min.
ThePCRproductswerepurifiedandsequencedinbothdirectionsonanautomatedsequencerABI3730XL(AppliedBiosystems,FosterCity,CA).
ThesequencedDNAfragmentswereassembledintocompletegenomesusingContigExpressprojectinVectorNTIversion11.
5.
SequencesimilaritiesamongstrainswerecalculatedusingBioEdit7.
2.
5.
ThefulllengthgenomicsequencesindicatedaboveweredepositedinGenBankundertheaccessionnumbersKJ541154-KJ541169.
All14CVA6genomesequenceswereusedinphylogeneticanalysisbasedonVP1,2Cand3Dregion.
Recombinationanalysis.
Thirteenfull-lengthgenomesequencesofrecentlyemergingEV-Astrainsincluding5039/CVA6/SH/CHN/2013,PF3/CVA6/SH/CHN/2013,5056/CVA6/SH/CHN/2013,4592/CVA6/SH/CHN/2012,5068/CVA6/SH/CHN/2013,4645/CVA6/SH/CHN/2012,PF19/CVA6/SH/CHN/2013,5047/CVA6/SH/CHN/2013,5084/CVA6/SH/CHN/2013,701/CVA4/SH/CHN/2010and1047/CVA4/SH/CHN/2010detectedinthepresentstudy,togetherwithHQ728260/CVA4/GD/CHN/2009andJF799986/EV71/GD/CHN/2009(aprogenyderivedfromtherecombinationofEV71subgenotypeC4andCVA4,whichwascloselyrelatedtorecombinantCVA6inthenon-structuralproteinregion)34whichareavailableinGenBankwerealignedwith12prototypesincludingCVA2~8,10,12,14,16,EV71whichwereconsideredtobethemostcloselyrelatedstrainsofEV-Abasedonnon-structuralproteinregion(SupplementalTable2).
ThesequencealignmentwasperformedusingClustalW2asdepictedpreviously5,andthensimilarityplotsamongthealignedsequencesweregeneratedusingSimPlotversion3.
5.
1.
Similaritywascalculatedineachwindowof200bpbytheKimura(2-parameter)distancemodelwithatransition-transversionratioof2.
0.
Thewindowwassuccessivelyadvancedalongthegenomealignmentin20bpincrements.
Forbootscananalysis,theneighbor-joiningalgorithmwasrunwith500bootstrapreplicates.
Athresholdof70%ormoreoftheobservedpermutedtreesindicatedpoten-tialrecombinationevents.
Ageneticalgorithmforthedetectionofarecombination(GARD)program(http://www.
datamonkey.
org/dataupload.
php)wasthenusedtoconfirmrecombinationandsearchforputativebreakpointswithaphylogeneticincongruencetestinwhichtheHKY85nucleotidesubstitu-tionbiasmodelwasselected.
SupportforrecombinationwasbasedonAICcgoodnessoffitandtheKHtestforphylogeneticincongruence.
HomologoussequencescorrespondingtodiversepartsofthegenomefromtherecombinantCVA6weresearchedwithinGenBankusingblastn.
Thesequencehomol-ogyofdiversepartsofthegenomebetweentherecombinantCVA6andthecloselyrelatedprototypeandrecentlyemergingEV-AstrainswerecalculatedseparatelybyBioEdit.
Differentiationofrecombinantandnon-recombinantCVA6strainsbytype-specificRT-PCR.
Todeterminetheproportionofrecombinantandnon-recombinantCVA6strainsinthissur-veillance,aone-stepduplexRT-PCRassaytargetingpartial2Band2CproteingenewithprimersspecifictorecombinantCVA6andnon-recombinantCVA6strains,respectively(SupplementalFigure1),wasdevelopedandappliedfordifferentiationofallCVA6isolatesinvolvedinthisstudy.
TheduplexRT-PCRwasperformedusingprimersRCA6F,RCA6R,NRCA6FandNRCA6R(SupplementalTable1)bywhichrecombinantCVA6yielded222-bpampliconandnon-recombinantCVA6yielded543-bpamplicon.
Thereactionvolumeof50μLcontained5μLgenomicRNA,25μL2*onestepRT-PCRbuffer,1μLTaKaRaExTaqHS,1μLPrimeScriptRTEnzymeMix,1μLofeachprimer(10μM),and16μLRNaseFreedH2O.
RT-PCRwasperformedinanEppendorfMastercyclerat42°Cfor15min,95°Cfor1min,followedby40cyclesof95°Cfor20s,56°Cfor30s,and72°Cfor30swithafinalextensionstepat72°Cfor7min.
PCRproductswereseparatedona1.
5%(wt/vol)agarosegelat140Vfor25min.
RecombinantCVA6isolatesidentifiedbyduplexRT-PCRwerefurtherconfirmedusingsequencingofthe2Cpartialregion.
Additionally,aCVA4type-specificPCRwasemployedtoruleoutthemixedinfectionwithCVA4insamplesinwhichrecombinantCVA6wasidentified(seeSupplementalTable4).
AmplificationandsequencingofVP1,2Cand3Dgenes.
ThecompleteVP1genewasamplifiedandsequencedusingprimers2333CA6Fand3530CA6R(SupplementalTable1).
Theone-stepRT-PCRwasperformedinafinalvolumeof50μLcontaining5μLgenomicRNA,25μL2*onestepRT-PCRbuffer,1μLTaKaRaExTaqHS,1μLPrimeScriptRTEnzymeMix,1μLofeachprimer(10μM),and16μLRNaseFreedH2O.
RT-PCRwasperformedinanEppendorfMastercyclerat42°Cfor15min,95°Cfor1min,followedby40cyclesof95°Cfor20s,56°Cfor30s,and72°Cfor1.
2minwithafinalextensionstepat72°Cfor10min.
The5′regionof2Cproteingeneafterthebreakpointwasamplifiedandsequencedwithprimers3714CA6Fand4579CA6R(SupplementalTable1).
Theone-stepRT-PCRmixtureisthesameasstated4above.
RT-PCRwasperformedinanEppendorfMastercyclerat42°Cfor15min,95°Cfor1min,fol-lowedby40cyclesof95°Cfor20s,56°Cfor30s,and72°Cfor50swithafinalextensionstepat72°Cfor10min.
Thepartial3Dgenewasamplifiedandsequencedwithprimers3DCA6Fand3DCA6R(SupplementalTable1).
Theone-stepRT-PCRmixtureisthesameasstatedabove.
RT-PCRwasperformedinanEppendorfMastercyclerat42°Cfor15min,95°Cfor1min,followedby40cyclesof95°Cfor20s,56°Cfor30s,and72°Cfor1minwithafinalextensionstepat72°Cfor10min.
ThecompleteVP1genewasamplifiedandsequencedfor61recombinantand101non-recombinantCVA6strains.
The61recombinantCVA6and33randomlyselectednon-recombinantCVA6strainswereamplifiedandsequencedforthepartial2Cand3Dgenes.
ThePCRproductswerepurifiedandsequencedonanautomatedsequencerABI3730XL(AppliedBiosystems,FosterCity,CA).
TheVP1,2Cand3DsequencesobtainedinthisstudyweredepositedinGenBankundertheacces-sionnumbersJX495118-JX495149,KC414726-KC414757,KF647875-KF647892,KJ541170-KJ541441,KP398832-KP398846.
Phylogeneticanalysis.
ThecapsidproteinVP1(915bp),non-structureproteins2C(425bp)and3D(812bp)fragmentswereselectedforphylogeneticanalyses.
Dendrogramswereproducedbyneighbor-joininganalysisusingsequencesalignmentwiththeKimura2-parametermethod.
Gapsweretreatedasacompletedeletion.
Statisticalsupportforeachcladewasassessedusingbootstrapanalysiswith500replicates.
Geneticdistanceswerecalculatedwiththep-distancemodel.
The61recombinantCVA6and33randomlyselectednon-recombinantCVA6strainstogetherwithEV-Aprototypes(CVA2~8,10,12,14,16,EV71)andcloselyrelatedEV-Astrainsweresubjectedtophy-logeneticanalysesbasedonVP1,2Cand3Dregions,respectively.
ThereferencestrainsofEVusedin3phylogenetictreeswereretrievedfromtheGenbankdatabase,whichsharedthehighestidentitywithoursequencesbyusingBLASTtools.
Additionally,the162CVA6strainsincluding61recombinantand101non-recombinantCVA6strainsinourstudyandotherCVA6strainsworldwideavailablefromGenBankweresubjectedtophylogeneticanalysisbasedonfulllengthsequenceoftheVP1gene.
Statisticalanalyses.
Discretevariableswereexpressedascounts(percentage)andcontinuousvaria-bleswereexpressedasmeans±standarddeviation(SD).
VariationsbetweengroupswereevaluatedusingtheT-testforage,durationoffeverandbodytemperature.
Differencesfordiscretevariables(gender,contacthistory,timingofskinlesion,clinicalcharacteristicsandlaboratorydata)wereanalyzedbyusingthechi-squaretestorFisher'sexacttest.
P<0.
05wasconsideredtobestatisticallysignificant.
ResultsSerotypingofentrovirusesassociatedwithHFMDcases.
FromJanuary2012toSeptember2013,atotalof626patientswithaclinicaldiagnosisofHFMDwereinvestigatedinthisstudy.
Adefiniteserotypewasidentifiedin609(97.
3%)casesandthemostcommonEV-AwasCVA6,whichaccountedfor292(46.
6%)cases,followedbyEV71(181;28.
9%),CVA16(96;15.
3%),CVA10(22;3.
5%),CVA4(5;0.
8%),CVA2(4;0.
6%),CVA5(2;0.
3%),andCVA12(1;0.
2%).
BeforeSeptember2012,EV71wasthedominantserotypeinHFMDcases(115;50.
9%),whereasCVA6dominated(281;70.
3%)afterOctober2012(Fig.
1).
FulllengthgenomicsequencingandrecombinationanalysisoftherecentlyemergingCVA6isolates.
ToelucidatethemolecularfeaturesofprevalentCVA6strainscausingtheoutbreakofCVA6-associatedHFMDs,fourteenCVA6strainsfrom14casesbeforeandduringtheCVA6outbreakperiodwererandomlyselectedandthewholegenomeofeachviruswasamplifieddirectlyfromtheclinicalspecimens.
Sequencesalignmentthroughoutthewholegenomeshowedthat,5039/CVA6/SH/CHN/2013,PF3/CVA6/SH/CHN/2013and5056/CVA6/SH/CHN/2013,whichwerecollectedduringtheCVA6outbreakperiod,exhibitedsignificantsequencedivergencefromallthepreviouslypublishednon-recombinantCVA6sinthenoncapsidproteinregion.
Theidentitiesofarepresentativestrain(PF3/CVA6/SH/CHN/2013)andotherEVstainsareshowninTable1.
PF3/CVA6/SH/CHN/2013hadthehighestsequenceidentity(94.
2%~96.
7%)withnon-recombinantCVA6strainsinthe5′UTR,VP4,VP2,VP3,VP1and2Aregions;howeverthesimilaritydroppedto87.
8%and82.
9%inthe2Band3Aregions,respectively.
Notably,inthe2C,3B,3Dand3′UTRregions,thisCVA6strainwasmoresimilartotheotherEV-Aserotypesthantothenon-recombinantCVA6strains,suggestingthatarecombinanteventoccurredwiththeseviruses(selectedisolateswhicharecloselyrelatedtorecombinantCVA6areshowninTable1).
TheP3regionoftherecombinantCVA6virusshowedlessthan91%ofsimilaritywithothersequencesincludingCVA6strainsavailablefromGenBank.
Tocorroborateourhypothesis,Simplotanalysisandsubsequentbootscananalysiswereemployed(Fig.
2showsonlythemostcloselyrelatedisolates).
Itwasfoundthatrecombinationhasprobablyoccurredatthe2Cregions.
TheputativerecombinantfragmentshowedhighestidentityandbootstrapsupportwithaCVA4straincirculatinginShanghai(1047/CVA4/SH/CHN/2010)ratherthanwithCVA6strains(Fig.
2).
IntheP3region,therewasnoreliablephylogeneticrelationshipbetweentherecombinantCVA6andotherserotypesofenterovirusesanalyzed.
5TherecombinationeventwasconfirmedbythedetectionofputativebreakpointswithingenomesusingGARD,andfurtherconfirmedbystatisticalanalysisoftheKHtest.
Theanalysisfound5putativebreakpoints,butonlythetopologyflankingpositionat4041bp(p=0.
01),whichislocatedwithinthe2Bgene,wassignificantlydiscordantviatheKHtest,supportingthatpositionasarecombinationbreak-point(SupplementalTable3).
BestGARDtreesbasedonsegmentspan3535to4041bpandsegmentspan4042to4470bpshowedmarkedtopologicincongruence.
Thus,theresultoftheGARDanalysiswasconsistentwiththeSimplotandbootscananalysisresults.
PrevalenceofthenovelrecombinantCVA6virusamongtheHFMDcases.
ToinvestigatetheprevalenceofthenovelrecombinantCVA6duringourobservationperiodanditscontributiontotheoutbreakofCVA6infection,allofthe292CVA6strainsweretestedbyaduplexRT-PCRwithtwopairsofprimerswhichtargetedthenon-recombinantandtherecombinantCVA6,respectively.
ThisshowedthattherecombinantCVA6strainwasfirstdetectedinNovember2012.
Amongthe292CVA6strains,21.
9%(64/292)weretypedastherecombinantCVA6whichwereidentifiedfrom64throatswabsand10vesicleswabs,and78.
1%(228/292)werenon-recombinantCVA6whichwereidentifiedfrom228throatswabs,16vesicleswabsand1feces.
TherewasnocasethatwaspositiveinbothPCRs.
SinceCVA4couldnotbedetectedbyCVA4type-specificRT-PCR(SupplementalTable4)inthe64recombinantCVA6-positivespecimens,CVA4-likesequenceswerenotlikelytohavearisenfromdualinfectionwithCVA4andCVA6.
Instead,recombinationbetweenthetwoserotypesmayexplainthefindings.
ComparingthetwowavesofHFMDoutbreakssincethenovelCVA6emerged,anincreasedpropor-tionoftherecombinanttypeinthewholepopulationofCVA6wasobserved(9%fromNovember2012toJanuary2013versus30.
9%fromMarchtoSeptember2013).
Theproportionoftherecombinanttypereacheditspeak(58.
8%)inJuly2013.
ThisresultsuggestedapartialcontributionofthenovelCVA6totheoutbreakofCVA6associatedHFMDinShanghaiduring2012and2013.
PhylogeneticanalysesoftheCVA6virusesbasedonVP1,2Cand3Dsequences.
Thecom-pleteVP1genewassuccessfullyamplifiedandsequencedfor61recombinantand101non-recombinantCVA6strains.
These162CVA6strainsfromourstudyandotherCVA6strainsworldwideavailablefromGenBankweresubjectedtophylogeneticanalysisbasedonfulllengthsequenceoftheVP1gene.
Thesestrainscouldbedividedinto4genotypicsubgroups,genotypesA,B,CandD,accordingtothecrite-riaestablishedforgenotypeEV71bycalculatinggeneticdistance9.
TheCVA6strainsduringtheCVA6outbreakperiodinourstudyclusteredwiththestrainsfromotherprovincesofmainlandChina,Japan,Taiwan,andFrance,andmadeuptwopredominantclusters,D6andD7.
AllrecombinantCVA6isolatestogetherwithsomeoftheGuangdongstrainsisolatedin2012–2013,Hunanin2012,Jiangsuin2013,Fujianin2012andShanxiin2013clusteredinaseparatedsub-clusterwithinD7(Fig.
3;SupplementalFigure2).
ThissuggestedthattherecentlyemergedlineageofCVA6mightcirculateindifferentareasofChina,althoughthereisalackofnon-structuredproteinsequencesfromthesestrains.
Figure1.
MonthlydistributionofEV-AinShanghai,China,fromJanuary2012toSeptember2013.
ThecontinuouslinedescribesthetotalnumberofspecimenscollectedeachmonthinShanghai;thehistogramdepictsthedistributionofrecombinantCVA6(R-CVA6),non-recombinantCVA6(NR-CVA6)andotherEV-Aserotypesidentifiedeachmonth.
6The61recombinantCVA6and33randomlyselectednon-recombinantCVA6strainswereamplifiedandsequencedforfulllengthsequencingoftheVP1gene,andpartial2Cand3Dgenes.
PhylogeneticanalysisbasedontheVP1geneshowedthatrecombinantCVA6strainsclusteredinoneclade,whichwascloselyrelatedtotheothernon-recombinantCVA6strains(Fig.
4a);however,phylogeneticanalysisbasedonthe2CregionshowedthatrecombinantCVA6strainsclusteredinoneclade,whichwascloselyrelatedtothecladecomposedofrecentlyemergingCVA4strainsratherthannon-recombinantCVA6strains(Fig.
4b).
Basedonthe3Dregion,recombinantCVA6strainsmadeupaseparatebranchandshowednocloserelationshipwithanyspecificserotype(Fig.
4c).
Thus,phylogeneticanalysessupportedthefindingsoftherecombinantanalysis.
Table1.
Pair-wisenucleotidesequenceidentitiesbetweenrecombinantCVA6strainandothercloselyrelatedEV-Astrains.
GenomicregionsshowingdiscrepantresultsregardingsequencesimilaritybetweenrecombinantCVA6(R-CVA6,therepresentativestrain:PF3/CVA6/SH/CHN/2013)andnon-recombinantCVA6(NR-CVA6,therepresentativestrain:5069/CVA6/SH/CHN/2013)aremarkedwithagraybackground.
RecombinantEV71(R-EV71:JF799986/EV71/GD/CHN/2009),recentlyemergingCVA4(E-CVA4:1047/CVA4/SH/CHN/2010),CVA4prototypestrain(P-CVA4:HighPoint),CVA14prototypestrain(P-CVA14:G-14),CVA16prototypestrain(P-CVA16:G-10)andCVA6prototypestrain(P-CVA6:Gdula)wereinvolvedinthisanalysis.
7Figure2.
SimplotandBootscanninganalysesoftherecombinantCVA6,non-recombinantCVA6,recombinantEV71andrecentlyemergingCVA4strainsonthebasisoffull-lengthgenomes.
RecombinantCVA6(PF3/CVA6/SH/CHN/2013)wasusedasthequerysequence.
RecombinantCVA6andnon-recombinantCVA6strainsweremarkedbyreddotsandbluedots,respectively.
ThisisaschematicrepresentationoftheamplifiedregionsoftheCVA6genome.
Primernamesandbindingsitesareshownbyshortarrows.
8ClinicalfeaturesoftheHFMDcasesinfectedwiththisnovelrecombinantCVA6virus.
Ofthe292patientsinfectedwithCVA6,283metourcriteriaandwereenrolledinclinicalanalyses.
Thedemo-graphicandclinicalcharacteristicsofthenon-recombinantCVA6,andrecombinantCVA6groupsareshowninTable2.
Weanalyzedtherelationshipsofdemographicandclinicalcharacteristicsbetweenthenon-recombinantCVA6group(n=223)andtherecombinantCVA6group(n=60).
Wefoundthatskinlesionsontheupperlimbs,lowerlimbsandanteriortrunkweremorefrequentintherecombinantCVA6group(p=0.
008,p=0.
019,p=0.
004,respectively)usingthechi-squaretest.
Thepercentagesofthegeneralizedformwithskinlesionsinfiveormoreanatomicsitesinvolvedinthenon-recombinantCVA6andrecombinantCVA6groupswere35.
4%and51.
7%respectively(p=0.
022)usingthechi-squaretest,whichindicatedthatthedistributionofskinlesionswasmorewidespreadinrecombinantCVA6-infectedcases.
Nosignificantdifferencewasdetectedindemographicparametersorotherclinicalorlaboratorydatabetweenthe2groups.
DiscussionNumerousepidemicsofHFMDhaveoccurredintheAsia-Pacificregioninthepastdecade,withEV71themostcommoncause7.
BecausesurveillanceofHFMDinmainlandChinahasfocusedprimarilyonEV71andCVA16,informationaboutthepathogenicroleofotherEVs,andtheirgeographicdistributionandepidemiologicaltraitsarestilllimited6.
Sincelate2012,CVA6hasbeenreportedasthepredominantpathogenresponsibleforHFMDinseveralprovincesofChina21,23,25–27.
Althoughepidemiologicanalyseswereperformedinthosestudies,therelationshipbetweenmolecularcharacterizationandclinicalfea-turesremaintobedemonstrated.
Inthisstudy,theCVA6serotype,previouslyaminorcauseofHFMD,wasfoundtobethepredominantstrainresponsibleforHFMDinShanghaisincelate2012.
Becauselimiteddatawereavailableaboutthisemergingvirus,weinvestigatedthemolecularepidemiologyandclinicalfeaturesoftheCVA6serotypetoevaluatethepotentialrisktopublichealthandtoguideintheclinicaldiagnosisandtreatment.
AnalysesofcompletegenomesofEV-AorBspeciessuggestedthatrecombinationusuallyoccurredwithinnoncapidregionsofthegenome32,35.
MultipleEV-AvirusesofidenticalordifferentserotypesFigure3.
Neighbor-joiningtreesconstructedonthebasisofVP1regionoftheCVA6strainsbyusingMEGA6.
05.
CVA6strainsisolatedfromShanghaiaremarkedwithdistinctcolorsaccordingtoyearsofisolation.
ThecladeinvolvingallrecombinantCVA6strainswasmarkedwithredcolor.
9couldco-infectanindividualandco-circulateinthesamegeographicalarea,whichgavevirusestheopportunitytoundergorecombination22,36,37.
Theco-circulationofCVA4andCVA6strainsinthesamegeographicregionisarelativelycommonphenomenoninChina22,37,andwouldfavortheoccurrenceofrecombinationbetweenthetwoserotypes.
Accordingtothemolecularanalyses,thenovelCVA6sharedgreatersimilaritywithotherEV-AstrainsthanwiththepreviouslyreportedCVA6strainsinthepartialnoncapid2CregionwhichweremostcloselyrelatedtotheCVA4strainsinShanghai.
Webelievethatrecombinationprobablyoccurredwithinthenoncapid2CregionbetweenthenovelCVA6virusandarecentlyemergingCVA4strain.
However,duetonocloselyrelatedsequencesfoundinGenBank,itwasspeculatedthattheP3regionoftherecombinantCVA6mayderivefromanunknownenteroviruswaitingtobediscovered.
ToclarifythegeneticrelationshipofCVA6outbreakstrainsbetweenthoseinShanghaiandotherareas,phylogeneticanalysisbasedontheVP1genewasperformed.
ResultsshowedthatallCVA6strainscollectedduringtheCVA6outbreakperiodinShanghaitogetherwiththosefromGuangdong,Shanxi,Jiangsu,FujianandHunanformedtwocloselyrelatedclustersnamelyD6andD7,suggestingthatthetwosubgenotypeshadbecomethemajorcirculatingstrainsintherecentHFMDepidemicinChina.
AllrecombinantCVA6strainsinourstudyclusteredtogetherandconstituteamonophyleticlineagewithsomeoftheGuangdong,Shanxi,Jiangsu,FujianandHunanstrainswithinsubgenotypeD7.
Thisindi-catesthattherecombinationeventoccurredrecentlyandthenewrecombinantCVA6isolatestogetherwiththestrainsfromotherprovincesmaybedescendantsofanunknowncommonrecombinantances-tor.
Unfortunately,thesequencesofotherpartsofthegenomeshavenotbeendeterminedintheCVA6strainsfromotherprovinces.
DuetothehighlysimilarVP1sequencesfoundintherecombinantCVA6virusinShanghaiandtheCVA6strainsfromotherprovincessincetheCVA6outbreakinmainlandChina,largescaleepidemiologicstudiesthroughoutthecountryarewarrantedtoillustratetheroleofthenovelrecombinantCVA6virusinthisHFMDoutbreak.
Moreover,wemustbealerttothepossibilitythatthisrecombinantCVA6viruswillcauseanewHFMDoutbreakinthenextfewyears.
Theclinicalcharacteristicsincludingtherashdistributionamongpatientsinfectedwithdistinctsero-typesincludingEV71,CVA6,CVA16andCVA10hadbeencomparedinpreviousreports15,25,27,28,37,sowefocusoncomparingtheclinicalcharacteristicsbetweenpatientsinfectedwiththerecombinantandnon-recombinantCVA6.
BecauseinfectionwiththerecombinantCVA6viruscouldresultinmoregen-eralizedskinlesionsthaninfectionwithnon-recombinantCVA6,andtheupperlimbs,lowerlimbsandanteriortrunkpartsweremorereadilyinvolvedinrecombinantCVA6-infectedcases,dermatologistsshouldpaymoreattentiontothissituationinclinicalpractice.
OurstudyprovidesstrongevidencethatthepreviouslyinfrequentlydetectedCVA6virushasbecomethepredominantHFMDpathogeninShanghai,China,andanovelrecombinantCVA6lineagewithFigure4.
Neighbor-joiningtreesconstructedonthebasisofVP1(a),2C(b)and3D(c)regionsoftherecombinantCVA6,non-recombinantCVA6,prototypeandrecentemergingEV-Astrains.
byusingMEGA6.
05,respectively.
CVA6strainsisolatedfromShanghaiaremarkedwithdistinctcolorsaccordingtoyearsofisolation.
RecombinantCVA6strainsareshownbyaredtriangle.
Bootstrapvaluesareindicatedforthemainbranches,inwhichvalueslowerthan70arenotshown.
10Table2.
DemographicandclinicalcharacteristicsofHFMDcasescausedbyrecombinantandnon-recombinantCVA6infections.
P<0.
05isshowninboldface;R-CVA6representsrecombinantCVA6;NR-CVA6representsnon-recombinantCVA6.
11specialclinicalfeaturesemergedandpartiallycontributedtotheCVA6outbreak,thushighlightingthenecessityofcomprehensivesurveillanceofitscirculationinHFMDepidemicsinChinaaswellasthroughouttherestoftheworld.
References1.
Chan,L.
G.
etal.
Deathsofchildrenduringanoutbreakofhand,foot,andmouthdiseaseinsarawak,malaysia:clinicalandpathologicalcharacteristicsofthedisease.
FortheOutbreakStudyGroup.
ClinInfectDis31,678–683(2000).
2.
Ho,M.
etal.
Anepidemicofenterovirus71infectioninTaiwan.
TaiwanEnterovirusEpidemicWorkingGroup.
NEnglJMed341,929–935(1999).
3.
Mao,L.
X.
etal.
Epidemiologyofhand,foot,andmouthdiseaseandgenotypecharacterizationofEnterovirus71inJiangsu,China.
JClinVirol49,100–104(2010).
4.
Yang,F.
etal.
Surveyofenterovirusinfectionsfromhand,footandmouthdiseaseoutbreakinChina,2009.
VirolJ8,508(2011).
5.
Zhang,Y.
etal.
CompletegenomeanalysisoftheC4subgenotypestrainsofenterovirus71:predominantrecombinationC4virusespersistentlycirculatinginChinafor14years.
PLoSOne8,e56341(2013).
6.
Xing,W.
etal.
Hand,foot,andmouthdiseaseinChina,2008-12:anepidemiologicalstudy.
LancetInfectDis14,308–318(2014).
7.
Solomon,T.
etal.
Virology,epidemiology,pathogenesis,andcontrolofenterovirus71.
LancetInfectDis10,778–790(2010).
8.
McMinn,P.
C.
Recentadvancesinthemolecularepidemiologyandcontrolofhumanenterovirus71infection.
CurrOpinVirol2,199–205(2012).
9.
Yip,C.
C.
,Lau,S.
K.
,Woo,P.
C.
&Yuen,K.
Y.
Humanenterovirus71epidemics:what'snextEmergHealthThreatsJ6,19780(2013).
10.
Hu,Y.
F.
etal.
CompletegenomesequenceofarecombinantcoxsackievirusB4fromapatientwithafatalcaseofhand,foot,andmouthdiseaseinGuangxi,China.
JVirol86,10901–10902(2012).
11.
Yip,C.
C.
etal.
RecombinantcoxsackievirusA2anddeathsofchildren,HongKong,2012.
EmergInfectDis19,1285–1288(2013).
12.
Li,J.
etal.
EvidencesforintertypicandintratypicrecombinanteventsinEV71ofhand,footandmouthdiseaseduringanepidemicinHubeiProvince,China,2011.
VirusRes169,195–202(2012).
13.
Osterback,R.
etal.
CoxsackievirusA6andhand,foot,andmouthdisease,Finland.
EmergInfectDis15,1485–1488(2009).
14.
Feder,H.
J.
,Bennett,N.
&Modlin,J.
F.
Atypicalhand,foot,andmouthdisease:avesiculobullouseruptioncausedbyCoxsackievirusA6.
LancetInfectDis14,83–86(2014).
15.
Cabrerizo,M.
etal.
Molecularepidemiologyofenterovirus71,coxsackievirusA16andA6associatedwithhand,footandmouthdiseaseinSpain.
ClinMicrobiolInfect20,O150–O156(2014).
16.
Wei,S.
H.
etal.
AnoutbreakofcoxsackievirusA6hand,foot,andmouthdiseaseassociatedwithonychomadesisinTaiwan,2010.
BmcInfectDis11,346(2011).
17.
Flett,K.
etal.
Hand,foot,andmouthdiseasecausedbycoxsackievirusa6.
EmergInfectDis18,1702–1704(2012).
18.
Fujimoto,T.
etal.
Hand,foot,andmouthdiseasecausedbycoxsackievirusA6,Japan,2011.
EmergInfectDis18,337–339(2012).
19.
Montes,M.
etal.
Hand,foot,andmouthdiseaseoutbreakandcoxsackievirusA6,northernSpain,2011.
EmergInfectDis19,676–678(2013).
20.
Puenpa,J.
etal.
Hand,foot,andmouthdiseasecausedbycoxsackievirusA6,Thailand,2012.
EmergInfectDis19,641–643(2013).
21.
Han,J.
F.
etal.
Hand,foot,andmouthdiseaseoutbreakcausedbycoxsackievirusA6,China,2013.
JInfect69,303–305(2014).
22.
He,Y.
Q.
etal.
Emergence,circulation,andspatiotemporalphylogeneticanalysisofcoxsackievirusa6-andcoxsackievirusa10-associatedhand,foot,andmouthdiseaseinfectionsfrom2008to2012inShenzhen,China.
JClinMicrobiol51,3560–3566(2013).
23.
Lu,J.
etal.
Hand,footandmouthdiseaseinGuangdong,China,in2013:newtrendsinthecontinuingepidemic.
ClinMicrobiolInfect20,O442–O445(2014).
24.
Sinclair,C.
etal.
Atypicalhand,foot,andmouthdiseaseassociatedwithcoxsackievirusA6infection,Edinburgh,UnitedKingdom,JanuarytoFebruary2014.
EuroSurveill19,20745(2014).
25.
Li,J.
L.
etal.
Epidemiccharacteristicsofhand,foot,andmouthdiseaseinsouthernChina,2013:coxsackievirusA6hasemergedasthepredominantcausativeagent.
JInfect69,299–303(2014).
26.
Hongyan,G.
etal.
Hand,footandmouthdiseasecausedbycoxsackievirusa6,beijing,2013.
PediatrInfectDisJ33,1302–1303(2014).
27.
Chen,J.
F.
,Zhang,R.
S.
,Ou,X.
H.
,Chen,F.
M.
&Sun,B.
C.
Theroleofenterovirus71andcoxsackievirusAstrainsinalargeoutbreakofhand,foot,andmouthdiseasein2012inChangsha,China.
IntJInfectDis28,17–25(2014).
28.
Hubiche,T.
etal.
Dermatologicalspectrumofhand,footandmouthdiseasefromclassicaltogeneralizedexanthema.
PediatrInfectDisJ33,e92–e98(2014).
29.
Verstrepen,W.
A.
,Bruynseels,P.
&Mertens,A.
H.
Evaluationofarapidreal-timeRT-PCRassayfordetectionofenterovirusRNAincerebrospinalfluidspecimens.
JClinVirol25Suppl1,S39–S43(2002).
30.
Xiao,X.
L.
etal.
Simultaneousdetectionofhumanenterovirus71andcoxsackievirusA16inclinicalspecimensbymultiplexreal-timePCRwithaninternalamplificationcontrol.
ArchVirol154,121–125(2009).
31.
Nix,W.
A.
,Oberste,M.
S.
&Pallansch,M.
A.
Sensitive,seminestedPCRamplificationofVP1sequencesfordirectidentificationofallenterovirusserotypesfromoriginalclinicalspecimens.
JClinMicrobiol44,2698–2704(2006).
32.
Hu,Y.
F.
etal.
CompletegenomeanalysisofcoxsackievirusA2,A4,A5,andA10strainsisolatedfromhand,foot,andmouthdiseasepatientsinChinarevealingfrequentrecombinationofhumanenterovirusA.
JClinMicrobiol49,2426–2434(2011).
33.
Chen,Y.
J.
etal.
ComparativegenomicanalysisofcoxsackievirusA6strainsofdifferentclinicaldiseaseentities.
PLoSOne7,e52432(2012).
34.
Guan,L.
L.
etal.
Recombinationanalysisofenterovirus71strainisolatedinGuangzhou,2009.
BingDuXueBao29,299–303(2013).
35.
Oberste,M.
S.
,Maher,K.
&Pallansch,M.
A.
EvidenceforfrequentrecombinationwithinspecieshumanenterovirusBbasedoncompletegenomicsequencesofallthirty-sevenserotypes.
JVirol78,855–867(2004).
36.
Blomqvist,S.
etal.
Co-circulationofcoxsackievirusesA6andA10inhand,footandmouthdiseaseoutbreakinFinland.
JClinVirol48,49–54(2010).
37.
Lu,Q.
B.
etal.
CirculationofCoxsackievirusA10andA6inhand-foot-mouthdiseaseinChina,2009-2011.
PLoSOne7,e52073(2012).
AcknowledgmentsThisstudywassupportedbytheNationalScienceandTechnologyMajorProjectofChina(2012ZX10004211and2013ZX10004-601),ShanghaiMunicipalHealthandFamilyPlanningCommission(20134184)andthekeyClinicalSpecialtyDisciplineConstructionProgramofChina.
12AuthorContributionsZ.
Y.
andY.
H.
designedthestudy.
X.
F.
,Y.
G.
,W.
G.
,Y.
H.
andZ.
Y.
wrotethepaper.
X.
F.
,Y.
G.
,H.
Y.
,M.
L.
,H.
Z.
,Z.
Z.
,J.
Z.
,Z.
L.
,H.
L.
,Y.
Z.
andY.
B.
gathereddata.
X.
F.
,W.
G.
,X.
Z.
,R.
C.
,Z.
W.
andY.
G.
analyzedthedata.
W.
G.
andX.
F.
isolatedviruses.
X.
F.
andW.
G.
didthemolecularanalyses.
AdditionalInformationSupplementaryinformationaccompaniesthispaperathttp://www.
nature.
com/srepCompetingfinancialinterests:Theauthorsdeclarenocompetingfinancialinterests.
Howtocitethisarticle:Feng,X.
etal.
Anovelrecombinantlineage'scontributiontotheoutbreakofcoxsackievirusA6-associatedhand,footandmouthdiseaseinShanghai,China,2012-2013.
Sci.
Rep.
5,11700;doi:10.
1038/srep11700(2015).
ThisworkislicensedunderaCreativeCommonsAttribution4.
0InternationalLicense.
Theimagesorotherthirdpartymaterialinthisarticleareincludedinthearticle'sCreativeCom-monslicense,unlessindicatedotherwiseinthecreditline;ifthematerialisnotincludedundertheCreativeCommonslicense,userswillneedtoobtainpermissionfromthelicenseholdertoreproducethematerial.
Toviewacopyofthislicense,visithttp://creativecommons.
org/licenses/by/4.
0/