account168post

Maoetal.
EmergingMicrobes&Infections(2018)7:185EmergingMicrobes&InfectionsDOI10.
1038/s41426-018-0186-ywww.
nature.
com/emiARTICLEOpenAccessAneonatalmousemodelofcentralnervoussysteminfectionscausedbyCoxsackievirusB5QunyingMao1,XiaotianHao1,YalinHu2,RuixiaoDu1,ShuhuiLang3,LianlianBian1,FanGao1,CeYang1,BopeiCui1,FengcaiZhu4,LianzhongShen3andZhenglunLiang1AbstractAsoneofthekeymembersofthecoxsackievirusBgroup,coxsackievirusB5(CV-B5)cancausemanycentralnervoussystemdiseases,suchasviralencephalitis,asepticmeningitis,andacuteaccidparalysis.
Notably,epidemiologicaldataindicatethatoutbreaksofCV-B5-associatedcentralnervoussystem(CNS)diseaseshavebeenreportedworldwidethroughouthistory.
Inthisstudy,whichwasconductedtopromoteCV-B5vaccineandanti-virusdrugresearch,a3-day-oldBALB/cmousemodelwasestablishedusingaCV-B5clinicalisolate(CV-B5/JS417)asthechallengestrain.
MicechallengedwithCV-B5/JS417exhibitedaseriesofneuralclinicalsymptomsanddeathwithnecrosisofneuronalcellsinthecerebralcortexandtheentirespinalcord,hindlimbmuscles,andcardiomyocytes.
Theviralloadofeachtissueatvariouspost-challengetimepointssuggestedthatCV-B5replicatedinthesmallintestineandwassubsequentlytransmittedtovariousorgansviaviremia;theviruspotentiallyenteredthebrainthroughthespinalaxons,causingneuronalcellnecrosis.
Inaddition,thismousemodelwasusedtoevaluatetheprotectiveeffectofaCV-B5vaccine.
TheresultsindicatedthatboththeinactivatedCV-B5vaccineandanti-CVB5serumsignicantlyprotectedmicefromalethalinfectionofCV-B5/JS417byproducingneutralizingantibodies.
Insummary,therstCV-B5neonatalmousemodelhasbeenestablishedandcansustainCNSinfectionsinamannersimilartothatobservedinhumans.
Thismodelwillbeausefultoolforstudiesonpathogenesis,vaccines,andanti-viraldrugevaluations.
IntroductionCoxsackievirusB5(CV-B5)belongstotheEnterovirusgenusinthePicornaviridaefamilyandisamajormemberofthecoxsackievirusBgroup.
CV-B5isanicosahedralsphericalparticlewithadiameterof24–30nm,whichconsistsofanonenvelopedcapsidandacoreofsingle-strandedRNAofapproximately7400nucleotides(nt)1.
InfectionswithCV-B5cancauseavarietyofcentralnervoussystem(CNS)diseases,suchasasepticmeningi-tis2–5,viralencephalitis6–9,acuteaccidparalysis10,myocarditis11,12,Type1diabetes13,14,transientaphasia15,herpangina16,andhand,foot,andmouthdisease(HFMD)17–19.
Numerousasepticmeningitisandence-phalitisoutbreaksassociatedwithCV-B5havebeenreportedworldwide,withclinicalcharacteristicsincludingacuteonset,severeclinicalsymptoms,andhighmortality,especiallyinthelastdecade20–30.
Thisvirusisapotentialthreattothehealthofinfantsandyoungchildren.
Todate,notmanydatafrombasicresearchonCV-B5infectionsareavailable,andcurrently,thereisnoeffectivetreatmentforthediseasescausedbyCV-B5.
Animalmodelsarethebasictoolsforpathogenesisresearch,vaccinedevelopment,anddrugscreeningforviralinfec-tiousdiseases.
Inthisstudy,weestablishedaneonatalmousemodelthatexhibitedsymptomssimilartothoseobservedinhumancases,e.
g.
,CNSinfectionsandTheAuthor(s)2018OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.
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Ifmaterialisnotincludedinthearticle'sCreativeCommonslicenseandyourintendeduseisnotpermittedbystatutoryregulationorexceedsthepermitteduse,youwillneedtoobtainpermissiondirectlyfromthecopyrightholder.
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org/licenses/by/4.
0/.
Correspondence:ZhenglunLiang(lzhenglun@126.
com)1InstituteforBiologicalProductsControl,NationalInstitutesforFoodandDrugControl,Beijing,China2QualityControlDepartment,HualanBiologicalEngineeringInc.
,Henan,ChinaFulllistofauthorinformationisavailableattheendofthearticleTheseauthorscontributedequally:QunyingMao,XiaotianHao1234567890():,;1234567890():,;1234567890():,;1234567890():,;myocarditis.
ThismousemodelprovidesavaluabletoolforCV-B5-relatedbasicresearchandvaccineevaluation.
ResultsEstablishmentofaCV-B5susceptiblemousemodelToselectthemostsusceptiblemousestrains,1-day-oldBALB/c,C57BL/6,KM,ICR,andNIHmicewereintra-peritoneally(i.
p.
)challengedwithCV-B5/JS417(3.
16*107CCID50/mouse).
Allmousestrainsdevelopedclinicalsymptoms,suchasinactivity,wasting,shivering,hairloss,hunching,hindlimbparalysis,andevendeath.
Theclinicalsymptomswererankedfrommildtosevere,asfollows:0-healthy;1-inactivityandwasting;2-shivering;3-hunchedpostureandhairloss;4-hind-limbparalysis;and5-moribundanddeath(S1Table).
ThesurvivalratesfortheBALB/c,C57BL/6,ICR,KMandNIHmicewere0,25,50,67%,and80%upto21dayspost-infection(dpi),respectively(Fig.
1a).
Thus,theBALB/cmousewasthemostsusceptiblestraintoCV-B5infection,andthisstrainwasselectedforfurtherstudy.
ToassessthesusceptibilityofmiceofvariousagestoCV-B5challenge,groupsofBALB/cmiceaged1,3,5,7,and14dayswereinoculated(i.
p.
)withCV-B5/JS417(3.
16*107CCID50/mouse).
AsshowninFig.
1b,allinfectedneonatalmicebecamesick,andsomemicediedduringtheexperiment.
However,thesurvivalrateincreasedwithincreasingage.
Allofthe1-day-oldand3-day-oldmicediedwithin4dpiand7dpi,respectively.
Thesurvivalratesfor5-,7-,and14-day-oldmicewere50,67,and67%onday21,respectively.
Con-sideringthati.
p.
administrationismoredifculttoper-formon1-day-oldmice—adifcultythatcouldpotentiallyresultintheadministrationofalessaccuratechallengedoseofCV-B5–3-day-oldmicewereselectedforfurtherstudy.
Toselectasuitableinoculationroute,3-day-oldBALB/cmicewerechallengedwithCV-B5/JS417viathei.
p.
,intracerebral(i.
c.
)orintragastric(i.
g.
)route.
AsshowninFig.
1c,allinoculatedmicebecamesick,andthemortalityratewas100%.
Miceinoculatedviathei.
c.
,i.
p.
,andi.
g.
routediedwithin3,9,and9dpi,respectively.
Amongthethreeroutesofadministration,i.
g.
ismostsimilartotheinfectionroutethatoccursinhumans.
However,thisprocedureisdifculttoperformona3-day-oldmouse.
Therefore,thei.
p.
routewaschosenforfurtherstudy.
Fig.
1CV-B5susceptiblemousemodelwithCV-B5/JS417.
aTheprocessofselectingasuitablemousestrain.
One-day-oldmiceofvariousstrains(BALB/c,C57BL/6,KM,ICR,andNIH)werei.
p.
challengedwithCV-B5/JS417(3.
16*107CCID50/mouse).
bTheprocessofselectingthesuitableage.
BALB/cmiceat1,3,5,7,and14daysofagewerei.
p.
challengedwithCV-B5/JS417(3.
16*107CCID50/mouse).
cTheprocessforselectingthesuitableinoculationroute.
Three-day-oldBALB/cmicewerechallengedwithCV-B5/JS417(3.
16*107CCID50/mouse)viathei.
p.
,intracerebral(i.
c.
),orintragastric(i.
g.
)route.
Thecontrolmiceweretreatedwithmediumviathesamecorrespondingroutes.
n=6to10miceforeachgroup.
Allmiceweremonitoreddailyforbodyweightandclinicalsymptomsfor21dpi.
Onerepresentationoftwoindependentexperimentswasshown.
TheMantel–Coxlog-ranktestwasusedtocomparethesurvivalofthepupsbetweeneachgroupandthemediumcontrolgroupat21dayspost-infection.
***p10)rstandthendecreasing,includedtheintestine,spleenandliver,andviralloadratioswere25to1318,19to1926,and43to100,respectively,fromhour6tohour72post-challenge;afterhour72,theVLdecreasedrapidly.
Theviralloadwasfoundtobeapproximately1300timeshigherintheintestinecomparedwiththeblood.
Fig.
4ImmunohistochemicalstainingofvarioustissuesfromtheBALB/cneonatalmicefollowingtheintraperitonealinjectionwithCV-B5/JS417.
a,c,e,gIHCstainingofthecerebralcortex,spinalcord,myocardium,andhindlimbmuscleinthecontrolgroup,respectively.
b,d,f,hIHCstainingofthecerebralcortex,spinalcord,myocardium,andhindlimbmuscleintheexperimentalgroup.
Positivestainingwasdetectedinthecerebralcortex(b,arrow),spinalcord(d,arrow),myocardium(f,arrow)andhindlimbmuscle(h,arrow)oftheneonatalmiceaftertheintraperitonealinjectionofCV-B5/JS417.
Magnications*40(a);magnications*100(b–h).
n=6–10micepergroup.
OnerepresentativeimageisshownMaoetal.
EmergingMicrobes&Infections(2018)7:185Page5of11Moreover,theVLoftheintestinewasmuchhigherthantheVLfoundinmostothertissuesathour72.
Giventhenegativeresultsobtainedinthepathologicalexaminationofthesetissues(Figs.
3and4),wespeculatedthattheintestinemaybetheprimarytargetorganofCV-B5invasionandreplication.
Afterthei.
p.
injection,CV-B5/JS417mayabsorbintothebloodstreamthroughthemesenteryandrapidlyentertheintestinaltissue,leadingtorapidviralamplicationandreplicationintheintestine.
Theviralreplicationpeakedathour72post-injectionandalargenumberofvirusesweresubsequentlyreleased,resultinginthesecondaryvirusinltration.
ThechangeofCV-B5viralloadratiosinthespleenandlivertissuesrelativetothebloodalsosuggestedthattheamountofvirusinthebloodrosecontinuouslyuntilhour72.
2.
TissueswithlowVL(<5)rstandthenincreasing,includedthespinalcord,brainandhindlimbmuscles.
Theratiosoftheviralloadintheseorgansremainedatalowleveluptohour24post-challengeandthenincreasedcontinuouslyfromhour72,120,or72tilldeath.
Atthetimeofdeath,theviralloadinthebrain,spinalcord,andhindlimbmuscleswas14,500,4000,and90timeshigherthanthatintheblood,respectively,andmuchhigherthantheVLfoundinotherorgans.
Takingintoaccounttheresultsofpathology,histochemistry,andtheclinicalsymptoms,itappearedthatthebrain,spinalcordandhindlimbmusclesmaybetheprivilegedtargetorgans.
ThedamagetothebrainandspinalcordmayberesponsiblefortheCNSclinicalmanifestations,whicheventuallyleadtoCV-B5-associateddeath.
3.
TissueswithlowVL(<5)rst,thenincreasingandnallydecreasing,includedtheheart,lung,andkidney.
TheVLinthesetissueswaslowinitially,thenpeakedathour72,andultimatelybecamelowattheendoftheexperiment.
Theseresultssuggestthatthevirusmaynotpersistinthesetissuesandmayincreaseinthecourseofthesecondaryviralinvasionintheblood.
Thepathologyandhistochemistryresultsrevealedthatthecardiomyocytenecrosisresultedfromanattackbythehighviralloadintheblood.
ActiveprotectionfromCV-B5vaccinationFemalemice(8weeksold)wereimmunizedwitha10-foldseriallydiluted,inactivatedCV-B5vaccineat1.
58*105to1.
58*108CCID50/mouse,twice,witha2-weekinterval.
Themicewereallowedtomate1haftertherstimmunization.
Theresultingpupswerei.
p.
chal-lengedwithCV-B5/JS417(26LD50)onday3afterbirth(Fig.
6).
Theneonatalmiceinthecontrolgroupstartedtodie5daysafterthechallenge,andallmicediedwithin10days.
Adose-dependentprotectionoftheCV-B5vaccinewasobservedintheneonatalmice;thesurvivalrateswere10044%,30and0%,correspondingtothedammiceimmunizedwithadoseof1.
58*108,1.
58*107,1.
58*106,and1.
58*105CCID50CV-B5vaccine,respectively.
Allmicebornfromthe1.
58*108CCID50-dosedamgroupexhibitednoclinicalsymptomsduringtheentireobservationperiod,andtheyallsurvived.
TheReed–Muenchcalculationrevealedthathalfoftheneonatalmicecouldhavebeenprotectedfromthelethalchallengeiftheirdamshadbeenimmunizedwith9.
58*106CCID50oftheinacti-vatedCV-B5vaccine.
Fig.
5Theviralloadratiosoftissue/seruminCV-B5-challengedmice.
Three-day-oldBALB/cmicewerei.
p.
inoculatedwithCV-B5/JS417(3.
16*103CCID50/mouse).
Toexcludetheimpactoftheviralloadintheserum,theviralloadratio(tissue/serum)wascalculatedbydividingthemeanviralloadofeachtissuewiththeviralloadoftheserumat6,12,24,72,120,and168hpost-challenge(n=3,pertimepoint)Maoetal.
EmergingMicrobes&Infections(2018)7:185Page6of11Passiveprotectionofanti-CVB5serumThree-foldseriallydilutedanti-CVB5serum(neu-tralizingantibody1:768)ormedium(control)wasincu-batedwithanequalvolumeof3.
16*103CCID50CV-B5/JS417at37°Cforonehour(seeMaterialandMethods).
3-day-oldBALB/cmicewerei.
p.
inoculatedwiththemixtureofvirus/anti-serum.
AsshowninFig.
7,theneonatalmicefromthecontrolgroupstartedtodieonday8,and100%mortalitywasreachedwithin10daysFig.
6EvaluationoftheprotectiveeffectoftheinactivatedCV-B5vaccineinthemousemodel.
AdultfemaleBALB/cmicewerevaccinatedwithformaldehyde-inactivatedCV-B5/JS417(experimentgroup)ormedium(controlgroup)twicewitha2-weekintervalandallowedtomate1haftertherstvaccination.
TheresultingpupswerechallengedwithCV-B5/JS417(3.
16*103CCID50/mouse)onday3afterbirth.
Themortality,clinicalsymptoms,andbodyweightweremonitoredandrecordeddailyaftertheinfection(n=6to10,pergroup).
Onerepresentativeresultisshown.
TheMantel–Coxlog-ranktestwasusedtocomparethesurvivalofthepupsbetweeneachvaccinegroupandthemediumcontrolgroupat21dayspost-infection.
***p<0.
001Fig.
7Evaluationoftheprotectiveeffectofanti-CVB5seruminthemousemodel.
Seriallydilutedanti-CVB5serum(neutralizingantibody1:768)ormediumwasincubatedwithanequalvolumeof3.
16*103CCID50CV-B5/JS417at37°Cfor1h.
3-day-oldBALB/cmice(n=6–10,pergroup)werei.
p.
inoculatedwiththedilutedserum.
Themortality,clinicalsymptoms,andbodyweightweremonitoredandrecordeddailyaftertheinfection.
Onerepresentativeresultoftwoindependentexperimentsisshown.
TheMantel–Coxlog-ranktestwasusedtocomparethesurvivalofthepupsbetweeneachanti-serumgroupandthemediumcontrolgroupat21dayspost-infection.
**p<0.
01,*p<0.
05Maoetal.
EmergingMicrobes&Infections(2018)7:185Page7of11post-inoculation.
Thesurvivalratesoftheneonatalmicewere100,83,67,29,and0%,whichcorrespondedtotheinoculatedmixturesofvirus/anti-serumwithratiosof1:15,1:45,1:135,1:405,and1:1215,respectively.
Theseresultsindicatethatanti-CVB5serumconferredprotec-tiontotheexperimentalanimalsinadose-dependentmanner.
AccordingtotheReed–Muenchcalculation,the1:200dilutionoftheanti-CVB5serumprotectedhalfoftheneonatalmice.
TheED50oftheneutralizingantibodytiteragainstCV-B5was1:3.
8.
DiscussionAnimalmodelsareessentialtoolsforvirologyresearch.
In2005,C3H/HeJmicewereusedtoinvestigatethehis-topathologicalchangesandthedistributionofCV-B5RNAintheheart,liver,andpancreasduringtheacutephaseofCV-B5infection31.
HistologicalexaminationrevealedCV-B5-inducedacutepancreatitiswithmassivelymphocyteinltrationandlossofacinarcells.
However,neitherclinicalsymptomsnordeathwerereportedinthatstudy.
In2009,aninvivostudywasconductedonarbidolactivityusingBALB/cmicechallengedwithCV-B532.
Themiceexhibiteddiminishedvitalityandweightloss,andtheydiedaftertheCV-B5challengewithlunglesionsandheartdiseases.
Inaddition,severalpigmodelshavealsobeenusedtostudyCV-B533,34.
However,noCNSinfec-tionsymptoms,whichhavebeenobservedinhumancases,werereportedinthesestudies.
Inthepresentstudy,aCV-B5mousemodelwasestablishedusing3-day-oldBALB/cmice.
Aclinicalstrain(CV-B5/JS417)isolatedfromanHFMDpatientinJiangsuProvinceofChinain2013waschosenasthechallengestrain,asthisstrainhasshownhightoxicityinmice.
Afterthei.
p.
challengewithCV-B5/JS417atadoseof26LD50/mouse,theanimalsexhibitednotonlyanti-feeding,weightloss,malaise,andhairlossbutalsoavarietyofneurologicalsymptoms,suchastrembling,hunching,hindlimbparalysis,andevendeath.
ThepathologicalandhistochemicalexaminationsrevealedeosinophilicnecrosisofnervecellsandthegliacytereactioninthecerebralcortexandthewholespinalcordinducedbyCV-B5.
Toourknowledge,thisistherstanimalmodelthatexhibitssevereacuteCNSinfectionsinducedbyCV-B5,therebyresemblingthehumandisease.
Thedegenerationandnecrosisofcardiomyocyteswerealsoobservedinthismodel.
Itisinterestingtonotethat,liketheothertwomainmembersofcoxsackievirusBgroup,CV-B3andCV-B4,CV-B5alsocausessevereacutepathologicallesionsintheheartandbrainofnewbornmice35–37.
However,CV-B5infectionwasnotshowntoresultinpericarditisorchronicautoimmunemyocarditis,andthesetwoaretheobvioussymptomscausedbyCV-B4andCV-B3inmice35,38.
Inthiscontext,thecharacteristicsofCV-B5infectionaremoresimilartothoseofEV71,anotherenterovirusthatcancauseasepticmeningitis,encephali-tis,andotherseriousneurologicalsymptomsininfantsandyoungchildren39–41.
EV71hasexhibitedawiderangeofneurotropiccellularpropertiesinbothmiceandmonkeysandhasbeenshowntobedistributedthrough-outthenervoussystem42.
StudyingtheVLintissueswillhopefullyhelptoclarifytheviraltranslation/distributioninanimals.
Inthisstudy,wefoundthatCV-B5rstappearedintheintestineathour6post-infectionandreacheditspeakathour72.
However,theVLinthespinalcord,brainandhindlimbmusclesremainedatlowlevelsuptohour24post-chal-lenge,andthentheVLcontinuedtoincreasefromhour72,120,and72,respectively.
Therefore,CV-B5maybeabsorbedintothebloodcirculationthroughthemesen-tery,whereuponitquicklyentersandinvadestheintest-inaltissues.
Afterreplication(peakingathour72)intheintestine,alargeamountofviruswasreleasedintothebloodcirculation,resultinginthesecondwaveofbloodinvasion.
Throughthebloodcirculation,thevirusenteredthespinalcordnervecells,hindlimbmuscles,andmyo-cardialcells,resultinginpathologicaldamage.
However,theVLinthebrainsignicantlyincreasedafterhour120,whichwaslaterthantheincreaseinVLinthespinalcordandhindlimbmuscles.
TheseresultssuggestthatCV-B5mayenterthebrainthroughthespinalaxonswhereitthencausesneuronalcellnecrosis.
ThesendingsarealsosimilartothemechanismoftheEV71andpoliovirus43–47.
In2004,themechanismofEV71infectionwasinvesti-gatedina1-day-oldmousemodelusingvirusisolationdetection.
TheEV71antigenwasdetectedinintestinal,thoracic,cervical,andbrainstemcellsathour6,24,50and78post-challenge,indicatingthatEV71crossedtheblood–brainbarrierthroughtheaxonaltransportneuralpathway43.
Furtherresearchintothepathwaythatfacil-itatesCV-B5transmissiontotheCNSiswarranted.
Vaccinesarethemosteffectiveandeconomicalmeansofpreventingandcontrollinginfectiousdiseases.
His-torically,thedevelopmentandsubsequentapplicationofvaccinesforpolioandhepatitisA,whichareimportantmembersofthePicornaviridae,havesignicantlycon-tributedtothepreventionandcontrolofpolioandhepatitisAonaglobalscale48,49.
Inrecentyears,EV71-inactivatedvaccineshavealsoshowngoodsafetyandimmunogenicitywithaprotectiveeffectofover90%,providingapowerfulweaponinghtingseriousHFMDintheAsian-Pacicregion50,51.
ToexplorethefeasibilityofaCV-B5vaccine,inthepresentstudy,8-week-oldfemalemicewereimmunizedwithanexperimentalvaccinefromtheinactivatedCV-B5virus,andtheirnewbornswerechallengedwithCV-B5/JS417.
Allofthepupsfromthecontrolgroupdevelopedclinicalsymptomsandeventuallydied;however,immunizationofthefemalemicewiththeexperimentalvaccineeffectivelyprotectedtheirnewbornsMaoetal.
EmergingMicrobes&Infections(2018)7:185Page8of11fromtheCV-B5/JS417challenge.
Furthermore,thepro-tectionwaspositivelylinkedtotheimmunizationdosageusedforthedams;thenewbornsexhibitedvaryingresponsesrangingfromnoclinicalsymptomstodelayedonsettodeath,astheimmunizationdosagedecreased.
TheseresultsindicatethattheinactivatedCV-B5vaccineeffectivelyprotectedthenewbornsfromtheCV-B5infectioninanimmunizationdose-dependentmanner.
ImmunizationofthedamswiththeinactivatedCV-B5at9.
58*106CCID50protected50%ofthepupsfromdeath.
Furtherresearchontheantibodyresponseindamsandthetransferofantibodiestothepupswouldbeinterest-ing.
TofurtherinvestigatetheprotectivemechanismoftheinactivatedCV-B5vaccine,thechallengeviruswasneutralizedwithaspecicCV-B5neutralizationantibody(titer1:768),andtheantibody/virusmixturewasinjectedintoneonatalmice.
Dose-dependentprotectionwasagainobserved.
SpecicCV-B5neutralizingantibodieswereshowntohaveanimportantroleinCV-B5prevention.
EpidemiologicalstudieshavealsoindicatedthatafterCV-B5epidemics,CV-B5neutralizationantibodiesinhumansincreasedsignicantly52.
Thesendingslayasolidfoun-dationforthefuturedevelopmentofaCV-B5vaccine.
Insummary,aCV-B5/JS417isolatewassuccessfullyusedtoestablishalethalCV-B5neonatalmousemodelwithgoodreliabilityandreproducibility;thismodelexhibitedthedevelopmentoftypicalCNSinfectionsymptomssimilartothoseobservedinthehumandisease.
ThisisthersttimethataninactivatedCV-B5vaccinehasbeendemonstratedtoprovideprotectionagainstCV-B5challengeinvivo,inwhichCV-B5neutralizinganti-bodiesmayhavethemostimportantrole.
ThismodelisareliableresearchtoolforCV-B5-relatedresearch,parti-cularlyforresearchintothedevelopmentofvaccinesanddrugs.
MaterialsandmethodsEthicsstatementAllanimalresearchprotocolswereapprovedbytheInstitutionalAnimalCareandUseCommitteeatNationalInstitutesforFoodandDrugControl,China(No.
2014-B-004),andtheseprotocolswereconductedinaccordancewiththeregulationsonthemanagementoflaboratoryanimals(NationalScienceandTechnologyCommissionno.
2ofOct.
31,1988)and"guidancenotesonthetreatmentofexperimentalanimals"(Chineseversion(2006)no.
398).
Allinstitutionalguidelinesforanimalcareandusewerestrictlyfollowedthroughouttheexperiments.
CellsandvirusesVerocells(AmericanTypeCultureCollection,Mana-ssas,VA)weremaintainedinEagle'sminimumessentialmediumcontaining2or10%fetalbovineserumplus2mML-glutamine,100IU/mLpenicillin,and100μg/mLstreptomycin.
TheCV-B5strainCV-B5/JS417(GenBank:KY303900)usedinthisstudywasisolatedfromahumanthroatswabsampleofa2-year-oldboy,wasobtainedfromapre-viouslydescribedclinicalHFMDcasecollectionatJiangsuProvinceCenterforDiseaseControlandPreventionandwasanonymizedinmainlandChinain201353,54.
TheCV-B5/JS417strainwasgrowninVerocells,withatiterof3.
16*108CCID50/mL.
MouseinfectionstudyThespecicpathogen-free(SPF)miceusedinthisstudyincludedinbredC57BL/6,BALB/c,ICR,NIHandKunming(KM)mice(NationalInstituteforFoodandDrugControl,Beijing,China).
First,toestablishtheCV-B5-susceptiblemousemodel,themostsusceptiblemousestrainwasidentied.
1-day-oldBALB/c,C57BL/6,KM,ICR,andNIHmicewerei.
p.
challengedwithCV-B5/JS417(3.
16*107CCID50/mouse).
Second,todeterminethemoresusceptibleage,BALB/cmiceat1,3,5,7,and14daysofagewerei.
p.
challengedwithCV-B5/JS417(3.
16*107CCID50/mouse).
Then,todeterminetheoptimalrouteofadministration,3-day-oldBALB/cmicewerechallengedwithCV-B5/JS417(3.
16*107CCID50/mouse)viathei.
p.
,i.
c.
,ori.
g.
route.
TodeterminetheoptimaldosageofCV-B5,3-day-oldBALB/cmicewerei.
p.
challengedwithCV-B5/JS417atvariousdoses(3.
16to3.
16*105CCID50/mouse).
Thecontrolmiceweremock-infectedwithmediumviathesameroute.
Eachgroupcontained6to10mice.
Allmiceweremonitoreddailyforbodyweightandclinicalsymptomsuntildeathor21dpi.
Duplicateexperimentswerecarriedoutforeachexperimentalconditionselection.
TheLD50wascalculatedusingthemethodreportedbyReedandMuench55.
Clinicalsymptomswererankedfrommildtosevere,asfollows:0-healthy;1-inactivityandwasting;2-shivering;3-hunchedpostureandhairloss;4-hind-limbparalysis;and5-moribundanddeath(S1Table).
Histopathologicalexaminationandimmunohistochemicalstaining(IHC)Sixtotenmiceintheexperimentalgroupwerei.
p.
challengedwithCV-B5/JS417(3.
16*103CCID50/mouse).
WhentheclinicalsymptomsofthechallengedmicereachedGrade5(S1Table),themiceinboththeexperimentalandcontrolgroupswereeuthanizedandsubjectedtohistopathologicalandimmunohistochemicalexaminations(IHC).
Thebrain,spinalcord,heart,lung,liver,spleen,kidney,intestines,pancreasandhindlimbmuscleswereisolated,xed,andembeddedinparafnforthehistopathologicexaminationandimmunohistochem-icalanalysis56.
IHCanalysiswasperformedusingtheTMHRP-PolymerAnti-MouseIHCKit(FuzhouMaixinMaoetal.
EmergingMicrobes&Infections(2018)7:185Page9of11BiotechnologyDevelopmentCo.
,Ltd.
,Fuzhou,China)accordingtothemanufacturer'srecommendations.
Amouseanti-CVB5antibodywasusedastheprimaryantibody(1:500dilution)fortheimmunohistochemicaltestinggroup,andthecontrolgroupwaschallengedwithmediumCV-B5/JS417.
VLsoftissuesinpost-challengedinfantmiceAfterbeingi.
p.
challengedwithCV-B5/JS417(3.
16*103CCID50/mouse)oruninfectedculturemedium,tis-suesandbloodsamplesfromtheexperimentalmice(n=3pertimepoint)werecollectedathour6,12,24,72,120,and168post-infectiontoassaytheviralloadsusingreal-timeqRT-PCR56.
BloodandhomogenizedtissueswereharvestedusingtheMagMaxTMviralisolationkit(AMBIONInc.
,Austin,TX,USA).
TheOneStepPrimeScriptTMRT-PCRKit(Takaracompany,Dalian,China)wasusedforRT-PCR.
Briey,cDNAwassynthesizedfromRNAbyreversetranscriptionfor5minat42°Cfollowedby10sat95°C.
ThecDNAwassubsequentlyampliedwith40cyclesof95°Cfor5s,and60°Cfor34s.
Theprimersusedwereasfollows:forward5′-CCCAGTGCCTACGAAAGTGA-3′;reverse5′-CATACGGGGTGGTGCACTC-3′;probe5′-FAM-TACAGCTGGCAGACGTCCACCAA-BQ1-3′.
Viralloadswereexpressedaslog10copies/mgoftissueorlog10copies/mLofblood.
ProtectiveefcacyoftheinactivatedCV-B5vaccineToevaluatetheprotectiveefcacyoftheCV-B5vac-cine,CV-B5/JS417(3.
16*108CCID50/mL)wasinacti-vatedwith37%formaldehyde(SinopharmGroup,Beijing,China)atanalconcentrationof1/2000at37°Cfor3days.
Theinactivatedviralsuspensionswerestoredat80°C.
NoliveviruswasdetectedaftertheculturewasrepeatedinVerocellsthreetimes.
Eight-week-oldfemaleBALB/cmice(n=2pergroup)werei.
p.
injectedtwiceat2-weekintervalswith0.
5mLof10-foldseriallydilutedinactivatedCV-B5/JS417ormediumalone(asacontrol).
Themicewereallowedtomate1haftertherstinjection.
Afterdelivery(5to10daysaftertheboost),thepupswerei.
p.
challengedwithCV-B5/JS417(3.
16*103CCID50/mouse)onday3postnatal.
Thebodyweightandclinicalsymptomsofthechallengedneonatalmiceweremon-itoreddailyuntildeathor21dpi.
Theexperimentswereperformedinduplicate.
TheED50wascalculatedbasedonthemethodreportedbyReedandMuench55.
ProtectiveefcacyofneutralizingantibodiesTounderstandtheprotectiveefcacyoftheCV-B5neutralizingantibody,neonatalmicewerepassivelyimmunizedtoprotectthemagainsttheCV-B5/JS417challengeusingmouseanti-CVB5serum(neutralizingantibody1:768).
Theanti-CVB5serumwas3-foldseriallydilutedfrom1:15to1:1215andthenmixedwithanequalvolumeofCV-B5/JS417virus(3.
16*103CCID50/mouse).
Mediumthatwastreatedinthesamemanner,ratherthananti-serum,wasusedasthecontrol.
Afterinoculationat37℃for1hinatube,3-day-oldBALB/cmice(n=6to10pergroup)werei.
p.
inoculatedwithseriallydilutedmixtures.
Themortalityandclinicalsymptomswerethenmonitoredandrecordeddailyuntil21dpi.
Theexperi-mentswereperformedinduplicate.
TheED50wascal-culatedbasedonthemethodreportedbyReedandMuench55.
StatisticalanalysisAllstatisticalanalyseswereperformedwithGraphPadPrismsoftware(version5.
01)forWindows.
Survivalcurveswerecomparedbythelog-rank(Mantel–Cox)test.
The50%lethaldose(LD50)andthedoserequiredfor50%ofthemaximaleffect(ED50)weredeterminedbythemethodsdescribedbyReedandMuench55.
Apvalueof<0.
05wasconsideredstatisticallysignicant.
AcknowledgementsThisworkwassupportedbytheMajorSpecialProjectsFundingProgram(Nos.
2016ZX09101120and2018ZX09737011)theMinistryofScienceandTechnologyofthePeople'sRepublicofChina.
Authordetails1InstituteforBiologicalProductsControl,NationalInstitutesforFoodandDrugControl,Beijing,China.
2QualityControlDepartment,HualanBiologicalEngineeringInc.
,Henan,China.
3ShandongXinboPharmaceuticalCo.
Ltd.
,Dezhou,China.
4VaccineClinicalEvaluationDepartment,JiangsuProvincialCenterforDiseaseControlandPrevention,Nanjing,ChinaConictofinterestY.
HuisafliatedwithHualanBiologicalEngineeringInc.
,S.
LangandL.
ShenareafliatedwithShandongXinboPharmaceuticalCo.
Ltd.
Theremainingauthorsdeclarethattheyhavenoconictofinterest.
Publisher'snoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalafliations.
SupplementaryInformationaccompaniesthispaperat(https://doi.
org/10.
1038/s41426-018-0186-y).
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