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  • Studiesofpathogenesis,innateimmunityandtherapeuticsofhumanentericvirusesingnotobioticpigs

    TammyBuiCastellucci

    DissertationsubmittedtothefacultyoftheVirginiaPolytechnicInstituteandStateUniversityin

    partialfulfillmentoftherequirementsforthedegreeof

    DoctorofPhilosophy

    In

    BiomedicalandVeterinarySciences

    LijuanYuan,Chair

    Xiang-JinMeng

    XinM.Luo

    LiwuLi

    April24th,2017

    BlacksburgVA

    Keywords:humanrotavirus,humannorovirus,gnotobioticpigs,pathogenesis,dendriticcells,

    anti-diarrhealdrugs

    CopyrightÓ2017TammyBuiCastellucci

  • Studiesofpathogenesis,innateimmunityandtherapeuticsofhumanentericvirusesingnotobioticpigs

    TammyBuiCastellucci

    ACADEMICABSTRACT

    Norovirusandrotavirusarethemostcommonviralcausesofacutegastroenteritis

    amongallagegroupsandinchildrenunder5yearsofage,respectively.Understandingthe

    pathogenesisofthevirusandcorrelatesofprotectiveimmunityisfundamentaltodeveloping

    effectivepreventionandtreatmentstrategies.Gnotobiotic(Gn)pigsareanattractiveanimal

    modelforstudyingentericvirusesduetotheirsimilaritiestohumans,particularlyinregardsto

    theimmunesystemandgastrointestinalanatomyandphysiology.Here,toestablishareliable

    Gnpigmodelofhumannorovirus(HuNoV)infectionanddisease,wedeterminedthemedian

    infectiousdose(ID50)ofaGII.42006bvariantinpigs.Wealsoevaluatedtheeffectsofageand

    administrationofthecholesterol-loweringdrugsimvastatinonsusceptibilitytoNoVinfection.

    Inneonatalpigs(4-5daysofage,theID50wasdeterminedtobe£2.74x103viralRNAcopies.

    TheID50wasincreasedin33-34dayoldpigs(6.43x104),butdecreasedto

  • weightcomparedtocontrolsduringthecourseofthestudy.Wealsodeterminedthatahigh

    doseoftheprobioticLactobacillusacidophilusNCFM(LA)wasabletoreduceRVdiarrhea

    severityanddurationcomparedtoalowdose.Thedifferenceintherapeuticpotentialwas

    attributedtodivergenteffectsininnateimmunitypre-andpost-challenge.HighdoseofLA

    (HiLA)inducedananti-inflammatorydendriticcell(DC)profile,characterizedprimarilyby

    upregulationofTLR2expressionandproductionofcytokineIL-10.Conversely,lowdoseofLA

    (LoLA)upregulatedTLR3andTLR9andincreasedsecretionofcytokineIL-6.Additionally,HiLA

    inducedbothIFN-aandTNF-aresponsesinDCs,butLoLAwasonlyabletoincreasethe

    frequencyofTNF-a-producingDCs.TheseresultsprovidefurthersupportofGnpigsasahighly

    applicableanimalmodelforstudyingpathogenesis,innateimmunityandtherapeuticsof

    humanentericviruses.

  • Studiesofpathogenesis,innateimmunityandtherapeuticsofhumanentericvirusesin

    gnotobioticpigs

    TammyBuiCastellucci

    GENERALAUDIENCEABSTRACT

    Norovirusandrotavirusarethemostcommonviralcausesofacutegastroenteritis

    amongallagegroupsandinchildrenunder5yearsofage,respectively.Understandingthe

    pathogenesisofthevirusandcorrelatesofprotectiveimmunityisfundamentaltodeveloping

    effectivepreventionandtreatmentstrategies.Gnotobiotic(Gn)pigsareanattractiveanimal

    modelforstudyingentericvirusesduetotheirsimilaritiestohumans,particularlyinregardsto

    theimmunesystemandgastrointestinalanatomyandphysiology.Here,weestablisheda

    reliableGnpigmodelofhumannorovirus(HuNoV)infectionanddisease.Overall,the

    developmentofdiarrhea,fecalvirussheddingandsmallintestinalcytopathologicalchanges

    confirmedtheusefulnessoftheGnpigasanappropriateanimalmodelforstudyingHuNoVs.

    Wealsoutilizedthewell-establishedGnpigmodelofhumanrotavirus(HRV)infectionand

    diseasetoevaluateadjunctivetreatmentoptionsforHRV-induceddiarrhea.Wedemonstrated

    thattheanti-secretorydrugracecadotrilwascapableofdiminishingclinicalsignsofHRV

    infectionandshorteningdurationofillness.Wealsodeterminedthatahighdoseofthe

    probioticLactobacillusacidophilusNCFM(LA)wasabletoreduceRVdiarrheaseverityand

    durationcomparedtoalowdose.TheseresultsprovidefurthersupportofGnpigsasahighly

    applicableanimalmodelforstudyingpathogenesis,innateimmunityandtherapeuticsof

    humanentericviruses.

  • v

    DedicatedtoErnestandtoallthosewholovedhim,becausethatprettymuchcoverseveryoneI

    careaboutaswell.

  • vi

    ACKNOWLEDGEMENTS

    Mygraduatecareerbeganinthefallof2008andhasprovidedmetheopportunitytomeetand

    interactwithnumerousintelligent,genuinelyhelpfulandabsolutelywonderfulindividuals.

    Completingthisdegreewouldnothavebeenpossiblewithouttheirassistance.Wordscannot

    fullyexpressmyappreciation,butIwillcertainlytry.

    Iowemuchthankstomyamazingadvisor,Dr.LijuanYuan.Shetookmeinasafairlynaïve

    graduatestudentandhasprovidedsomanylessons,bothinthelabandinlife.Herguidance

    andencouragementhavebeenintegraltomydevelopmentasascientist.Amorebrilliant,

    dependable,caringandsupportivementorisdifficulttoimagine.Thankyoufortheopportunity

    tolearnfromyouandtoconductresearchinyourlab.

    IhavethoroughlyenjoyedworkingwitheveryindividualwhohaspassedthroughtheYuanLab

    throughtheyears.IwouldespeciallyliketooffersincereappreciationtoDr.GuohuaLi,Dr.Ke

    Wen,Dr.FangningLiu,Dr.JacobKocher,Dr.YanruLi,Dr.XingdongYang,Dr.EricaTwitchell,

    MariahWeiss,AshwinRamesh,ShaohuaLei,ChristineTinandRichardKevorkian.Thankyouall

    foryouradvice,assistanceandfriendship.IalsobelieveIhaveaskedeachoneofyouatone

    timeoranothertocheckifIhadleftafridge/freezerdooropen.Thankyouforacceptingmy

    absentmindednessandhumoringmyparanoia.

    GnotobioticpigworkwouldnotberemotelyfeasiblewithouttheTRACSSstaff,includingPete

    Jobst,KarenHall,MariahWeiss,KimAllen,AndreaPulliam,MelissaSquib,LauraThomas,Cassi

  • vii

    BashamandJillStafford.Iwouldalsoliketothanktheveterinarystaff,includingDrs.Sherrie

    Clark-Deener,KevinPelzer,NicoleLindstromandMarliceVonck.Thankyoufortheexcellent

    animalcareandforallyoureffortsinmakingourpigprojectssuccessful.

    Muchgratitudeisalsoduetomyadvisorycommitteememberspastandpresent,includingDrs.

    Meng,Subbiah,Myles,LuoandLi.Yourmentorship,insightfuldiscussionsandsupporthave

    beeninvaluable.

    IwouldalsoliketothankthefacultyandstaffoftheVirginia-MarylandCollegeofVeterinary

    MedicineandtheBMVSprogram,includingDr.AnsarAhmed,Dr.RogerAvery,BeckyJones,

    CindyBoothandSusanRosebrough.Thankyouforprovidingthefoundationforsucha

    wonderfulgraduateprogram.

    IwouldliketothankourcollaboratorsatCincinnatiChildren’sHospitalMedicalCenterand

    PATH,includingXiJiang,MingTan,MingXia,WeimingZhongandRobertChoy.These

    individualsgraciouslyprovidedtheirtime,scientificexpertiseandreagents.Iwouldalsoliketo

    acknowledgethefollowingfundingsourcesfortheirsupport:NationalInstituteofAllergyand

    InfectiousDiseases,NIH(R01AI089634-01);PATH;NationalCenterofComplementaryand

    AlternativeMedicine,NIH(R01AT004789).

    Myparents,sisterandfriendshavebeenamazingintheirsupportandformaintainingmy

    humorandsanity.Specialthanksarereservedformylovingandimmenselypatienthusband,

  • viii

    Matt.He’slistenedtomevent,helpedmetroubleshootvariouslabequipment-related

    emergencies,fedmechocolateandallowedmetoadoptsixanimalsinthespanof8years.

    There’snooneelseI’dwanttoshareabathroomwithwhilesufferingfromgastroenteritis.

  • ix

    ATTRIBUTIONS

    Severalcolleaguescontributedtotheprojects,research,writing,andeditingofeachchapterof

    thisdissertation.

    Chapters2through4:

    LijuanYuan,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)iscurrentlyan

    AssociateProfessoratVMCVMandisthecorrespondingauthoronthesemanuscripts.She

    aidedinprojectdevelopment,writingandeditingofthemanuscripts.

    GuohuaLi,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthe

    manuscriptsandiscurrentlyaResearchScientistatCincinnatiChildren’sHospitalMedical

    Center.Hecontributedtoprojectdevelopment,samplecollection,sampleprocessing,flow

    cytometricanalysis,datainterpretationandeditingofthemanuscripts.

    KeWen,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthe

    manuscriptsandiscurrentlyaPost-doctoralResearchAssociateatTexasTechUniversityHealth

    SciencesCenter.Hecontributedtosamplecollection,sampleprocessingandeditingofthe

    manuscripts.

    XingdongYang,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoron

    themanuscriptsandiscurrentlyaPost-doctoralResearchAssociateattheNationalInstitutesof

    Health.Hecontributedtosamplecollectionandeditingofthemanuscripts.

    Chapters2and4:

    JacobKocher,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoron

    thesemanuscriptsandiscurrentlyaPost-doctoralResearchAssociateattheUniversityofNorth

    CarolinaatChapelHill.Hecontributedtoprojectdevelopment,samplecollectionandeditingof

    themanuscripts.

    FangningLiu,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoron

    thesemanuscriptsandcontributedtosamplecollectionandeditingofthemanuscripts.

    Chapters3and4:

    MariahWeiss(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthese

    manuscriptsandiscurrentlytheProjectandLabManagerwiththeTeaching&ResearchAnimal

    CareSupportServiceatVMCVM.Shecontributedtosamplecollectionandeditingofthe

    manuscripts.

    Chapter2:

  • x

    YanruLi,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthis

    manuscriptandcontributedtosamplecollection,histopathologicalreviewandeditingofthe

    manuscript.

    TanyaLeRoith,DVM,Ph.D(DepartmentofBiomedicalSciencesandPathobiology)isaco-author

    onthismanuscriptandiscurrentlyaClinicalAssociateProfessoratVMCVM.Shecontributedto

    samplecollection,histopathologicalreviewandeditingofthemanuscript.

    MingTan,Ph.D.(CincinnatiChildren’sHospitalMedicalcenter)isaco-authoronthis

    manuscriptandprovidedmaterialsandcontributedtoeditingofthemanuscript.

    MingXia,Ph.D.(CincinnatiChildren’sHospitalMedicalcenter)isaco-authoronthismanuscript

    andprovidedmaterialsandcontributedtoeditingofthemanuscript.

    WeimingZhong(CincinnatiChildren’sHospitalMedicalcenter)isaco-authoronthismanuscript

    andprovidedmaterialsandcontributedtoeditingofthemanuscript.

    XiJiang,Ph.D.(CincinnatiChildren’sHospitalMedicalcenter)isaco-authoronthismanuscript

    andaidedinprojectdevelopment,providingmaterialsandeditingofthemanuscript.

    Chapter3:

    InyoungKim,Ph.D.(DepartmentofStatistics)isaco-authoronthismanuscriptandiscurrently

    anAssociateProfessoratVirginiaTech.Shecontributedtostatisticalanalysisandeditingofthe

    manuscript.

    EricaTwitchell,DVM(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoron

    thismanuscriptandiscurrentlyaGraduateResearchAssociateatVirginiaTech.She

    contributedtosamplecollectionandeditingofthemanuscripts.

    ShaohuaLei(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthis

    manuscriptandiscurrentlyaGraduateResearchAssociateatVirginiaTech.Hecontributedto

    samplecollectionandeditingofthemanuscripts.

    AshwinRamesh(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoronthis

    manuscriptandiscurrentlyaGraduateResearchAssociateatVirginiaTech.Hecontributedto

    samplecollectionandeditingofthemanuscripts.

    SherrieClark-Deener,DVM,Ph.D.(DepartmentofLargeAnimalClinicalSciences)isaco-author

    onthismanuscriptandiscurrentlyanAssociateProfessoratVMCVM.Shecontributedwith

    animalderivationandeditingofthemanuscript.

  • xi

    RobertK.M.Choy,Ph.D.(PATH)isaco-authoronthismanuscript.Hecontributedwithproject

    development,datainterpretationandeditingofthemanuscript.

    Chapter4:

    HaifengWang,Ph.D.(DepartmentofBiomedicalSciencesandPathobiology)isaco-authoron

    thismanuscriptandcontributedtosamplecollectionandeditingofthemanuscript.

    KevinPelzer,DVM,MPVM(DepartmentofLargeAnimalClinicalSciences)isaco-authoronthis

    manuscriptandiscurrentlyaProfessoratVMCVM.Hecontributedwithanimalderivationand

    editingofthemanuscript.

  • xii

    TABLEOFCONTENTSAbstract...................................................................................................................................................iiDedication...............................................................................................................................................vAcknowledgements.................................................................................................................................viAttributions.............................................................................................................................................ixListofTables............................................................................................................................................xviListofFigures..........................................................................................................................................xvii

    Chapter1:Literaturereview

    Acuteviralgastroenteritisduetonorovirusandrotavirusinfections:Studiesofpathogenesis,

    innateimmunity,andtherapeuticsinagnotobioticpigmodel

    1.1INTRODUCTION:ACUTEVIRALGASTROENTERITIS 2

    1.2NOROVIRUS(NOV) 2

    1.2.1CLINICALSIGNSOFNOVINFECTION 31.2.2PATHOGENESISOFNOVINFECTION 5

    1.3ROTAVIRUS(RV) 11

    1.3.1CLINICALSIGNSOFRVINFECTION 111.3.2PATHOGENESISOFRVINFECTION 13

    1.4GNOTOBIOTIC(GN)PIGMODELSFORACUTEVIRALGASTROENTERITIS 17

    1.4.1GENERALOVERVIEW 171.4.2GNPIGMODELOFHUNOVINFECTION 191.4.3GNPIGMODELOFHRVINFECTION 21

    1.5ROLEOFDENDRITICCELLSININNATEIMMUNITYAGAINSTACUTEVIRALGASTROENTERITIS 22

    1.5.1GENERALOVERVIEW 221.5.2DENDRITICCELLRESPONSESTONOVINFECTION 241.5.3DENDRITICCELLRESPONSESTORVINFECTION 24

    1.6PREVENTIONOFACUTEVIRALGASTROENTERITIS 25

    1.6.1DISINFECTIONMETHODS 251.6.2NOVVACCINEDEVELOPMENT 261.6.3RVVACCINEDEVELOPMENT 29

    1.7TREATMENTOFACUTEVIRALGASTROENTERITIS 33

    1.7.1ORALREHYDRATIONSOLUTIONS 331.7.2GENERALANTI-DIARRHEALANDANTI-VIRALAGENTS 341.7.3RV-SPECIFICADJUNCTIVETREATMENTS 361.7.4NOV-SPECIFICADJUNCTIVETREATMENTS 36

  • xiii

    1.8CONCLUDINGREMARKS 37

    1.9REFERENCES 39

    Chapter2

    MedianinfectiousdoseofhumannorovirusGII.4ingnotobioticpigsisdecreasedbysimvastatin

    treatmentandincreasedbyage

    2.1ABSTRACT 69

    2.2INTRODUCTION 70

    2.3MATERIALSANDMETHODS 71

    2.3.1VIRUS 712.3.2INOCULATIONOFGNPIGS 722.3.3SIMVASTATIN 722.3.4BLOODTYPINGOFGNPIGSBYPCRANDIMMUNOFLUORESCENCEASSAY 732.3.5ASSESSMENTOFNOVDIARRHEA 742.3.6DETECTIONOFNOVSHEDDINGBYRT-PCR 742.3.7DETERMINATIONOFNOVSHEDDINGTITERSBYTAQMANREAL-TIMERT-PCR 752.3.8DETERMINATIONOFID50FORNOVGII.4VARIANT092895 762.3.9DETECTIONOFNOVANTIGENININTESTINALTISSUESBYINDIRECTIMMUNOFLUORESCENCE 762.3.10EVALUATIONOFCYTOPATHOLOGICALCHANGESBYTRANSMISSIONELECTRONMICROSCOPY 772.3.11STATISTICALANALYSIS 77

    2.4RESULTS 77

    2.4.1SIMVASTATINFEEDINGLOWERSTHESERUMCHOLESTEROLLEVELS 772.4.2OBSERVATIONOFDIARRHEAINBOTHAGEGROUPSOFGNPIGSFOLLOWINGINOCULATIONWITHNOV 792.4.3GNPIGSSHEDVIRUSINFECESASDETERMINEDBYRT-PCRANDQUANTIFIEDBYTAQMANREAL-TIMERT-PCR 792.4.4VIRUSAMPLIFICATIONINGNPIGS 842.4.5SIMVASTATININCREASESSUSCEPTIBILITYOFNOVINFECTIONANDTHEINCIDENCEOFDIARRHEAINGNPIGS 842.4.6DETERMINATIONOFID50FORTHEGII.4092895VARIANTINNEONATALANDOLDERPIGS,ANDLOWERINGOFTHEID50INTHEOLDERAGEGROUPBYSIMVASTATIN 862.4.7DETECTIONOFNOVANTIGENINTHEDUODENALVILLIOFINFECTEDPIGS 862.4.8CYTOPATHOLOGICALCHANGESINTHEDUODENUMANDJEJUNUMININFECTEDPIGS 88

    2.5DISCUSSION 88

    2.6REFERENCES 96

  • xiv

    Chapter3

    Effectsofracecadotrilonweightlossanddiarrheaduetohumanrotavirusinneonatal

    gnotobioticpigs(Susscrofadomesticus)

    3.1ABSTRACT 101

    3.2INTRODUCTION 101

    3.3MATERIALSANDMETHODS 103

    3.3.1ANIMALS,DIETS,HOUSINGCONDITIONS,ANDHEALTHMONITORING 1033.3.2STERILITY 1053.3.3VIRUS 1053.3.4DRUGTREATMENTGROUPSANDINOCULATIONOFGNOTOBIOTICPIGS 1053.3.5SAMPLECOLLECTION 1063.3.6ASSESSMENTOFROTAVIRUSDIARRHEAANDVIRUSSHEDDING 1073.3.7STATISTICALANALYSIS 109

    3.4RESULTS 110

    3.4.1DETECTIONOFTHIORPHANINPLASMAAFTERRACECADOTRILADMINISTRATION 1103.4.2EFFECTSOFRACECADOTRILONTHEDURATIONANDSEVERITYOFDIARRHEAINGNOTOBIOTICPIGSINFECTEDWITHHRV 1103.4.3EFFICACYOFRACECADOTRILINREDUCINGDEHYDRATIONINGNOTOBIOTICPIGSINFECTEDWITHHRV 113

    3.5DISCUSSION 118

    3.6REFERENCES 124

    Chapter4

    DivergentimpactsofLactobacillusacidophilusdosageonintestinalandsystemicdendriticcellsingnotobioticpigsinfectedwithhumanrotavirus

    4.1ABSTRACT 128

    4.2INTRODUCTION 129

    4.3MATERIALSANDMETHODS 131

    4.3.1VIRUSANDPROBIOTICS 1314.3.2TREATMENTGROUPSANDINOCULATIONOFGNPIGS 1314.3.3ASSESSMENTOFSTERILITY 1334.3.4ASSESSMENTOFLACOLONIZATION 1334.3.5ASSESSMENTOFROTAVIRUSDIARRHEAANDVIRUSSHEDDING 1344.3.6ASSESSMENTOFFREQUENCIESOFCYTOKINEANDTLREXPRESSIONINPDCSANDCDCSBYFLOWCYTOMETRY 1344.3.7STATISTICALANALYSIS 138

  • xv

    4.4RESULTS 138

    4.4.1LACOLONIZATION 1384.4.2ROTAVIRUSSHEDDINGANDDIARRHEA 1404.4.3HIGHEROVERALLTLR2RESPONSESWEREOBSERVEDINHILAPIGS,WHILELOLAINDUCEDHIGHERTLR3ANDTLR9RESPONSESINILEUMANDSPLEEN 1404.4.4HIGHERIL-6RESPONSESWEREOBSERVEDINLOLAPIGSPRE-CHALLENGEANDINHILAPIGSPOST-CHALLENGE,WHILEIL-10WASMORECONSISTENTLYUPREGULATEDINDCSOFTHEHILAGROUP 1454.4.5INCREASEDPRODUCTIONOFIFN-ΑBYDCPOPULATIONSOCCURREDMOREFREQUENTLYINHILAPIGSPOST-CHALLENGE,WHILETNF-ΑPRODUCTIONWASOBSERVEDINBOTHLOLAANDHILAPIGSPRE-ANDPOST-CHALLENGE 148

    4.5DISCUSSION 148

    4.6REFERENCES 158

    Chapter5

    Generalconclusions

    5.1GENERALCONCLUSIONS 163

    5.2FUTUREDIRECTIONS 166

    5.3REFERENCES 168

  • xvi

    LISTOFTABLES

    Chapter2

    MedianInfectiousdoseofhumannorovirusGII.4ingnotobioticpigsisdecreasedbysimvastatintreatmentandincreasedbyageTable1:Cholesterollevels(mg/dL)after11daysofsimvastatinfeeding(8mg/day)............78 Table2:SummaryofdiarrheaandnorovirussheddinginGnpigschallengedwithGII.42006b

    variant(inoculum092895)at4-5daysofage.............................................................80Table3:SummaryofnorovirussheddinginGnpigschallengedwithGII.42006bvariant

    (inoculum092895)at33-34daysofagewithandwithoutsimvastatinfeeding........81Table4:VirusamplificationinolderGnpigswithandwithoutsimvastatinfeeding.............85

    Chapter3

    EffectsofracecadotrilonweightlossanddiarrheaduetohumanrotavirusinneonatalgnotobioticpigsTable1:PlasmathiorphanconcentrationsinGnpigstreatedwithracecadotril(dose80mg/kg)

    ...................................................................................................................................111Table2:Percentageofdayswithdiarrhea(relativetoPID1;mean±SEM)ingnotobioticpigs

    infectedwithhumanrotavirusandtreatedwithPBS(mock),racecadotril,or

    chlorpromazine...........................................................................................................112Table3:Summaryofdiarrheadataanalysisusinglinearmixed-effectsmodel......................112Table4:Bodyweights(%relativetoPID0,mean±SEM)ingnotobioticpigsinfectedwithhuman

    rotavirusandtreatedwithPBS(mock),racecadotril,orchlorpromazine...................117

    Chapter4

    DivergentimpactsofLactobacillusacidophilusdosageonintestinalandsystemicdendriticcellsingnotobioticpigsTable1:ProbioticdoseandfeedingregimensandVirHRVchallenge.....................................132Table2:ProtectionagainstrotavirusdiarrheaafterVirHRVchallenge...................................141

  • xvii

    LISTOFFIGURES

    Chapter2

    MedianInfectiousdoseofhumannorovirusGII.4ingnotobioticpigsisdecreasedbysimvastatintreatmentandincreasedbyageFigure1:RT-PCRtodetectNoVsheddinginlargeintestinalcontents(LIC)...........................83Figure2:DetectionofNoVantigeninintestinaltissuebyimmunohistochemistry................87Figure3:CytopathologicalchangesintheduodenumandjejunumofGnpigsinfectedwithNoV

    092895......................................................................................................................89

    Chapter3

    EffectsofracecadotrilonweightlossanddiarrheaduetohumanrotavirusinneonatalgnotobioticpigsFigure1:MeandiarrheascoreafterinfectionwithhumanrotavirusandtreatmentwithPBS

    (mock),racecadotril,orchlorpromazine.....................................................................114Figure2:Changesinbodyweight(kg)afterinfectionwithhumanrotavirusandtreatmentwith

    PBS(mock),racecadotril,orchlorpromazine..............................................................115

    Chapter4

    DivergentimpactsofLactobacillusacidophilusdosageonintestinalandsystemicdendriticcellsingnotobioticpigsFigure1:RepresentativedotplotsoffrequenciesofTLRs,cytokines,CD80/86andMHCIIamong

    SWC3a+CD4+(pDC)andSWC3a+CD11R1+(cDC)cells................................................135Figure2:LAcolonizationinLoLAandHiLAgroups..................................................................139Figure3:TLRexpressionprofileofDCsfromileum,spleenandPBLofGnpigspre-(PCD0)and

    post-challenge(PCD7)withVirHRV............................................................................142Figure4:IL-6andIL-10expressionprofileofDCsfromileum,spleenandPBLofGnpigspre-

    (PCD0)andpost-challenge(PCD7)withVirHRV........................................................146Figure5:IFN-aandTNF-aexpressionprofileofDCsfromileum,spleenandPBLofGnpigspre-

    (PCD0)andpost-challenge(PCD7)withVirHRV........................................................149

  • 1

    Chapter1

    Acuteviralgastroenteritisduetonorovirusandrotavirusinfections:Studiesof

    pathogenesis,innateimmunity,andtherapeuticsinagnotobioticpigmodel

    T.Castellucci1

    1DepartmentofBiomedicalSciencesandPathobiology,Virginia-MarylandRegionalCollegeof

    VeterinaryMedicine,VirginiaTech,IntegratedLifeScienceBuilding,1981KraftDr,Blacksburg,

    VA24061-0913,USA.

  • 2

    1.1Introduction:Acuteviralgastroenteritis

    Acutegastroenteritis(AGE)presentsasasuddenonsetofdiarrheawithorwithout

    vomiting,andcanincludesymptomsofnauseaandabdominalpainaswell(1).Whiletypically

    self-limitinginduration,casesofAGEstillhaveasignificantimpactonhealthcareand

    economiccosts.Furthermore,diarrheaiscurrentlythefourthleadingcauseofmortalityamong

    allagesglobally,causingapproximately1.3milliondeathsin2015(2,3).Youngchildrenare

    especiallyaffectedbyinfectiouscausesofAGE;diarrheaisthesecondmostcommoncauseof

    deathduetocommunicablediseaseworldwideinindividualsunder5yearsofage,withan

    estimated578,000deathsoccurringin2013(4,5).Amonginfectiouscausesofdiarrhea,

    norovirus(NoV)androtavirus(RV)arethepredominantviralagents.NoVsareimplicatedinthe

    majorityofAGEcasesinallagegroupsworldwide,whileRVsarespecificallythemostcommon

    causeinchildrenunder5yearsofage(6,7).

    1.2Norovirus(NoV)

    NoVsbelongtotheCaliciviridaefamilyandaresmall,non-enveloped,positive-stranded

    RNAviruses(8-12).Theprototypeofthevirus,Norwalkvirus(GI.1),wasdiscoveredfollowing

    anoutbreakofgastroenteritisinNorwalk,Ohioin1968(13,14).Currently,sixgenogroupsand

    >40genotypeshavebeenidentified,withGII.4strainsbeingresponsibleforthemajorityof

    humanNoV(HuNoV)infections(9).MorerecentoutbreakshavealsoincludedGII.17strains

    (15-18).AsidefromGII,othergenogroupsthatareknowntoinfecthumansincludeGIandGIV

    (12).OtheranimalNoVsincludethosethatinfectmice(GV),pigs(GII),cattle(GIII)anddogs

    (GIV,GVI)(19,20).

  • 3

    1.2.1ClinicalsignsofNoVinfection

    TransmissionoftheNoVprimarilyoccursthroughafecal-oralroutefromcontaminated

    food,waterandpersons(21,22).However,NoVinvomitusmaybeacontributingfactorin

    spreadingofthevirusaswell,eitherbyaerosolizationorcontaminationoffomites(23,24).

    Additionally,detectionofNoVinnasopharyngealswabsraisesthequestionofwhether

    respiratorytransmissionispossible(25).Overall,theinfectiousdosehasbeenreportedtobe

    fairlylowat10-100viralparticles(26).

    Afterashortincubationperiodof12-48hours(8,9,11,21,27),symptomsmanifestas

    vomitingandnon-bloodydiarrheawithsomeindividualsalsoexperiencing,nausea,abdominal

    pain,mildfeverandmyalgia(8-10,12,21,27,28).Mostcasesofnorovirusinhealthy,

    immunocompetentindividualsareself-limitingandsymptomsceaseafteratypicalperiodof2-3

    days,butcanlastforupto6days(8-11).Virussheddinginfecescanstartbeforeclinical

    presentation(asearlyas18hoursafterinfection)andtendstopersistformuchlonger,though

    peaksheddinghasbeenreportedatfourdayspost-infection(28).Ingeneral,virussheddingcan

    continueforthreetoeightweeks(8,11,27,28).Asymptomaticsheddingisalsopossibleand

    hasbeenreportedinapproximately1/3ofcases(8,29).Inbothsymptomaticand

    asymptomaticinfections,highsheddingtitersbetween105-10

    11viralcopiespergramoffeces

    canbeexpected(29).Inarecentstudy,NoVtitersinvomituswerereportedas8.0x105and3.9

    x104viralcopiespermilliliterforGIandGIIviruses,respectively(24).Thecombinationof

    sheddinginhightiters,oflongdurationandinasymptomaticindividuals,alongwithlow

    infectiousdosefacilitateseaseofNoVtransmission.

  • 4

    WhileNoVsarecapableofinfectingallagegroups,moreseverepresentationsandeven

    mortalitiescanoccurintheyoung(65yearsofage)or

    immunocompromised(9,30,31).Aswithmostcausesofdiarrhea,severedehydrationisa

    commoncomplicatingfactor.IntheUS,childrenunder5yearsofagehavehigherratesof

    clinicalvisitspertainingtoNoV-relatedillnesses(32).Longerdurationofsymptoms(7versus3.5

    days)andgreaterseverityofdiarrheahasbeenreportedinchildren

  • 5

    (44,54).Inadditiontoseveredehydration,complicationsarisingfromchronicdiarrheacan

    includedisruptionoftheintestinalbarrier,malnutrition,acutekidneyinjuryand

    transplant/graftfailure(48,55).Anincreasedoccurrenceofpneumatosisintestinalis,orgas

    withintheintestinalwall,hasbeenreportedaswell.Fouroutoffiveimmunocompromised

    individualswithpneumatosisintestinaliswereNoV-positiveinoneretrospectivestudy(56).

    Virussheddingisprolongedcomparedtohealthy,immunocompetentpatientsandhas

    continuedforoverayearincertaincases(44,57).Suchlongperiodsofsheddingcanpossibly

    actasareservoirforinfectionandallowfortheemergenceofnewvariants(55,58).

    NoVinfectionshavealsobeenrarelyassociatedwithvariousotherunique

    presentations.Asidefromnecrotizingenterocolitisandneurologicalmanifestationsinyoung

    children,theseincludehemolytic-uremicsyndrome(59),ischemiccolitis(60),flare-upsof

    inflammatoryboweldisease(61),post-infectiousirritablebowelsyndrome(62)and

    spontaneousbowelperforation(63).ViremiaisnotacommonoccurrencewithNoVinfections

    andmayexplainthepredominanceofintestinaldisease.Viremiahasnotbeenreportedin

    healthyadults;onlyinchildren(64),immunocompromisedindividuals(65)andgnotobioticpigs

    (66,67).

    1.2.2PathogenesisofNoVinfection

    Historically,thelackofanefficientcellculturesystemandsmallanimalmodelfor

    humanNoVs(HuNoVs)haslimitedtheunderstandingofpathogenesis.Themostwell-studied

    aspectislikelythebindingofNoVstohisto-bloodgroupantigens(HBGAs).HBGAsarecomplex

    carbohydratesthatcanbefoundonerythrocytesandvariousepithelialtissues,aswellas

  • 6

    secretedfreelyinbiologicalfluidsofsecretors(seebelow)(68).Thespecificphenotypic

    expressionofHBGAsinanindividualisdependentondifferentfunctioningglycosyltransferases.

    Inhumans,ABO,secretorandLewisfamiliesareinvolvedinthebiosynthesispathwaysthat

    producethemajorHBGAs(69).TheFUT1geneencodesana1,2fucosyltransferasethat

    synthesizesH(O)antigenonerythrocytes,whiletheFUT2geneisresponsibleforthea1,2

    fucosyltransferasethatresultsinHantigenexpressiononmucosalepithelialsurfacesandas

    freeoligosaccharidesinsaliva,milkandintestinalcontents(70,71).Individualswithatleastone

    functionalFUT2allelearedesignatedas‘secretors’.AandBantigensareproducedthrough

    subsequentmodificationoftheHantigenbyAandBglycosyltransferases(ABOgene),

    respectively(71).FurtheradditionofcarbohydratemoietiesbytheFUT3gene-encodeda1,3/4

    fucosyltransferaseleadstoexpressionofLewisantigensinsecretors(Leb,Le

    y)andnon-

    secretors(Lea,Le

    x)(68,69,72).Altogether,individualscanbeclassifiedbasedontheirABO

    blood-type,aswellaswhethertheyaresecretorsornon-secretorsandLewis-positiveor

    negative.

    Basedontheknowledgethatrabbithemorrhagicdiseasevirus,whichisacalicivirus,

    bindstoaHGBA(73,74),itwaspostulatedthatNoVsmayusesimilarantigensascellular

    receptors.Indeed,studiesdemonstratedHuNoVvirus-likeparticles(VLPs)hemagglutinating

    andbindingtoHBGASinsalivaandonintestinalepithelialcellsofsecretors(75-78).CanineNoV

    VLPshavealsobeenshowntobindHBGAsinvitro(20).TheuseofHBGAsascellularligandsfor

    NoVbindingwasfirstsupportedinvivobyastudyreportingassociationofNorwalkvirus(GI.1)

    infectioninhumanswithABOhisto-bloodtype.Thestudyrevealedanincreasedriskof

    infectioninthosewithOblood-typecomparedtoBblood-type(79).Furthercharacterizationof

  • 7

    susceptibilityconfirmedthat,inadditiontoblood-type,secretorstatuswasanequally

    importantdeterminantofsymptomaticinfectionsasnon-secretorswereresistanttoinfection

    withaGIINoVinonestudy(80).Manyotherinvestigationssincethenhavealsosupported

    increasedsusceptibilitytoNoVinfectioninpersonswhoaresecretors(17,72,81-86).

    Resistanceinsomesecretorindividuals,aswellassymptomaticinfectioninnon-secretors(87-

    89),hasbeenexplainedbyrecognitionofseveralstrain-specificbindingprofilestoABOand

    Lewisantigensthroughhumanstudies,invitroassaysandstructuralanalysisoftheNoVcapsid

    protein(71,90-94).Ingeneral,twomajorclassificationsofHBGAbindinghavebeenproposed

    andincludethosestrainsthatbindA/BantigensandthosethatbindLewisantigens(90).

    ThroughX-raycrystallography,ithasbeendeterminedthattheP2subdomainofthe

    NoVcapsidprotein(VP1)istheprimarysiteofbindinginteractionswithHBGAs.TheNoVcapsid

    proteiniscomprisedofshell(S)andprotruding(P)domains,withthePdomainbeingfurther

    subdividedintoP1andP2.TheP2subdomainisfoundontheoutermostsurfaceofVP1,making

    itanidealtargetasareceptorinterface.IthasbeensuggestedthatthepolymorphismofHBGA

    phenotypesinthehumanpopulationandmutationsoftheP2domaininfluenceNoVevolution

    (95-97)

    OtherpotentialreceptorcandidatesforHuNoVinfectionhaveincludedheparinsulfate

    (98),sialicacid(99)andb-galactosylceramide(100).FormurineNoVs(MNV),inadditionto

    sialicacid(101),glycolipidandglycoproteinreceptors(102),recentevidenceforproteinaceous

    cellreceptors,CD300lfandCD300ld,hasbeendemonstrated(103,104).

    ThecellulartropismforNoVsremainsincompletelyunderstood,withbothintestinal

    epithelialcells(IECs)orenterocytesandimmunecellsbeingviablecandidates(105-109).

  • 8

    Initially,basedonthemultitudeofevidenceforHBGAsascellularligandsandthenatureof

    NoVsasentericviruses,itwasspeculatedthatIECsexpressingHBGAsweretheprimarytargets

    ofNoVinfection.DetectionofNoVantigeninIECshasbeendemonstratedinchronically

    infectedhumantransplantpatients(110),Gnpigs(66),Gncalves(111)andSTAT1-/-mice(112),

    indicatingthatthesecellscanatleastbeinfectedinimmunocompromisedindividuals.Yet,until

    recently,multipleattemptstocultureHuNoVinepithelialcelllineshavefailed(113-116),even

    withthepresenceofHBGAs(117).SuccessfulHuNoVreplicationhasnowbeenestablished

    utilizingstemcell-derivedhumanintestinalenteroidswithandwithouttheinclusionofbileacid

    supplementation(118),thoughroutineuseofthismodelremainstobeimplemented.Still,the

    enteroidcellculturesystemprovidesstrongevidenceforthecontinuednotionthat,similarto

    otherentericviruses,NoVscaninfectenterocytes.

    Meanwhile,MNVshavebeenreadilyculturedindendriticcells,macrophagesandBcells

    (119,120).Invivo,MNVantigenhasalsobeendetectedintheaforementionedimmunecells

    (112,119-121)andintestinalviraltiterswerelowerinBcell-deficientmice(122),further

    suggestingtheimportanceofimmunecellsinthepathogenesisofMNVinfection.Ithasbeen

    proposedthat,inordertotraversetheintestinalepithelialbarrierandaccessimmunecells,

    virusistrancytosedbymicrofold(M)cells(106,107,123).Mcellsarespecializedintestinalcells

    thatareheavilyinvolvedinimmunosurveillancethroughtheengulfmentofmicroscopic

    materialfromthelumen,whichalsomakesthemakeyentryportalforpathogens(124).In

    micethatweredepletedofMcells,reducedviraltiterswereobservedcomparedtocontrols

    (125).

    EvidenceofHuNoVsinfectingimmunecellsalsoexists,thoughismoreconflicting.Inthe

  • 9

    previouslymentionedstudydemonstratingviralantigendetectioninenterocytesof

    immunocompromisedhumantransplantpatients(110),antigenwasalsodetectedin

    macrophages,dendriticcellsandTcellsinintestinalbiopsies.Yet,Norwalkvirus(GI.1)was

    incapableofreplicatinginhumanmonocyte-derivedmacrophagesanddendriticcells(126).In

    termsofBcellinfection,onlytheGII.4SydneystrainamongHuNoVshasbeenshownto

    replicateinBcellsinvitro(120,127).Thesituationinvivoislikelyevenmorecomplicated,as

    similarincidenceofNoVinfectionoccurredinBcellcompetent(60%)anddeficient(63%)

    childrenwithseverecombinedimmunodeficiency(SCID)(128).However,theviraltiterinBcell

    deficientindividuals,whilestillonthescaleofmillionsofgenomiccopiespermilliliter,was

    significantlyreducedcomparedtothosepatientswithBcells.Incontrast,infectionwithaGII.4

    HuNoVinRAG2/IL2RGdeficientGnpigs,whichlackBcells,Tcellsandnaturalkillercells,

    provedtobeofhighertiterandlongerdurationversuswildtypecontrols(67).Aswithprevious

    studiesinGnpigs,detectionofviralantigenoccurredinenteroctyesofthesmallintestine(66,

    129).Overall,itappearsthatregulationofNoVcelltropismiscomplex,possiblydependingon

    virusstrain,hostspecies,immunestatusandotherpotentiallyundeterminedinnateor

    environmentalfactors.

    Interestingly,theabilityofNoVtoreplicateintheBJABhumanBcelllinewasdependent

    onthepresenceofHBGA-expressingentericbacteriaasuseoffilteredstoolsamplesforculture

    resultedindecreasedtiters(120).AdditionofHtype-positiveEnterobactercloacaetocultures

    subsequentlyreestablishedNoVinfectivity.Inthesamestudy,antibiotictreatmentofmice

    priortoMNVinfectionsignificantlyreducedviraltiters.Anotherrecentstudyalsorecognized

    GII.4andGI.6NoVbindingtoentericbacteria,againpossiblyduetointeractionswithHBGA-like

  • 10

    moieties(130).Thus,HBGAsinthecontextofintestinalmicrobiotamaybeinvolvedin

    regulatingNoVpathogenesisaswell.However,theexactinteractionbetweenvirusand

    bacteriaisstilluncertain.ArecentstudyinGnpigsdemonstratedreducedviraltitersinpigs

    colonizedwithE.cloacaeandsubsequentlyinfectedwithaGII.4humannorovirus(131).The

    authorssuggestedthatHBGA-expressingbacteriamayfunctiontoactasdecoyreceptors,thus

    blockingratherthanenablingbindingtoviralreceptors.Furtherstudieswillneedtobe

    conductedtoclarifytheroleofHBGA-expressingbacteriainNoVinfection.

    AnotherunansweredquestionregardingNoVpathogenesisinvolvesreconcilingthe

    relativelackofintestinalpathologyandinflammationwiththeseverityofdiarrheathatcan

    occur(108,109).Moststudiesinbothhumans(132-134)andanimalmodels(66,111,135)

    reportfairlymilddisruptionoftheintestinalepithelium,withbluntingofvillibeingmost

    common.Otherchangesobservedincludemicrovillidestruction,mitochondrialdistension,

    cytoplasmicvacuolization,intercellularedemaandcellapoptosis(129).Therefore,itis

    reasonabletoassumethatothermechanismsofdiarrheamaybeinvolved.Onestudyofhuman

    duodenalbiopsiesreportedadecreaseintransepithelialresistancefollowingNoVinfection,

    likelysecondarytoareductionintightjunctionproteinsoccludin,claudin-4andclaudin-5(133).

    SuchfunctionalimpairmentsmaycontributemoretoNoVdiarrheathanstructuraldamageof

    enterocytes.FurtherstudiesareneededtounderstandthepathophysiologyofNoVdiarrhea.

  • 11

    1.3Rotavirus(RV)

    RVsbelongtotheReoviridaefamilyandarenon-enveloped,segmented,double-

    strandedRNAviruses(136,137).BasedonthestructuralproteinVP6,RVsareclassifiedintoat

    least8differentgroups,denotedwithlettersA-H(136,138).RVA,RVBandRVCcauseinfection

    inhumansandcertainanimals,whileRVD-RVHareexclusivelyassociatedwithanimal

    infections.Recently,newcanineandbatspeciesofRVhavebeensuggestedtocompriseRVI

    andRVJgroups,respectively(139,140).Withingroups,RVsarefurtherclassifiedbasedon

    capsidproteinsVP7(glycoprotein-Gtype)andVP4(proteasesensitiveprotein-Ptype)(137).

    AmongtheRVAgroup,whichisthemostcommonlyinvolvedinhumaninfections,G1P[8],

    G2P[4],G3P[8],G4P[8],G9P[8]andG12P[8]arethemostprevalentstrains(141).

    1.3.1ClinicalsignsofRVinfection

    RVinfectionismostprevalentininfantsandyoungchildrenandischaracterizedbynon-

    specificsymptomsofacutegastroenteritis.Vomitingfollowedbywatery,non-bloodydiarrhea

    typicallyoccurswithin48-72hoursofinfectionviathefecal-oralrouteandcanbeaccompanied

    byfever,abdominalcramps,nauseaandheadaches(136,142-146).Severityofsymptomscan

    varyfrommildtodebilitatinganddiarrheacanpersistforuptooneweek(146,147).

    Duetotheonsetofbothvomitinganddiarrhea,deathcanoccursubsequenttoextreme

    dehydration,electrolyteimbalancesandcardiovascularfailureifmedicalinterventionisnot

    available(136,142).DehydrationismorecommonwithRVinfectioncomparedtoother

    bacterialorviralagentsofgastroenteritisinchildren(143).Casesthatrequirehospitalization

    andeitheroralorintravenousrehydrationtherapyareintheminoritythough,andRVinfection

  • 12

    ismoreoftenself-limiting(147).Childrenbetweenthreemonthstothreeyearsofagearemore

    likelytopresentwithsevereillness(145).Mortalityratesarehighestinunderdeveloped

    regions,suchasinSaharanAfricaandsoutheastAsia,whereaccesstoappropriatemedicalcare

    canbelimitedandmalnutritionorcoinfectionswithotherenteropathogensexacerbateclinical

    symptoms(136,147-149).

    Virussheddingoftenprecedesonsetofclinicalsymptomsandcanusuallybeexpected

    tocontinueforupto10days,withanaverageoffourdaysduration(145,150).Peaklevelsof

    sheddinginyoungchildrencanproduceasmuchas1010-11

    viralparticlespergramoffeces

    (142).Prolongedsheddinghasbeenreportedwithsevereinfections(150)andin

    immunocompromisedindividuals(145,150,151).

    AsymptomaticRVinfectionsareknowntooccur,bothinyoungchildrenandinadults.

    Thoughsevereinfectionscanoccurinneonates,infectionsatthisagearetypically

    asymptomaticduetomaternalantibodyprotectionviaplacentaltransferorbreastfeeding(145,

    152,153).Furthermore,individualsencountermultipleRVinfectionswithageandhaveless

    severesymptomswithsuccessiveinfectionsduetopartialimmunity(145,154-156).Fecalvirus

    sheddingcontinuestooccurintheseinfections,butoftenatmuchlowertitersthanwith

    symptomaticcases(142,157).

    However,notallRVre-infectionsintheadultpopulationareasymptomatic(158-160).In

    arecentstudyofadultsrequiringhospitalizationforacutegastroenteritisinDenmark,RVwas

    determinedtobethesecond-mostcommon(25/265patientsor9.4%)enteropathogen

    implicated(161).Similarly,intheUS,RVwasdetectedasthecauseofdiarrheain18%ofcases

    (19/106)fromseveralemergencycenters(162).Althoughelderlyandimmunocompromised

  • 13

    individualsareunderstandablymorepredisposedtoinfection(163,164),outbreakshavebeen

    reportedinyoungadultsaswell(159).Thesefindingssuggestthatrotavirusshouldstillbe

    consideredasacausativeagentinpresentationsofacutegastroenteritisinadults.Symptomsin

    adultsaresimilartothoseobservedinyoungchildren(142).

    RVreplicatesinthesmallintestineandexpectedlyresultsinlocaldisease,butthereare

    severalreportedinstancesofextraintestinalmanifestations.Seizuresarethemostcommonly

    observedpresentationofsystemicinfection(165-167),withbothfebrileandafebrileseizures

    beingpossibleandgenerallyoccurringwithoutlastingconsequences.Meningitisand

    encephalitisareamongtheotherprevalentneurologicaldiseasesoccurringwithRVinfection

    (165,168,169).Thoughevenrarer,tentativeassociationshavealsobeenproposedforcasesof

    pneumonia(170),disseminatedintravascularcoagulation(171),exanthema(172),

    haemophagocyticlymphohistiocytosis(173)andautoimmunediseasessuchdiabetesmellitus

    amongothers(174).

    Ingeneral,ithasbecomeacceptedthatantigenemiaandviremiafrequentlyoccurwith

    acuteRVinfections,eitherwithorwithoutdiarrhea(168,175-178).Althoughusuallyclinically

    insignificantinthecourseofillness,thisevidencemayexplaintheabilityofRVtocausedisease

    outsideofthegastrointestinaltract.

    1.3.2PathogenesisofRVinfection

    Rotavirusiswidelyknowntoinfectandreplicateinmatureenterocytesofsmall

    intestinalvilli(136,179,180).HowexactlyRVbindstothesecellsandinduceswaterydiarrhea

  • 14

    andvomitinghasbeenthesubjectofmultiplestudies.Fromtheseobservations,itisclearthat

    thepathogenesisofRVinfectionismultifactorialandpossiblyincompletelyunderstood.

    ThemaincapsidproteinsofRVareVP4andVP7,withVP7comprisingtheoutershellof

    thecapsidandVP4formingthespikeprotein(137).Assuch,VP4,isprimarilyresponsibleforRV

    attachmenttoenterocytes.VP4isenzymaticallycleavedbytrypsinintoVP5*andVP8*,with

    VP5*formingthemainstemandVP8*formingtheheadofthespikethatdirectlyencounters

    receptorsonsmallintestinalcells(136,181).Initially,sialicacidmoietieswerethoughttobe

    involvedinRVbinding,sinceinteractionofsomeanimalRVstrainswithcellsurfacesis

    interruptedbysialidasetreatment(182).However,itwaseventuallydeterminedthathuman

    andmostanimalRVstrainsweresialidase-resistantandlikelynotdependentonsialicacidfor

    cellularentry(181,183).Basedonmultidisciplinarystudiesinvolvingstructuralbiologyand

    glycobiolity,itbecameevidentthatRVscanuseHBGAsascellularreceptors(181,184-188).

    Asmentioned,theseligandshavepreviouslybeendeterminedtoberesponsibleforthe

    cellularbindingofNoV(76,92).SinceNoVcaninfectenterocytes,itwasspeculatedthatRV

    mayproceedsimilarly.Indeed,frominvitroassaysusinghumansaliva,milksynthetic

    oligosaccharides,rotavirusVP8*wasdemonstratedtobindtoHBGAs(186).Currentknowledge

    indicatesRVstrain-specificbindingtoA,Htype1and/orLewisbantigens(185,186).Most

    studieshavealsodeterminedincreasedsusceptibilitytosymptomaticinfectioninsecretor-

    positiveindividuals(82,189-191),thoughconflictingevidenceofinfectioninnon-secretors

    doesexist(192).Yet,arecentstudydiddemonstrateincreasedRV-specificsalivaryIgAtitersin

    secretors(193).

  • 15

    HBGAsarenottheonlyglycansthathavebeendemonstratedtointeractwithRVs.VP8*

    alsointeractswithgangliosidesinastrain-dependentmanner(194).Asstatedpreviously,some

    animalstrainsdobindsialicacidasconfirmedbytheirsensitivitytosialidases.Thesestrainsare

    abletobindthesialicacidfoundasaterminalmoietyonthegangliosideGM3(195,196)andas

    terminalandinternalmoietiesongangliosideGD1a(197).Incontrast,sialidase-insensitiveRVs,

    whichincludemosthumanstrains,appeartointeractwiththegangliosideGM1athatlacksa

    terminalsialicacid(197).Ofnote,GM1acontainsaninternalsialicacidmoietythatcaninteract

    withVP8*anditisnotaffectedbysialidases.

    InadditiontoVP8*,VP5*andVP7havealsobeenimplicatedfortheirroleincellular

    binding.VP5*hasbeendemonstratedtobindintegrina2b1(198),whileVP7bindsintegrins

    axb2andavb3(198,199).Heatshockcognateproteinhsc70(200)andtightjunctionprotein

    JAM-A(201)arealsoinvolvedinRV-hostcellinteraction.Onceboundtoenterocytes,RVhas

    beenshowntoenterthecellviaendocytosis,thoughtheexactmechanismappearstobestrain

    dependent(202-204).

    MalabsorptionisoneofthefundamentalmechanismsofRV-induceddiarrhea.Inthe

    smallintestine,maturevillusenterocytesarenon-replicatingcellswithnumerousmicrovillithat

    areinvolvedinbothactiveandpassiveabsorptionoffluidandelectrolytes,aswellasenzymatic

    digestionoffood.Conversely,intestinalcryptcellsproliferateinself-renewalofintestinal

    epitheliumandmaintainasecretorycapacity,releasingchlorideionsintotheintestinallumen

    (179).WithRVinfection,structuraldamagetotheabsorptivematureenterocyteshaslongbeen

    acceptedasamajorcontributortothepresenceofwaterydiarrhea.Earlyhistologicalstudyof

    duodenumsamplesformchildrendiagnosedwithRVinfectionidentifiedbluntingofvilli,

  • 16

    vacuolizationofepithelialcells,increaseincuboidalversuscolumnarepithelium,intestinal

    crypthyperplasiaandamildinfluxofinflammatorycells(136,179,205-208).Intestinalvilli

    damagehasalsobeenreportedduetoischemiainmice,thoughtherelevancetohumansis

    unclear(209).Combined,thereducedabsorptivecapacityduetoenterocytelossresultsin

    increasedintestinalfluid.

    However,theselesionsarenotalwaysconsistentlyobservedandoftendonot

    necessarilycorrelatewithclinicalsymptoms(146,179).Assuch,amaldigestioncomponenthas

    alsobeenimplicatedasacauseofRVdiarrhea.Adecreaseindigestivefunctionhasbeen

    reportedsubsequenttoalossofdisaccharidasesthatarelocatedatvillitips(205,208,210,

    211).Thisproducesanabundanceofcarbohydrates,whichactasosmoticallyactiveparticles

    andfurtheraddstothewaterbeingdrawnintotheintestinallumen.

    AuniqueaspectofRV-induceddiarrheaistheroleofnonstructuralprotein,NSP4,which

    actsasanenterotoxin.NSP4isproducedbyinfectedenterocytes(146,179,208,212,213)and

    peptidesaresecretedintotheintestinallumentofurtheraffectneighboringcells(214).NSP4

    appearstohavemultipleeffectsonenterocytesthatcollectivelycontributetointestinalfluid

    lossandsecretion.ItactstoreleaseintracellularCa2+fromcells,whichcaninducechangesin

    cellulariontransportandpermeability(208,215,216).IthasbeensuggestedthattheCa2+

    signaltransductionproducesamild,netchloridesecretionthroughacysticfibrosis

    transmembraneconductanceregulator(CFTR)-independentprocess(208,212,217).NSP4has

    alsobeendemonstratedtoinhibitthesodium-glucosetransporterSGLT1,reducingwater

    reabsorption(218).Additionally,thereissomeevidencethatthissignalingcanmodifyactin

  • 17

    filamentsofmicrovillianddisruptintestinaltightjunctions,possiblyincreasingintestinal

    permeability(216,219,220).

    Furthermore,Ca2+signalingcaninciteproductionofcytokines,prostaglandinsand

    reactiveoxygenspeciesthathavebeenpostulatedtostimulatetheentericnervoussystem

    (ENS)(208).Locatedintheintestinalwall,theENSisconvenientlyabletosensechangesinthe

    intestinalenvironmentandaltersecretionoffluidandelectrolytesinresponse(221).Serotonin

    (5-hydroxytryptamineor5-HT)isanimportantneurotransmitteroftheENSandissecreted

    exclusivelyinthesmallintestinebyenterochromaffin(EC)cells(207).Intermsof

    gastrointestinaleffects,itisresponsibleforgutmotility,intestinalsecretion,bloodflowand

    vagally-mediatedvomiting(207,222).Recently,RVhasdemonstratedcapabilityofinfectingEC

    cellsandsubsequentNSP4releaseactivatesserotoninsynthesistoinfluencediarrhea,nausea

    andvomiting(207,222).Additionally,serotoninantagonistshavebeenshowntoreduceRV

    diarrheainmice,furtherconfirmingtheroleofENSinRVpathogenesis(207,223).Intermsof

    ENSinvolvementinintestinalmotility,thedrugsloperamideandatropine,whichfunctionto

    increaseintestinaltransittimeandallowforagreaterperiodoffluidabsorption,havebeen

    showntoattenuatediarrheainducedbyRVinfection(224).

    1.4Gnotobiotic(Gn)pigmodelsforacuteviralgastroenteritis

    1.4.1Generaloverview

    Domesticpigs(Susscrofadomesticus)arebeingincreasinglyutilizedasananimalmodel

    inbiomedicalresearch.Whilepigs,evenminipigbreeds,representagreaterhusbandry

  • 18

    challengeduetotheirsize,thereareseveralfactorsthatmakethemmoreidealthanrodentor

    otheranimalmodelsforstudyinghumandiseases.Inparticular,pigsareanatomically,

    physiologicallyandimmunologicallyfairlyequivalenttohumans(225-227).Manystudiesthus

    haveexploitedthesimilaritieswiththeintegumentary,cardiovascular,urinaryanddigestive

    systems(225).Additionally,thereproductivecharacteristics(shortgenerationinterval,large

    littersize),easeofadequatesamplecollectionandavailabilityofnumerousoutbredandinbred

    linesarealsoadvantageousqualities(227).

    Intermsofutilizingpigsasananimalmodelforstudyingentericviruses,the

    resemblancewiththehumangastrointestinaltractandimmunesystemareespecially

    appropriate.Therearegrossanatomicaldifferencesinthestomach,smallandlargeintestine

    (225),butthedigestivephysiology,aswellassusceptibilityandresponsetodiseaseare

    comparable(227-229).Furthermore,pigshavesimilarintestinalvillistructureandcell

    classification(228)(230),enablingstudiesofdiseasepathophysiology.Forimmunological

    evaluation,itisespeciallybeneficialthat>80%oftheimmuneparametersassessedare

    analogoustohumans(231)andthatallimmunecellpopulationsaresimilarlypresentinpigs

    (227).

    Germ-freeanimalsarethosethataredeemedmicrobiologicallysterilebyspecific

    testing,whilegnotobiotic(Gn)animalsarethosethatareassociatedwithknownand

    identifiableorganisms(232).Technically,germ-freeanimalscanalsobecalledGnbecausethe

    statusofmicroorganismsisknown.Gnmammalsarederivedbyaseptichysterectomyinto

    sterileisolators,wheretheyarefedasterilized,commercialmilkdiet.Gnpigshavebeen

    availableforbiomedicalresearchsincethe1960’sandpresentauniqueopportunitytoevaluate

  • 19

    host-pathogeninteractionsandrelatedimmunity.Unlikehumansandmice,pigshaveadiffuse

    epitheliochorialplacentation,whichpreventsthetransferofmaternalantibodiesduring

    intrauterinedevelopment(225,233).Neonatalpigsacquirepassiveimmunityviaingestionof

    colostrumwithinthefirst36hoursafterbirth(234)toaidinprotectionagainstpathogenswhile

    theirimmunesystemsarestilldeveloping.Thelackofmaternalantibodyinterference,

    intestinalmicrobiotaandunknownpathogensallowsforspecificevaluationofprimaryimmune

    responsestochosenentericmicrobesandvaccines.

    Still,recentstudieshavehighlightedtheimportanceoftheintestinalmicrobiotain

    influencingentericimmunityandresponsetoinfections(235,236).Tobetterelucidatethese

    relationships,Gnpigshavebeensuccessfullytransplantedwithhumangutmicrobiota(235-

    237).Additionally,withthedevelopmentoftheCRISPR/Cas9systemforgeneediting,the

    manipulabilityoftheGnpigmodelhasbroadenedevenfurther(67).

    Currently,inadditiontoseveralotherGnpigmodelsofentericpathogens(238-240),

    modelsforHuNoVandHRVhavebeenwell-established.

    1.4.2GnpigmodelofHuNoVinfection

    Asstated,efficientcellculturesystemsforHuNoVarelacking,makinganimalmodels

    evenmorevitalforstudyingpathogenesis,immunityandresponsetovaccinesand

    therapeutics.AsidefromGnpigs,otheranimalmodelsincludemice(241),Gncalves(111),and

    non-humanprimates(135,242).However,Gnpigshavebeensuggestedasamoresuitable

    modelduetopresenceofclinicalsigns,i.e.diarrhea,comparedtomiceandnon-human

  • 20

    primates.Additionally,pigshavealsobeendemonstratedtohavesimilarintestinalHBGA

    expressionandNoVbindingashumans(243).

    TheGnpigmodelofHuNoVinfectionanddiseasewasfirstdescribedin2006usinga

    GII.4strain(HS66)(66).Theauthorsreportedmilddiarrheain74%ofinfectedpigs,aswellas

    fecalvirussheddingasdeterminedbyRT-PCRandELISA.NoVcapsidantigenwasdetectedin

    enterocytesofboththeduodenumandjejunum,supportingoccurrenceofviralreplication.

    Similartofindingsinhumans,onlymildhistopathologicallesionsoftheintestineswere

    observed,mainlyconsistingofduodenalvillousatrophyandedemaofthelaminapropria.Ina

    follow-upstudywiththesameGII.4strain,thegroupevaluatedcytokineandantibody

    responsespost-infection(244).Itwasnotedthatpigswithgreaterseverityofdiarrheawere

    morelikelytoseroconvertandhadhigherintestinalIgAandIgGtiters.Intermsofcytokine

    secretion,HuNoVinfectioninducedabalancedTh1/Th2(IFN-g,IL-12,IL-4,IL-10)anddelayed

    IFN-aresponsesystemically,whileonlyIFN-aandIL-12weredetectedlocallyinintestinal

    contents.

    GnpigscontinuetobeusedtoevaluateNoVvaccinecandidates,includingbothVLPand

    Pparticle-basedvaccines(245,246).Otherstudieshaveutilizedthemodeltoassessefficacyof

    immunomodulatorsandtherapeutics,suchasIFN-a,probioticsandricebran(247,248).As

    previouslymentioned,recentpathogenesisstudiesalsodemonstratedthat,incontrasttoMNV,

    HuNoVdoesnothaveatropismforBcellsinGnpigsandthatE.cloacaeinhibitsinfectivity

    instead(67,131,249).

  • 21

    1.4.3GnpigmodelofHRVinfection

    IncontrasttoHuNoVs,HRVscanbegrownefficientlyincellcultureandcommercial

    vaccinesarereadilyavailable.However,thesevaccineshaveloweredefficacyindeveloping

    countries,thustheGnpigmodelisstillfairlyrelevantforstudyingRVs.SimilartoHuNoVs,

    infectionwithHRViscapableofcausingclinicaldiseaseinGnpigs.Additionally,unlikemiceand

    rabbitmodels,GnpigscontinuetobesusceptibletoRV-induceddiarrheauntil6weeksofage

    (250).

    GnpigswerefirstutilizedtostudyHRVsstartinginthe1970’s(251,252),demonstrating

    developmentofdiarrheaandfecalvirusshedding.Yet,itwasnotuntil1996whentheGnpig

    modelofHRVinfectionanddiseasewasfullyestablished(253).Thismodelwasdevelopedusing

    apig-adaptedHRVWastrain(G1P1A[P8]),whichinduceddiarrheainallpigsatadoseof~105

    focus-formingunits(FFU)approximately13hourspost-inoculationandlastingforanaverageof

    fourdays.Thediarrheawasassociatedwithhistopathologicallesionsintheduodenumand

    jejunum(villousatrophy),seroconversionandfecalvirussheddingupto2x107FFU/ml.

    Sincethattime,themodelhasbeenutilizedtodetermineimmuneresponsestoHRV

    infectionandevaluationofvaccineandtherapeuticcandidates.AstudyconductedinGnpigs

    wasthefirsttodemonstrateintestinalIgAantibody-secretingcells(ASCs)asacorrelateof

    protectiveimmunityinanHRVanimalmodel(254).TwootherGnpigstudiessuggestedthat

    vaccinecandidatesforregionswithinfantshavinghighmaternalantibodylevels(developing

    countries)mayrequirespecializedformulationsand/ordosingregimenstocircumvent

    maternalantibodyinterferenceonthedevelopmentofprotectiveASCresponses(255,256).An

    additionalstudyhighlightedtheimportanceofabalancedTh1/Th2responseinducedbyWa

  • 22

    HRVinprovidingprotectionagainstsubsequentre-challenge(257).PotentialRVvaccinesand

    therapeuticsevaluatedinGnpigsincludeVLP-basedvaccines(258,259),chickeneggyolkIgY

    antibodies(260),llama-derivedantibodies(261),probiotics(262-265)andricebran(266,267).

    Recently,aGnpigmodelofentericdysbiosiswasestablishedtoelucidatetheeffectsofhuman

    gutmicrobiotaonimmuneresponsesinducedbyRVvaccination(235).

    1.5Roleofdendriticcellsininnateimmunityagainstacuteviral

    gastroenteritis

    1.5.1Generaloverview

    Dendriticcells(DCs)arespecializedcellsoftheinnateimmunesystemthatare

    particularlyadaptedforantigencaptureintheirimmaturestate(268,269).Recognitionof

    antigenicstimulibyDCsarecontrolledbypatternrecognitionreceptors(PRRs)thatare

    associatedwithconservedpathogenassociatedmolecularpatterns(PAMPs)ofmicrobes(268,

    270).Thiscontactpromptsthesecretionofcytokinesandchemokinesthatcanattractother

    innateimmunecells(268).Uponmaturation,theactivatedDCsmigratewiththecaptured

    antigenstolymphoidtissuesandinteractwithT,BandNKcells(271).Significantly,DC

    presentationofantigenstoTcellsresultsinguidanceoftheadaptiveimmuneresponseto

    pathogens,inducingeitheranimmunogenicortolerogenicstate(270).DCsaregenerally

    classifiedaseithermyeloid/conventionalDCs(cDCs)orplasmacytoidDCs(pDCs)(272).cDCsare

    recognizedprimarilyforantigenpresentation,whilepDCsareknownaspotentsecretorsof

    typeIinterferons(IFN-aandIFN-b)(273).Inmice,cDCsarecomprisedoftwomajorsubsets,

  • 23

    CD103+CD11b

    -andCD11b

    +cells(274,275).TwocDCsubsetsarealsopresentinhumansandare

    mainlycharacterizedaseitherCD1c+orCD141

    +(275,276).AsforpDCs,thoseinhumansare

    knowntoexpressCD303(CLEC4C),CD304(neuropilin-1),andCD123(IL3RA),whilethosein

    miceexpressB220,sialicacid-bindingimmunoglobulin-likelectinH(SiglecH),andintermediate

    levelsofCD11c(275,277).cDCandpDCpopulationsinpigshavebeendefinedas

    CD172a(SWC3a)+CD4

    -CD11R1

    +andCD172a(SWC3a)

    +CD4

    +CD11R1

    -,respectively(278).Itis

    importanttonotethatCD11R1inpigs,whichisrecognizedbymouseanti-pigCD11R1antibody

    cloneMIL4,isanalogoustoCD11binhumansandthusalsorecognizedbythecross-reactive

    anti-humanCD11bcloneTMG6-5.

    DCsarefoundinseverallocationsthroughoutthebodywherethereisahigher

    incidenceofencounteringpathogens.Specifically,inthegastrointestinaltract,DCsarelocated

    inPeyer’spatchesofthesmallintestineandthroughoutthelaminapropria(279,280).Inmice,

    themajorityoftheDCsinthelaminapropriaofthesmallintestinearecharacterizedas

    CD103+CD11b

    +,whilethoseinthecolonarecomprisedofCD103

    +CD11b

    -andCD103

    -CD11b

    +

    populations(281).StudiesofintestinalDCpopulationsinhumanshavedemonstratedthat

    CD103+CD172a

    +DCsarecloselyrelatedtomurineCD103

    +CD11b

    +DCs.Similarly,CD103

    +CD172a

    -

    DCsinhumansarehomologoustoCD103+CD11b

    -DCsinmice(282).Inpigs,DCsinthelamina

    propriaaredefinedasCD11b+CD172a

    +,whileDCsinPeyer’spatchesaredividedintoCD11b

    -

    CD172a+insubepithelialdomesandCD11b

    -CD172a

    -ininterfollicularregions.Afourthsubsetin

    pigsisrepresentedbyCD11b+CD172a

    -DCsinmesentericlymphnodes(283).Asthe

    gastrointestinaltractisaprimesiteforinteractionswithentericpathogens,recentstudieshave

    beenconductedtounderstandtheroleofDCsinNoV-andRV-inducedimmunity.

  • 24

    1.5.2DendriticcellresponsestoNoVinfection

    StudiesofDCresponsestoNoVinfectionarelimitedandhaveprimarilybeenconducted

    inmicethusfar.InadditiontotheirpermissivenesstoMNVinfection,DCshavealsobeen

    implicatedinclearanceofvirusandinfluenceofantibodyresponses.OnestudydepletedcDCs

    frommicepriortoMNVinfectionandreportedincreasedvirussheddinganddecreasedMNV-

    specificIgGtiters(279).AmorerecentstudyassociatedtheabilityofDCstocontrolMNV

    replicationwiththesecretionofTypeIIFN(284).MicewithDCsthatwereunabletorespondto

    TypeIIFNduetoareceptordeficiencydemonstratedpersistenceofMNVinfection.This

    persistenceoccurreddespitecompensatoryincreasedhumoralresponses.

    1.5.3DendriticcellresponsestoRVinfection

    WhilenumerousstudieshaveadvancedunderstandingofadaptiveimmunityagainstRV

    infection,knowledgeofinnateimmunityintermsofDCresponsesisstillincomplete.Two

    featuresseemtobewell-acceptedfromrecentstudiesthough.First,DCsareclearlycapableof

    beingactivatedbyRVinfectionasdemonstratedbyup-regulationofCD40,CD86,MHCII,TLR3

    andTLR4,cytokinesecretion,andreducedbindingtoVLPs(285,286).Second,TypeIinterferon

    secretionbypDCs(272)isimportantforimmunedefensesagainstRVinfection.pDCsecretion

    ofIFN-ahasbeenassociatedwithstimulationofIFN-g-producingTcellresponsesinvitro(287).

    AnotherinvitrostudyrevealedthenecessityofTypeIIFNproductionbypDCsinregardstoB

    cellactivation(288).ThesamestudyalsodemonstratedthatmicewithoutfunctionalpDCs

  • 25

    wereunabletomountsufficientRV-specificantibodyresponsestocontrolinfectionasthese

    micehadincreasedvirusshedding.

    1.6Preventionofacuteviralgastroenteritis

    1.6.1Disinfectionmethods

    BothNoVandRVarespreadthroughthefecal-oralrouteandarefairlyeasily

    transmittedfrompersontopersonviacontaminatedfood,waterandfomitesifproperhygiene

    anddisinfectionarenotemployed.Numerousstudieshavebeenconductedtodeterminethe

    bestsanitationpracticestoreducespreadofeachvirus.

    DuetothelackofanefficientcellculturesystemforHuNoVs,moststudiesevaluating

    disinfectionmethodseitherutilizeothercalicviruses(MNV,FCV)assurrogates(289,290)or

    measurereductionofHuNoVsbyPCRmethods(291,292).NoVappearstobemostsuccessfully

    removedfromhandsviawashingwithregularsoapandwater(291-293).Different

    concentrationsofalcohol-basedhandsanitizershaveproveneitherineffective(291,292)or

    possiblyevendetrimental.Asurveyoflong-termcarefacilitiesfoundanassociationwiththe

    useofalcohol-basedhandsanitizersandanincreasedriskofNoVoutbreaks(294).

    ChlorhexidineisalsoconsideredineffectualasahandsanitizeragainstNoVs(295).Intermsof

    surfacedisinfectants,itseemsthatNoVsarefairlyresistanttoquaternaryammonium

    compounds(293).Sodiumhypochloriteorbleachataminimumconcentrationof500-1000

    ppmhasproventobeeffectiveinNoVdecontamination(296-298).

  • 26

    Incontrast,simplehandwashingwithregularsoapisnotnecessarilyeffectiveat

    removingRV.Studieshaveshownthatalcohol-basedhandsanitizersaremorereliableat

    reducingRVloadsfromhumanhands(145,299,300)andshouldbeutilizedinfoodpreparation

    andwhencontactwithinfectedindividualsorcontaminatedfomitesissuspected.Asfaras

    surfacedisinfectantsareconcerned,theseshouldalsocontainalcoholinhighconcentrationas

    alcoholhasbeenshowntodamagetheoutercapsidofRV(291,301).Chlorhexidinegluconate

    0.5%in70%ethanol,quaternaryammoniumcompoundscontaining>40%isopropylalcohol,

    0.1%o-phenylphenolwith79%ethanolor95%ethanolalonearecapableofinactivatingRV

    (145,302).DomesticbleachalsohasacceptableactivityagainstRV(302).

    1.6.2NoVvaccinedevelopment

    ThereiscurrentlynolicensedNoVvaccineavailable.Thedifficultyindevelopinga

    suitableNoVvaccineismultifactorialandincludestheinabilitytoefficientlycultureHuNoVs

    efficientlyandcost-effectively,thelackofappropriateanimalmodelsandthesignificant

    antigenicdiversityofNoVs(2,303,304).Additionally,thediversitybetweengenogroupsand

    thecontinualevolutionofthemoreprevalentGII.4genotypehascloudedunderstandingof

    howpreviousexposuretomultipleNoVstrainsmayinfluencetheresponsetovaccinesand

    whetherlong-lastingimmunityexists(303,305).Studieshavesuggestedimmuneprotection

    maylapseafter2monthsto2years(306)ormaypersistforupto8years(307).

    Progresshassteadilybeencontinuingtoaddresstheseissuesthoughandseveral

    candidatesarebeingevaluatedinpre-clinicalandclinicaltrials(304,305,308,309).Since

    HuNoVcellculturesystemsarenotwell-established,recombinantNoVvaccinesarebeing

  • 27

    developedatthistime.ParticularinteresthasbeenaffordedtoVLPvaccines.VLPsare

    producedbyexpressionofthemajorcapsidproteinVP1inanappropriatesystem(304,309),

    includinginsectcells(310),mammaliancelllines(311),E.coli(312),yeast(313),potatoes(314),

    tobacco(315)andtomatoes(316).Allleadtoformationofparticlesthataremorphologically

    andantigenicallycomparabletotheactualviruscapsiditself(304,317).Whenadministered

    eitherorally(318,319),intranasally(320)orintramuscularly(321),VLPvaccineshavebeen

    capableofinducinghomotypicandheterotypichumoral,mucosalandcellularimmunitiestoa

    varyingdegree(309).Variousadjuvants,includingLT(R192G)toxinfromE.coli(320),

    monophosphoryllipidA(MPL)andchitosan(322),havethusbeenutilizedtoincreaseimmune

    responsestoVLP-basedvaccines.Severalpreclinicalstudieshavealsoconsideredthedelivery

    ofVLPvaccinesviaaviralvectortoinducegreaterimmunity(323-325),thoughconcerns

    regardingbiosafetyandefficacyrelatedtoprevioushostexposurehavereducedtherelevance

    ofsuchvaccines(309,326).

    OfthecurrentNoVvaccinecandidates,aVLP-basedvaccinefromTakeda

    Pharmaceuticalsisthemostadvancedinclinicaltrialprogress.Toovercomethehurdleof

    antigenicdiversityamongNoVs,designofmultivalentvaccinesisconsideredimperative(327).

    Thephase2clinicaltrialcandidateisabivalentGI.1/GII.4vaccinethatisadjuvantedwithMPL

    andaluminumhydroxide;itisadministeredintramuscularlyintwodoses,28daysapart(328-

    332).ThevaccineiscomprisedoftheNorwalkGI.1strainVLPandaGII.4VLPdevelopedfroma

    consensussequenceofthreeGII.4variants.Variousdosageformulationshavebeenevaluated,

    withmorerecentresultsindicatinga15/50(GI.1/GII.4)formulationproducingrobust

    serologicalresponsesfollowingjustonedoseofvaccine,whilemaintainingtolerability(331).

  • 28

    Aftervaccinationofhumanadults,IgAandIgGASCresponsespeakedatday7post1stdoseof

    vaccine.Allvaccinerecipientsshowed>4-foldrisesinASCsfrompre-vaccinationlevelsatthis

    timepointandminimalincreasesinASCswereobservedfollowingasecondvaccinedose,

    indicatingtheimmuneresponsesareanamnesticinnature.GI.1-andGII.4-specificIgGmemory

    Bcellsweredetected180dayspost-vaccination.Butitremainstobeevaluatedifthese

    responsesfunctionasvaccine-inducedcorrelatesofprotection(332).Overall,thehighestGI.1

    antibodylevels(pan-Ig,IgAandHBGA-blockingtiters)wereseenwitha50/50formulation,but

    the15/50formulationunexpectedlyproducedbothgoodGI.1responsesandevenbetterGII.4

    responses.AsGII.4NoVsarethepredominantcirculatingstrains,thiswillmostlikelybethe

    formulationmovingforwardwithfuturestudies.Currentplansincludedeterminingwhether

    theMPLadjuvantisabsolutelyessential.

    The other recombinant protein utilized inNoV vaccine development is the P particle,

    whichisasubviralparticleformedfromexpressionoftheVP1PdomaininE.colioryeast(333).

    Asmentionedpreviously,thePdomain,specificallytheP2subdomain, isresponsibleforhost

    bindinginteractions(334).AnadvantageofthePparticleistheeaseofproduction,makingthem

    likelyamoreeconomicalvaccineoptionthanVLPs(334).StudiesindicatePparticlesretainHBGA-

    binding properties (333) and are immunogenic in mice (335) and Gn pigs (246) following

    intranasal administration. The study conducted in pigs demonstrated 47% cross-variant

    protectionagainstdiarrheacompared to60%with thesameGII.4strain-derivedVLPvaccine.

    However,theauthorsnotedthatimmunizationwithPparticlesinducedgreaterTcellresponses

    thanwithVLPs.Conversely,anotherstudyrevealedthatVLPs,butnotPparticles,werecapable

    ofprimingTcellsforIFN-gproductionandinducingcross-reactiveTandBcellresponsesinmice

  • 29

    (321).QuestionableformationofthePparticlesusedinthemousestudymayhaveaffectedthe

    immunological responses though (326, 336). Further studies are likely required to better

    characterizetheimmunityinducedbyPparticlevaccination.

    Asmultipleentericpathogenscancauseacutegastroenteritis,therehasbeen

    considerableinterestindevelopingcombinationvaccinesthatincludeNoV.Inparticular,

    inclusionofRVrecombinantVP6proteinwithNoVVLPshasbeenevaluatedinseveralstudies

    (337-339).Additionally,thePparticlehasproventobeanexcellentplatformforpresentation

    ofadditionalantigens(340);PparticleshavebeendevelopedwithadditionofRV(340),

    hepatitisEvirus(341),influenzavirus(342)andastrovirus(343)antigens.

    ItremainstobeseenwhetherNoVvaccineswillbecapableofproducingdurableand

    adequatehomotypicandheterotypicimmuneresponses.ItispossiblethatNoVvaccineswill

    needtobereformulatedasemergentstrainsappear.

    1.6.3RVvaccinedevelopment

    RVvaccinedevelopmenthasbeenanimportantcontributortoreducingdiseaseburden.

    ThefirstlicensedoralRVvaccinewasatetravalentreassortantofarhesusRVstrain(G3)with

    VP7fromhumanG1,G2,andG4(137,344).RotaShieldorRRV-TVwaslicensedin1998after

    provingtobesafeandefficaciousinstudiesconductedintheUS,FinlandandVenezuela(137,

    344-347).Unfortunately,itwasbelievedtobeassociatedwithanincreasedriskof

    intussusception,aconditionwhenapartoftheintestinallumenfoldsintoitselfandmayresult

    inobstruction,ischemiaandperforation.Theincidencewasgreatestinthe3-7daysfollowing

    thefirstdoseandmoreoftenwhenthevaccinewasadministeredtochildrenbetween3and9

  • 30

    monthsofage(344,348,349).Duetosafetyconcerns,RotaShieldwasvoluntarilywithdrawn

    fromthemarketin1999,lessthanayearafterlicensure.

    Since2006,twonewlive,attenuatedoralRVvaccineshavebeencommerciallyavailable

    –RotaTeq(approved2006)andRotarix(approved2008).Rotarix(GlaxoSmithKlineBiologicals)

    isamonovalenthumanG1P1A[8]strainvaccinethatwasattenuatedthroughserialcellculture

    passage(136,137,344).Upondevelopment,thesafetyandefficacywastestedin60,000

    infantsinEuropeandLatinAmerica(147,350,351).Thevaccineisadministeredin2dosesat2

    and4monthsofage.Duetothehumanstrainoriginofthevaccine,intestinalreplicationdoes

    occurandsheddingfollowingthefirstdoseiswell-observed.Immunityshouldbehighenough

    bytheseconddosethatsheddingshouldbeveryminimal(147,344).

    RotaTeq(Merck)isapentavalent,reassortantvaccinecontainingabovineRVstrain

    (WC-3,G6P[5])backbonewithVP7ofhumanG1,G2,G3andG4strainsandVP4ofaP1A[8]

    strain(344).SimilartoclinicaltrialsofRotarix,RotaTeqwasevaluatedin>60,000infantsinthe

    USandEurope(147,352).MostlikelyduetolessintestinalreplicationthanRotarix,RotaTeq

    requiresa3-dosescheduleofadministrationat2,4and6monthsofage.Inaccordancewith

    thelimitedreplication,muchlowerincidencesofsheddingofthevaccinestrainoccuraswell

    (137,147).

    RegionalRVvaccinesincludetheLanzhoulambRVvaccine(LLR)licensedinChina,a

    monovalenthuman-bovinevaccine(Rotavac)inIndiaandanattenuatedG1P[8]strainvaccine

    (Rotavin)inVietnam(353-356).Morerecently,anoralbovine-humanreassortantpentavalent

    vaccine(Rotasiil)demonstrateda66.7%efficacyagainstsevereRVgastroenteritiswith3doses

    inarandomized,placebo-controlledtrial(357).ThevaccinecontainshumanRVserotypesG1,

  • 31

    G2,G3,G4,andG9onabovineRVG6P[7]backbone(BRV-PV).Ofimportanceforusein

    resource-limitedcountries,thevaccineisheat-stablefor2yearsatatemperatureof37°Cand

    for6monthsat40°C.

    BothRotaTeqandRotarixweredemonstratedtoinducegoodimmunityinlicensing

    trials,withefficacyagainstsevereRVgastroenteritisrangingfrom85-98%thatseemstopersist

    forafewyearsfollowingimmunization,atleastindevelopedcountries(350,352,358-362).As

    countriescontinuetoincludeRVvaccinationinstandardimmunizationprotocolsas

    recommendedbytheWorldHealthOrganization(WHO),thetrueclinicalimpactisbecoming

    betterunderstood.AsofMay2016,81countrieshaveincorporatedRVvaccinationintotheir

    nationalimmunizationprograms(363).

    DeclineincasesofRVdiarrheaandrelatedhospitalizationhasbeenobservedinthe

    majorityofvaccinestudiesinbothdevelopedanddevelopingcountries.OnestudyintheUS

    reporteda74-90%decreaseinthenumberofpositiveRVtestsbetween2010-2012compared

    toprevaccinebaselines(2000-2006)(364).Additionally,RV-associatedhospitalizationsinthe

    USweredecreasedbetween60-94%from2007-2011whencomparedwithratesobserved

    from2001-2006(365).Similarly,hospitalizationratesinEuropewereestimatedtodeclineby

    65-84%whenreviewingdatafrom2006-2012(366).Furthermore,evidenceofherdimmunity

    hasemergedasdeclineinall-causediarrheahospitalizationshaveoccurredinolderchildren

    andadultssincereleaseofcurrentRVvaccines(367,368).RegardingreductionofRVburdenin

    developingcountries,arecentreportfromRwandaindicatedthatintroductionofRotaTeq

    vaccinationreducedRV-relatedhospitalizationsby61-70%between2011to2014(369).

  • 32

    Likewise,astudyfromGhanaconductedduringthefirstthreeyearsfollowingRotarixvaccine

    introductiondemonstrateda49%reductioninhospitalizationsduetoRV(370).

    DespitethepromisingresultsfromimplementationofRVvaccineusageindeveloping

    countries,overallvaccineefficacy(50-64%)stillremainslowerthanthatofdevelopedregions

    (354,363).Reasonsforthisobservationappeartobemultifactorialandencompass

    interferencebymaternalantibodies,concurrentadministrationoforalpoliovaccine,

    malnutrition,coinfectionswithotherentericpathogensanddifferencesingutmicrobiome

    (147,353,371).Addressingthisissuemayrequirealterationsofvaccineadministration

    schedulesordevelopmentofparenteralvaccinecandidates(147).

    SincetheintroductionofRotaTeqandRotarix,acoupleofsafetyconcernshavearisen,

    thoughneitherhavebeenseriousenoughtopermanentlyremoveeithervaccinefromthe

    market.Despitecarefulsafetyevaluationinclinicaltrials,bothvaccineshavepossiblybeen

    associatedwithaslightincreaseinintussusceptionriskinpostlicensurestudiesinseveral

    countries,includingtheUS,Australia,andMexico(372-375).SimilartoRotashield,the

    incidenceismostlikelytooccurinthefirstweekfollowinginitiationofthevaccineseries(376).

    However,theoverallincidenceismuchlowerthanobservedwithRotashield,equaling1-5

    excesscasesforevery100,000childrenvaccinated(147,376).

    Another,thoughtemporary,concernwasthediscoveryofporcinecircovirusI(PCV-1)

    DNAinRotarixandPCV-1andPCV-2DNAfragmentsinRotaTeqin2010.PCV-1DNAwasfound

    originatinginthemasterseedvirusofRotarix,whilecontaminatedtrypsinusedinthe

    productionofRotaTeqwasimplicated(344).Sinceporcinecircovirusdoesnotinfecthumans,

    useofbothvaccineswasallowedtocontinuewhileproductionofPCV-freevaccineswas

  • 33

    initiated.Overallthough,thebenefitsofRVvaccinationinreductionofmorbidityandmortality

    outweighanyperceivedrisks.

    1.7Treatmentofacuteviralgastroenteritis

    1.7.1Oralrehydrationsolutions

    DuetothelackofavailabilityofNoVvaccinesandthestillunder-utilizedRVvaccines,

    adjunctivetreatmentsremainsignificant.MosttreatmentmodalitiesforNoVandRVareaimed

    atreducingclinicalsymptomsofdiseaseandarethussimilarbetweenthetwo.Themost

    importantandbeneficialaspectoftreatmentisappropriaterehydrationtherapy.Fatalities

    occurwhenintestinalwaterlossissevereenoughtocauseelectrolyteimbalancesand

    cardiovascularfailure.WHOrecommendstheuseoforalrehydrationsolution(ORS)in

    treatmentofacutewaterydiarrheaofallcauses.UseofORShelpspreventlife-threatening

    dehydration,thoughitdoesnotalleviatesymptomsofdiarrheaorshortenduration.ORS

    containsglucose,electrolytesandcitrateinanhypo-osmolarformulationtoreplenishlosses

    andreduceacidosisassociatedwithdiarrheaandvomiting(377).ThebasisofORStherapyis

    thatglucosestimulateswaterandsaltabsorptioninthesmallintestineviatheNa+/glucose

    cotransporterSGLT1(378).Insituationswhereoralrehydrationisnotadequateenoughto

    counteractdehydration,thenintravenousfluidtherapyisnecessary(379).

    Since2004,WHOhasalsorecommendedtheuseofzincsupplementationfortwoweeks

    inconjunctionwithORS(380,381).Zinchasbeenshowntobeimportantformucosalintegrity

    oftheintestineandinhibitionofpotassiumchannelsinvolvedincyclicadenosine

  • 34

    monophosphate(cAMP)mediatedchloridesecretion(146,382,383).Consequently,low

    plasmazinclevelshavereportedlybeenlinkedwithincreasedriskofmorbidityduetosevere

    diarrhea(384)andsupplementationwithORScanreducediarrheavolumeandduration(385).

    1.7.2Generalanti-diarrhealandanti-viralagents

    SinceORSonlyservestooffsetdehydration,non-specificadjunctivetreatments

    continuetobeproposedandstudiedwiththeaimofeitherreducingdiarrheaorlimitingviral

    replication.Probioticsarewell-knownfortheirgastrointestinalhealthybenefit.Multiple

    probioticstrainshavebeenevaluatedfortheirabilitytodecreasedurationofdiarrheawith

    generallypositive,thoughvariablestrainanddose-dependentresponses(263,386,387).The

    mostcommonlyadministratedstrainsincludeLactobacillusandBifidobacteriumspecies(388).

    Themechanismofactionofprobioticsisnotfullydefined,butmayinvolveimprovinggut

    barrierfunction,regulatingtheintestinalenvironmentandotherentericmicrobesand/or

    influencingthehostimmunesystem(389,390).

    Inparticular,LactobacillusrhamnosusGG(LGG)hasgarneredthemostsupportive

    evidenceforlesseningclinicalsymptomsassociatedwithacutegastroenteritis(391).Inaclinical

    trialinPakistan,LGGsupplementationfollowingrehydrationelicitedaresponsebythesecond

    dayoftreatmentinchildrenwithacute,non-bloodygastroenteritis(392).Bythistime,31%of

    childrenintheLGGgrouphadcontinueddiarrheacomparedtothe75%intheplacebogroup.

    Similarly,inaclinicalstudyconductedinEurope,LGGadministrationinconjunctionwithORS

    reducedthedurationofdiarrheainchildrenwithassumedviralgastroenteritis(noidentifiable

    bacterialpathogensdetected)(393).LGGhasalsodemonstratedefficacyinpreventing

  • 35

    nosocomialdiarrheainyoungchildren(394).StudiesinGnpigshavealsoconfirmedspecific

    efficacyofLGGagainstbothHRV(265,395)andNoV-induceddiarrhea(248).

    Inrelationtoprobioticadministration,theprebioticricebranhasalsobeenreportedto

    ameliorateacute,waterydiarrhea.Comprisingtheouterlayerofrice,ricebranhasbeen

    identifiedasadietarysourceofbioactivecompoundsandhasbeensuggestedtobebeneficial

    inintestinalhealthaswell(396,397).Themechanismofactionincludespromotingthegrowth

    ofdiarrhea-reducingprobioticspecies,maintaininggutbarrierintegrityandimprovinginnate

    immunity(266).InGnpigsinfectedwithHuNoVandcolonizedwithLGGandE.coliNissle1917,

    ricebranfeedingreducedtheincidenceanddurationofdiarrhea,aswellasvirusshedding

    (248).SimilarlyinHRV-infectedGnpigs,ricebranfeedingalonewasabletodecreasethe

    incidenceandseverityofdiarrhea,thoughdidnotaffectvirusshedding(266).

    Commercialdrugsarealsoavailableforthetreatmentofacutegastroenteritis.Theanti-

    secretorydrugracecadotrilhasproventobeeffectiveindecreasingintestinalwaterlossand

    hasbeenlicensedforuseinEurope(398,399).Racecadotrilactstoinhibittheenzymeneutral

    endopeptidaseand,indoingso,increasestheintestinalconcentrationofenkephalins,which

    havenaturalanti-secretoryproperties(400).Anotherdrugthathasbeenevaluatedisthe

    broad-spectrumanti-infectivenitazoxanide(401,402).Nitazoxanidewasinitiallydevelopedas

    ananti-parasiticagent,buthasalsoshownactivityagainstanaerobicbacteria(403)andviruses,

    includingNoV(401,404)andRV(401,402,405).ItsuseisparticularlyapplicableincasesofNoV

    infectioninimmunocompromisedindividualswherereductioninimmunosuppressivetherapy

    oradministrationofintravenousimmunoglobulinisnotpossible(404).InRVinfectionatleast,

  • 36

    itappearsthatnitazoxanideinhibitsVP7maturation(406);themodeofactionagainstNoVis

    unknown(407).

    Recombinantllama-derivedsinglechainantibodyfragments(VHH)areanadditional

    therapeuticagentthathavebeenproposedforbothNoVandRV-induceddiarrhea.Theseso-

    callednanoantibodiesareabletobeproducedagainstaspecificantigen,includingVP6ofRV

    (261)andVP1ofNoV(408,409),andrepresentapotentialimmunotherapy.

    1.7.3RV-specificadjunctivetreatments

    ForRVinfection,othernovelapproachestoinducepassiveimmunityincludetheuseof

    hyperimmunebovinecolostrum(410)andhyperimmunechickeneggyolkimmunoglobulin

    (260).AdditionalpotentiallyeffectivetreatmentsincludeN-acetyl-cysteineandergoferon.The

    antioxidantN-acetyl-cysteinehasbeendemonstratedtoreduceRVinfectivityincellculture

    (411)andresolveRVdiarrheainchildrenafterjusttwodaysoforaladministrationat60

    mg/kg/day(412).Ergoferon,whichiscomposedofrelease-activepolyclonalantibodiesagainst

    IFN-g,CD4andhistamine,wasrecentlytestedforantiviralactivityagainstRVincellculturewith

    promisingresults(413).InvivostudiesevaluatingErgoferonhaveyettobeconducted.

    1.7.4NoV-specificadjunctivetreatments

    VariouscompoundshavebeenevaluatedinattempttodisrupttheNoVreplicationcycle

    (407).Inresponsetothewell-establisheduseofHBGAsascellularreceptorsbyNoVs,therehas

    beenconsiderableinterestinblockingthisbindinginteractiontolimitinfection(414-417).

  • 37

    Otheranti-NoVdrugsaimtoreducereplicationbytargetingtheviralprotease(418-420)or

    RNA-dependentRNApolymerase(421-423).

    1.8Concludingremarks

    Mydissertationresearchinvolvesinvestigationsofpathogenesis,innateimmunityand

    therapeuticsinGnpigmodelsofNoVandRVinfectionanddiarrhea.Asdiscussed,pigsarea

    particularlysuitableanimalmodelforstudyingthepathogenesisofentericvirusesduetotheir

    similaritiesinanatomy,physiologyandimmunefunctionwithhumans(225,227-229).

    UtilizationofGnpigsenablesevaluationofprimaryimmuneresponsestovirus,vaccination

    and/ortherapeuticswithoutconfoundingvariablessuchasmaternalantibodyinterference,

    priorantigenicexposureandpossibleco-infectionswithotherentericpathogens.

    Thefirstphaseofmydissertationresearchwasfocusedontheestablishmentofa

    reliableGnpigmodelofNoVinfectionanddisease.Ithaspreviouslybeendemonstratedthat

    GnpigsareindeedsusceptibletoinfectionwithHuNoVsusingGII.4andGII.12strains(66,424).

    However,thesepriormodelsfailedtodeterminethemedianinfectiousdose(ID50)ofthe

    strainsused.Thischaracterizationenablesmoreconsistentincidenceofinfectionfor

    appropriatelydeterminingprotectiveefficacyinvaccineandtherapeutictrials.Basedonthe

    predominanceofGII.4strainsinoutbreaksofNoVgastroenteritis(8),aGII.42006bvariantwas

    selectedasthechallengestrain.TodeterminetheeffectofageontheID50,bothneonatal(4-5

    daysofage)andolder(33-34daysofage)wereinfected.Wealsoconfirmedwhether

  • 38

    administrationofacholesterol-loweringdrug,simvastatin,wasabletoincreasesusceptibilityto

    NoVinfectionasreportedpreviously(247,425).

    Thesecondphaseofmydissertationresearchinvolvedutilizingthewell-establishedGn

    pigmodelofRVinfectionanddiseasetoevaluateadjunctivetreatmentsforRV-induced

    diarrhea.WhileRVvaccinationhasreducedmorbidityandmortality,theefficacyofRotarixand

    RotaTeqisstillunderwhelmingindevelopingcountries(354).Aneffectivetherapeuticto

    reducediarrheaandshortendurationofillnesswouldthusbeespeciallybeneficial.

    Racecadotrilisanattractiveanti-diarrhealdrugcandidateduetoitsefficacyand

    tolerability(399,400).Asitdecreasesintestinalsecretion,itaddressesoneofthepossible

    mechanismsofdiarrheaduetoRVinfection(208).Previousstudieshavereportedgenerally

    favorableresultsinall-causeandRV-induceddiarrhea,thoughconflictingevidenceexists(426,

    427),possiblyduetoconfoundingvariablessuchaspatientcomplianceandco-infectionswith

    otherentericpathogens.Subsequently,theGnpigmodelpresentsanidealopportunityto

    determinethespecificefficacyofracecadotrilagainstRV-induceddiarrhea.Suchevaluationis

    prudentbeforestandardizationofitsuseintreatmentprotocols.

    Probioticshavealsobeenhighlystudiedfortheiranti-diarrhealeffects.However,

    numerousstrainsareavailableanditiswell-acceptedthatresultsmaybebothstrain-and

    dose-dependent(263).Furthermore,theexactmechanismofactionisill-defined,though

    immunomodulationislikelyinvolved(389).Again,theimmunologicallynaïvebackgroundofGn

    pigsfacilitateselucidationofimmuneresponseselicitedbyprobioticadministrationandthe

    possibleassociationwithdiarrheareduction.Ourpreviousstudiesevaluatedthedose-

    dependenteffectsofLactobacillusacidophilusNCFM(LA)onTcellresponsesinGnpigs

  • 39

    receivinganoralHRVvaccine(428).Currently,limitedstudieshavebeenconductedregarding

    theroleofDCsininnateimmunitytoRVinfection.AsDCsinthegastrointestinaltractwould

    expectedlyencounterbothprobioticsandRVs,weelectedtoexplorethisinteractioninGnpigs

    administeredeitheraloworhighdoseofLA,bothbeforeandafterHRVinfection.

    1.9References

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    theUnitedStates,2012-2013.PLoSOne11:e0148395.2. LopmanBA,SteeleD,KirkwoodCD,ParasharUD.2016.TheVastandVariedGlobal

    BurdenofNorovirus:ProspectsforPreventionandControl.PLoSMed13:e1001999.3. MortalityGBD,CausesofDeathC.2016.Global,regional,andnationallifeexpectancy,

    all-causemortality,andcause-specificmortalityfor249causesofdeath,1980-2015:a

    systematicanalysisfortheGlobalBurdenofDiseaseStudy2015.Lancet388:1459-15444. LanataCF,Fischer-WalkerCL,OlascoagaAC,TorresCX,AryeeMJ,BlackRE,Child

    HealthEpidemiologyReferenceGroupoftheWorldHealthO,Unicef.2013.Globalcausesofdiarrhealdiseasemortalityinchildren

  • 40

    13. KapikianAZ.2000.Thediscoveryofthe27-nmNorwalkvirus:anhistoricperspective.JInfectDis181Suppl2:S295-302.

    14. KapikianAZ,WyattRG,DolinR,ThornhillTS,KalicaAR,ChanockRM.1972.Visualizationbyimmuneelectronmicroscopyofa27-nmparticleassociatedwithacute

    infectiousnonbacterialgastroenteritis.JVirol10:1075-1081.15. LuJ,FangL,ZhengH,LaoJ,YangF,SunL,XiaoJ,LinJ,SongT,NiT,RaghwaniJ,KeC,

    FariaNR,BowdenTA,PybusOG,LiH.2016.TheEvolutionandTransmissionofEpidemicGII.17Noroviruses.JInfectDis214:556-564.

    16. PanL,XueC,FuH,LiuD,ZhuL,CuiC,ZhuW,FuY,QiaoS.2016.ThenovelnorovirusgenotypeGII.17isthepredominantstrainindiarrhealpatientsinShanghai,China.Gut

    Pathog8:49.17. ZhangXF,HuangQ,LongY,JiangX,ZhangT,TanM,ZhangQL,HuangZY,LiYH,Ding

    YQ,HuGF,TangS,DaiYC.2015.AnoutbreakcausedbyGII.17noroviruswithawidespectrumofHBGA-associatedsusceptibility.SciRep5:17687.

    18. ChanMC,LeeN,HungTN,KwokK,CheungK,TinEK,LaiRW,NelsonEA,LeungTF,ChanPK.2015.Rapidemergenceandpredominanceofabroadlyrecognizingandfast-evolvingnorovirusGII.17variantinlate2014.NatCommun6:10061.

    19. ScipioniA,MauroyA,VinjeJ,ThiryE.2008.Animalnoroviruses.VetJ178:32-45.20. CaddyS,BreimanA,lePenduJ,GoodfellowI.2014.GenogroupIVandVIcanine

    norovirusesinteractwithhisto-bloodgroupantigens.JVirol88:10377-10391.21. deGraafM,vanBeekJ,KoopmansMP.2016.Humannorovirustransmissionand

    evolutioninachangingworld.NatRevMicrobiol14:421-433.22. deGraafM,VillabrunaN,KoopmansMP.2017.Capturingnorovirustransmission.Curr

    OpinVirol22:64-70.23. MarksPJ,VipondIB,CarlisleD,DeakinD,FeyRE,CaulEO.2000.Evidenceforairborne

    transmissionofNorwalk-likevirus(NLV)inahotelrestaurant.EpidemiolInfect124:481-487.

    24. KirbyAE,StrebyA,MoeCL.2016.VomitingasaSymptomandTransmissionRiskinNorovirusIllness:EvidencefromHumanChallengeStudies.PLoSOne11:e0143759.

    25. DabillaN,NunesVieiraAlmeidaT,CarvalhoReboucasOliveiraA,KipnisA,NeresSilvaT,SouzaFiaccadoriF,TeixeiradeSousaT,dePaulaCardosoDD,SouzaM.2017.Norovirusinfecesandnasopharyngealswabofchildrenwithandwithoutacute

    gastroenteritissymptoms:FirstreportofGI.5inBrazilandGI.3innasopharyngealswab.

    JClinVirol87:60-66.26. SaidMA,PerlTM,SearsCL.2008.Healthcareepidemiology:gastrointestinalflu:

    norovirusinhealthcareandlong-termcarefacilities.ClinInfectDis47:1202-1208.27. EstesMK,PrasadBV,AtmarRL.2006.Noroviruseseverywhere:hassomething

    changed?CurrOpinInfectDis19:467-474.28. AtmarRL,OpekunAR,GilgerMA,EstesMK,CrawfordSE,NeillFH,GrahamDY.2008.

    Norwalkvirussheddingafterexperimentalhumaninfection.EmergInfectDis14:1553-1557.

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    30. MattnerF,SohrD,HeimA,GastmeierP,VennemaH,KoopmansM.2006.Riskgroupsforclinicalcomplicationsofnorovirusinfections:anoutbreakinvestigation.Clin

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