Alyssa Bennett Evans Iowa State University. Iowa State University Capstones, Theses and Dissertations. Graduate Theses and Dissertations

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1 Graduate Theses and Dissertations Iowa State University Capstones, Theses and Dissertations 2016 Characterization of the molecular mechanisms underlying neutralization, immune escape, and inhibition of porcine reproductive and respiratory syndrome virus Alyssa Bennett Evans Iowa State University Follow this and additional works at: Part of the Genetics Commons, and the Virology Commons Recommended Citation Evans, Alyssa Bennett, "Characterization of the molecular mechanisms underlying neutralization, immune escape, and inhibition of porcine reproductive and respiratory syndrome virus" (2016). Graduate Theses and Dissertations This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact

2 Characterization of the molecular mechanisms underlying neutralization, immune escape, and inhibition of porcine reproductive and respiratory syndrome virus by Alyssa Bennett Evans A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics Program of Study Committee: Susan Carpenter, Major Professor Jack C.M. Dekkers Karin S. Dorman Susan J. Lamont Cathy L. Miller Iowa State University Ames, Iowa 2016 Copyright Alyssa Bennett Evans, All rights reserved.

3 ii TABLE&OF&CONTENTS& LIST&OF&TABLES& LIST&OF&FIGURES& ACKNOWLEDGEMENTS& ABSTRACT& iv& v& vi& viii& CHAPTER&1:&GENERAL&INTRODUCTION& 1& Introduction 1 OverallGoal 3 SpecificAims 3 DissertationOrganization 4 LiteratureCited 5 CHAPTER&2:&LITERATURE&REVIEW& 9& ViralVariationanditsConsequenceonInfection 9 ThequasispeciesstructureofRNAviruspopulations 9 Antigenicvariationandimmuneescape 11 Antiviraldrugresistance 16 Emergenceofnewviraldiseases 17 Summaryofviralvariation 20 PorcineReproductiveandRespiratorySyndromeVirus 20 Genomeandvirionstructure 21 Replicationandmolecularpathogenesis 22 ImmuneresponsetoPRRSV 27 PRRSVantigenicvariationandimmuneescape 29 Vaccinesandantivirals 32 Summaryofporcinereproductiveandrespiratorysyndromevirus 33 LiteratureCited 33 CHAPTER&3:&PORCINE&REPRODUCTIVE&AND&RESPIRATORY&SYNDROME& VIRUS&UTILIZES&MULTIPLE&GENETIC&PATHWAYS&OF&IMMUNE&ESCAPE& DURING&INFECTION&IN&VIVO& 52& Abstract 52 Importance 53 Introduction 54 MaterialsandMethods 56 Results 64 Discussion 76 LiteratureCited 81 Tables 87 Figures 93

4 iii CHAPTER&4:&CHARACTERIZATION&OF&PORCINE&REPRODUCTIVE&AND& RESPIRATORY&SYNDROME&VIRUS&NEUTRALIZATION&AND&REPLICATION& PHENOTYPES&OF&NEUTRALIZING&ANTIBODY&ESCAPE&VARIANTS& 100& Abstract 100 Introduction 101 MaterialsandMethods 105 Results 110 Discussion 116 LiteratureCited 121 Tables 125 Figures 126 CHAPTER&5:&IDENTIFICATION&AND&CHARACTERIZATION&OF&SMALL& MOLECULE&INHIBITORS&OF&PORCINE&REPRODUCTIVE&AND&RESPIRATORY& SYNDROME&VIRUS& 130& Abstract 130 Introduction 131 MaterialsandMethods 134 Results 140 Discussion 147 LiteratureCited 150 Tables 154 Figures 156 CHAPTER&6:&GENERAL&DISCUSSION& 164& GeneralSummary 164 Importance 164 LimitationsandAlternativeStrategies 169 FutureWork 173 GeneralConclusions 175 LiteratureCited 176 & & & & & &

5 iv LIST&OF&TABLES& Table&3.1:Summaryofsequencedclonesandviralvariation 87 Table&3.2:Summaryofsignificantnucleotidevariantsandassociatedaminoacid changesinorf2u6andnsp2 88 Table&3.3:PredominantORF2U6haplotypeswithinlatedayvirussamples 90 Table&4.1:Summaryofchimericviruses 125 Table&5.1:SummaryofantiUPRRSVcompounds potencyandcytotoxicity 154 Table&5.2:SummaryofantiUPRRSVpotencyofcompoundsupto10 5 FFUvirus 155 & & & & & & & & & & & & & & &

6 v LIST&OF&FIGURES& Figure&3.1:Viremiaandneutralizationprofilesinselectedpigs 93 Figure&3.2:Temporalchangesinthepopulationstructureinnsp2andORF2U6 94 Figure&3.3:ORF2U6quasispeciesstructureandlatedayvirushaplotypes 95 Figure&3.4:Neutralizationphenotypesofreboundhaplotypesagainstautologous reboundsera 96 Figure&3.5:TheeffectofsingleaminoacidvariantsfromORF2U6rebound haplotypesonresistancetoautologousneutralizingserum 98 Figure&3.6:Immuneescapevariantsaresensitivetoneutralizationbybroadly neutralizingsera 99 Figure&4.1:NeutralizingactivityofPHGCpooledserumversusFL12,escape,and nonuescapeviruses 126 Figure&4.2:AttachmentandPRRSVRNAreplicationassays±pooledserum 127 Figure&4.3:ParticletoinfectivityratiosforFL12andtheNAbescapeandnonU escapeviruses 128 Figure&4.4:ReplicationkineticsassaysmeasuringvirionproductionandFFU productionoffl12andthenabescapevariants 129 & Figure&5.1:Synthesisroutesforatractylodinolandanalogs 156 & Figure&5.2:AntiUPRRSVactivityscreensofthefifteensynthesizedcompounds 159 & Figure&5.3:InhibitionassaystodetermineIC50softheeightcompoundswith inhibitoryactivityofprrsv 160 & Figure&5.4:PotencyassaysoftheeightantiUPRRSVcompoundsupto10 5 FFU 161 & Figure&5.5:Mechanismofactionofcompound & & & &

7 vi ACKNOWLEDGEMENTS& & TherehavebeenmorepeoplethanIcouldpossiblylistinthisacknowledgements sectionwhohavehelpedmegettowhereiamtoday,butiwouldliketotakethetimeto thankatleastafewofyouwhohavebeeninstrumentalinmylifeandgraduateschool career.firstandforemost,iwouldliketothankmyhusbandandmyfamily.momanddad, thankyoufornevergivingmeanyideathaticouldn tdowhateveriwanted(careeruwise, atleast),aslongasiworkedhardforit.itmeansmoretomethanicaneverexpressto havehadyourfullsupportsincethedayiwasborn,andforalwaysbelievinginme.tomy husband,daveevans,whohasbeensosupportivesincethedaywemet,evenwheni movedusacrossthecountrytoiowa.thankyouforalwayslovingandsupportingme,and foralwaysbeingabletomakemelaugh.jarodbennett,thankyouforpushingmetobemy bestandalwaysmakingsureiknowhowproudyouareofme.iwouldalsoliketothank mygrandparents,andnumerousaunts,uncles,andcousinsforalltheirsupportand encouragementthroughouttheyears.itmeansagreatdealtometohavesuchanamazing groupofpeoplecheeringforme. Iwouldliketoespeciallythankmymajorprofessor,Dr.SusanCarpenter,forgoing throughallthehighsandlowsofgradschoolwithme.thankyouforalwayspushingme andchallengingmetobethebestresearchericanbe,forencouragingme,andmostofall forremindingmethatgradschoolshouldbefun,too.atleastmostofthetime.iwouldalso liketothankthemembers,pastandpresent,ofthecarpenterlabwhohavehelpedme immenselyonthisdissertationwork,andforbeingagreatgroupofpeopletoworkwith. HyeleeLoyd,Ican tthankyouenoughforallofyourhelpovertheyears.fromtrainingme

8 vii duringmyrotation,tohelpingdevelopandtroubleshootassays,toourmanyafternoon coffeebreaks,youhavehelpedmeinsomanyways.tomytwoamazing(former) undergrads,sarahvantolandmarcusbolton:thankyouforbeingsuchsmart,capable,and funpeopletoworkwith.therearemanyaspectsofthisdissertationworkthatwouldhave takenmuchlongerwithoutyourhardwork.andtodr.chijiokeumunnakwe,thanksfor alwaysbeingajoytobearoundandforallyouradvice. Iwouldalsoliketothankmycommitteemembersfortheirguidanceandadvice.Dr. KarinDorman,thankyouforalwayspatientlyansweringmymanystatisticalquestions, andcollaboratingonlargeportionsofmyresearch.dr.cathymiller,thankyouforsharing yourmolecularvirologyknowledgewithme,andalwaysprovidinghelpfuladvice.dr.jack Dekkers,thankyouforyourinsightsandyourmanyhelpfulsuggestionsformyresearch projects.dr.suelamont,thankyousomuchforbeingmyfellowshipadvisorand encouragingmetothinkaboutmyprojectsfromdifferentangles.ifeelveryfortunateto havehadsuchasmartandcaringcommitteetoguidemethroughgraduateschool. Finally,Iwouldliketothankafewofthefriendswhohaveencouraged, commiseratedwith,andsupportedmethroughoutgradschool,thoughtherearemany morethanicouldpossiblylisthere.jordanbaumbachandjoegallagher,thankyoufor beingmyfirstfriendsinames,andforhelpingtoformourformidable WolfPack.Ellie Powell,thankyouforourmanylunchesandalwayslettingmeknowthatyoubelieveinme. StaceyBarnesandCarrieBerg,thankyouforallthelaughsanddinners.Idonotknowwhat Iwouldhavedonewithoutallofyou,andgradschoolcertainlywouldnothavebeenasfun withoutyouall.tomybestfriend,nonavalancius,eventhoughyouareathousandmiles away,iappreciatealwaysbeingabletocountonyouforanything.

9 viii ABSTRACT& TherapidmutationrateofRNAvirusesleadstosignificantgeneticandantigenic variation.thisdiversitypresentsgreatchallengesforthedevelopmentofeffectivevaccines andtreatments.thesechallengeshighlighttheneedforabetterunderstandingofthe molecularmechanismsunderlyingantigenicvariationandalternativetreatmentstrategies. Whilesignificantresearchtoaddresstheseproblemshasbeenundertakeninhuman diseases,lessisknownabouttheseprocessesinmanyveterinarydiseases,includingthe economicallysignificantporcinereproductiveandrespiratorysyndromevirus(prrsv). AlthoughimmuneselectionhasbeenaproposedmechanismforthediversityinPRRSVand itscontinualcirculationwithinandbetweenherds,directevidenceofimmuneescape variantsarisingduringthecourseofinfectioninvivohasneverbeenshown.toexamine theroleofimmuneselectionandescapeduringprrsvinfection,weexaminedgeneticand antigenicvariationofprrsvusingretrospective,sequentialsamplesfromfive experimentallyinfectedpigswithdifferingvirologicalandimmunologicaloutcomes, includingthreepigsthatexperiencedareboundinviremia,suggestingviralescapefrom hostimmunecontrol.sequenceanalysesrevealedlimitedgeneticvariationduringacute PRRSVinfection.However,distinctenvelopeproteinUencodingORF2U6haplotypeswere identifiedineachpig slatedayvirus.chimericvirusescontainingallorpartof predominantorf2u6haplotypesweretestedinvirusneutralizationassaysusing autologoussera,andinfourviruses,geneticvariationinorf2u6resultedinantigenic variationandimmuneescape.immuneescapemappedtomultipleenvelopeproteins, includinggp5and/orgp2,gp3,andgp4andusually,butnotalways,requireda

10 ix combinationofaminoacidchanges.importantly,thekeyaminoacidchangesand/or combinationofchangesthatmediatedescapedifferedbypigandbyvirushaplotype.to testifthemutationsthatmediatedimmuneescaperesultedinareductioninreplication fitnessoftheviruses,weperformedaseriesofinfectivityandreplicationkineticsassays usingtheescapevariantsandtheparentalvirus.ourresultsshowedthatoneofthefour escapevariantshadreducedinfectivitycomparedtotheparentalvirus,butnoneofthe escapevariantshadmarkedlyreducedgrowthratesinmarcu145cells.prrsvpooled neutralizingantibodywasfoundtoprimarilytargetapostuattachment,preugenome synthesisstepofreplication.thesedatademonstratethatprrsvenvelopeproteinsare underimmuneselectionthatcontributestotheemergenceofreplicationufitimmune escapevariants.thediversestrategiesofimmuneescapelikelycontributetodifficultiesin producingeffectivevaccinesforprrsv,andthereforealternativecontrolstrategiesare needed.antiviralshaveproventobeanextremelysuccessfultreatmentstrategyforseveral humanviraldiseases,andhavethepotentialforuseinthetreatmentofprrsv.previously, thenaturalcompoundatractylodinolwasreportedtohaveantiuprrsvactivityinvitro. AtractylodinolandfourteenanalogsweresynthesizedandtheirantiUPRRSVactivitywas characterizedinvitro.sevenoftheanalogshadpotentinhibitoryactivityagainst5ulog10 infectiousunitsofprrsvatlowμmconcentrations.analogcompound19wasshownto inhibitprrsvprimarilyatapostuattachmentstepduringprrsventry.theseresults provideevidencethattheatractylodinolanalogsarepromisingantiviralcandidatesfor trialsinpigs.overall,theworkfromthisdissertationindicatesthatselectivepressureby theimmunesystemcontributestothevastgeneticdiversityofprrsv,andthereis potentialforthetreatmentofprrsvoutbreakswithantivirals.

11 1 CHAPTER&1:&GENERAL&INTRODUCTION& & Introduction& & & Therapidrateofviralevolutionandquickaccumulationofmutationsisamajor contributingfactortothelackofeffectivevaccinesformanyviraldiseases,bothforhuman andanimalinfections.someofthemostresearchedhumandiseases,includinghuman immunodeficiencyvirustype1(hivu1),hepatitiscvirus(hcv),andinfluenzavirus,still lackeffectivevaccinesduetothehighmutationrateofthesevirusesandtheirabilityto evadethehostimmuneresponse(1u5).thehighamountofgeneticvariationwithinthese virusesmakesdevelopingvaccinesthatareeffectiveagainstdiversestrainsofthevirus extremelychallenging.however,inrecentyearsclassesofbroadlyneutralizingantibody (bnab)havebeendiscoveredtargetedagainsthivu1orinfluenzaathatarecapableof neutralizingdiversestrainswithintheseviruses(6).thebroadlyactingnatureofthe bnabsisduetotheirtargetingofepitopeswithincriticalfunctionaldomainsofthevirus, suchasthehighlyconservedstemregionofinfluenza shemagglutinin(ha)envelope proteinthatiscriticalforfusionoftheviruswiththehost sendosomalmembrane(7,8), andthereceptorandcoureceptorbindingsitesofhivu1 senvelopeprotein(6,9,10).the discoveryofthesebnabsprovideshopeforthedevelopmentof universalvaccines againsthivu1,influenza,andothervirusesthathavethusfarbeenelusive.however,bnab escapemutantsdoarise,andinfactarecommonlyfoundinchronicallyinfectedhivu1 patients.howeverbecauseescapeismediatedthroughviralvariationinfunctionally importantdomains,thebnabescapevariantsareusuallyassociatedwithdecreased

12 2 replicationfitness(6,11).therefore,understandingthespecificinteractionsbetween virusesandhoststhatmediateimmuneescape,andtheeffectthesemutationshaveonviral replicationwillprovidecrucialinformationintoviralpathogenesisthatcanleadtobetter treatmentandvaccinestrategies. Porcinereproductiveandrespiratorysyndromevirus(PRRSV)hasremainedan economicallysignificantdiseaseofswinesinceitsemergenceinbotheuropeandthe UnitedStatesinthelate1980 s(12u14).despitenearlythirtyyearsofresearch,thereis currentlynoprrsvvaccineavailablethatiseffectiveagainstheterologousstrainsofthe virus(15,16).aswithhivu1andinfluenzaa,thelackofaneffectivevaccineislargelydue toahighdegreeofgeneticvariationamongprrsvstrains(17).however,unlikeinhivu1 andinfluenzaa,theroleofimmuneescapevariantsininfectionispoorlyunderstood,and nodirectevidenceofimmuneescapeinprrsvinfectioninvivocurrentlyexists.thereis someindirectevidenceofimmuneselectionviacharacterizationofinvitroescapevariants andbioinformaticsubasedapproaches(18u20).however,severalstudiesofprrsvvariation invivohavefoundnoevidenceofimmuneselectionorimmuneescape(21u23).thelackof evidenceofimmuneselectionandimmuneescapemaybedueinparttotheweakadaptive immuneresponseelicitedbyprrsv(24,25).inordertoevaluatetheexistenceofprrsv immuneescapevariants,theworkdescribedinthisdissertationutilizedretrospective samplesavailablefromtheprrshostgeneticsconsortiumfromexperimentallyinfected pigsthatexperiencedreboundinviremiafollowinganinitialclearanceofthevirusfrom thebloodstream(26,27).reboundviremiasuggestedescapefromtheinitialhostimmune control,providingauniquesamplesettoidentifyimmuneselectionpressuresandescape variants.furthercharacterizationoftheidentifiedescapevariantswascarriedoutinorder

13 3 toidentifythemechanismofneutralizationofprrsv,andtheeffectofimmuneescapeon viralreplicationfitness.inaddition,becauseoftheweakimmuneresponseanddifficultyin makingeffectivevaccinesagainstprrsv,theuseofantiviraldrugsmaybeaviable treatmentoption.todate,fewantiuprrsvcompoundshavebeenidentified,andcurrently noneareusedinthefield.therefore,weidentifiedandcharacterizedsmallmoleculesfor antiuprrsvactivityinvitro. Overall&Goal& ToaddresstheknowledgegapintheroleofimmuneescapeinPRRSVandtoincrease potentialtreatmentoptionsforprrsvinfection,theoverallgoalofthisdissertation researchwastounderstandtheimmuneselectivepressuresunderlyingprrsvvariation, theconsequencesofvariationonviralpathogenesiswithinpigsduringinfection,andthe susceptibilityofprrsvtosynthesizedsmallmoleculecompounds. Specific&Aims& & Inordertoachievetheoverallgoal,thespecificaimsofthisdissertationresearchare: 1. CharacterizegeneticvariationandimmuneselectionofPRRSVwithin experimentallyinfectedpigs. 2. EvaluatePRRSVenvelopeproteinvariantsforescapefromneutralizingantibody. 3. Characterizethemechanismofneutralizationandconsequencesofneutralizing antibodyescapeonvirusreplicationfitness.

14 4 4. Identifyandcharacterizesmallmoleculecompoundsforinhibitoryactivityof PRRSV. & Dissertation&Organization& & Thisdissertationisorganizedintosixchapters.Chapter1includesageneral introduction,andtheoverallgoalsandspecificaimsofthedissertationresearch.areview oftheliteraturepertainingtoviralvariationandprrsvpathogenesisisdescribedin Chapter2.AmanuscriptsubmittedtotheJournalofVirologydescribingtheevaluationof immuneselectionduringprrsvinfectionandtheidentificationofprrsvimmuneescape variantsispresentedinchapter3.alyssab.evansperformedthecloning,sequencing, haplotyping,constructionofchimericviruses,andneutralizingantibodyassays,aswellas helpeddesignexperiments,andwroteandpreparedthemanuscript;karins.dorman performedthedapc,cmh,andallassociatedstatisticalanalyses,andhelpedwriteandedit themanuscript;sarahvantolhelpedconstructchimericvirusclones;jackc.m.dekkers providedcriticalanalysesandreviewofthemanuscript;andsusancarpenterhelpedinthe experimentaldesign,manuscriptwritingandediting,andgeneraloversightoftheproject. Chapter4describesfurthercharacterizationofthemechanismofPRRSVneutralizationby neutralizingantibodyandtheeffectofimmuneescapeonviralreplicationfitness.alyssab. Evansdesignedandoversawallexperiments,performeddataanalyses,andwroteand preparedthemanuscript;marcusboltonperformedtheinfectivityandreplicationkinetics assays;hyeleeloydperformedneutralizationmechanismassays;andsusancarpenter oversawtheexperimentaldesign,analysisofresults,andeditingofthemanuscript.

15 5 Chapter5reportstheresultsofthesynthesis,identification,andcharacterizationofsmall moleculecompoundswithinhibitoryactivityagainstprrsvinvitro.thisprojectwasdone incollaborationwiththegeorgekrauslabinthedepartmentofchemistryatiowastate University.PengfeiDongfromtheKrauslabwasresponsibleforthesynthesisofallofthe syntheticcompounds,andgeorgekrausoversawthecompoundsynthesis.alyssaevans designed,performed,andanalyzedbiologicalandvirologicalassays,wroteandprepared themanuscript;hyeleeloydperformedbindingandreplicationassays;susancarpenter helpedintheexperimentaldesign,dataanalyses,generaloversightoftheproject,and reviewofthemanuscript.chapter6discussesthegeneralconclusions,implicationsand limitationsofthisdissertationwork,andpresentspotentialstudiesforfuturework. Literature&Cited& & & 1. Bouvier&NM,&Palese&P.2008.Thebiologyofinfluenzaviruses.Vaccine26&Suppl& 4:D49U Wei&X,&Decker&JM,&Wang&S,&Hui&H,&Kappes&JC,&Wu&X,&SalazarZGonzalez&JF,&Salazar& MG,&Kilby&JM,&Saag&MS,&Komarova&NL,&Nowak&MA,&Hahn&BH,&Kwong&PD,&Shaw& GM.2003.AntibodyneutralizationandescapebyHIVU1.Nature422:307U Liao&HX,&Lynch&R,&Zhou&T,&Gao&F,&Alam&SM,&Boyd&SD,&Fire&AZ,&Roskin&KM,& Schramm&CA,&Zhang&Z,&Zhu&J,&Shapiro&L,&Program&NCS,&Mullikin&JC,&Gnanakaran& S,&Hraber&P,&Wiehe&K,&Kelsoe&G,&Yang&G,&Xia&SM,&Montefiori&DC,&Parks&R,&Lloyd& KE,&Scearce&RM,&Soderberg&KA,&Cohen&M,&Kamanga&G,&Louder&MK,&Tran&LM,& Chen&Y,&Cai&F,&Chen&S,&Moquin&S,&Du&X,&Joyce&MG,&Srivatsan&S,&Zhang&B,&Zheng&A,& Shaw&GM,&Hahn&BH,&Kepler&TB,&Korber&BT,&Kwong&PD,&Mascola&JR,&Haynes&BF CoUevolutionofabroadlyneutralizingHIVU1antibodyandfoundervirus. Nature496:469U Kolls&JK,&Szabo&G.2015.ThegeneticsofhepatitisCvirusunderlieitsabilityto escapehumoralimmunity.thejournalofclinicalinvestigation125:97u Keck&ZY,&Angus&AG,&Wang&W,&Lau&P,&Wang&Y,&Gatherer&D,&Patel&AH,&Foung&SK NonUrandomescapepathwaysfromabroadlyneutralizinghumanmonoclonal

16 6 antibodymaptoahighlyconservedregiononthehepatitiscviruse2glycoprotein encompassingaminoacids412u423.plospathogens10:e Corti&D,&Lanzavecchia&A.2013.Broadlyneutralizingantiviralantibodies.Annual reviewofimmunology31:705u Corti&D,&Suguitan&AL,&Jr.,&Pinna&D,&Silacci&C,&FernandezZRodriguez&BM,&Vanzetta& F,&Santos&C,&Luke&CJ,&TorresZVelez&FJ,&Temperton&NJ,&Weiss&RA,&Sallusto&F,& Subbarao&K,&Lanzavecchia&A.2010.Heterosubtypicneutralizingantibodiesare producedbyindividualsimmunizedwithaseasonalinfluenzavaccine.thejournal ofclinicalinvestigation120:1663u Cho&A,&Wrammert&J.2016.Implicationsofbroadlyneutralizingantibodiesinthe developmentofauniversalinfluenzavaccine.currentopinioninvirology17:110u Burton&DR,&Pyati&J,&Koduri&R,&Sharp&SJ,&Thornton&GB,&Parren&PW,&Sawyer&LS,& Hendry&RM,&Dunlop&N,&Nara&PL,&et&al.1994.Efficientneutralizationofprimary isolatesofhivu1byarecombinanthumanmonoclonalantibody.science266:1024u Binley&JM,&Wrin&T,&Korber&B,&Zwick&MB,&Wang&M,&Chappey&C,&Stiegler&G,&Kunert& R,&ZollaZPazner&S,&Katinger&H,&Petropoulos&CJ,&Burton&DR.2004.Comprehensive crossucladeneutralizationanalysisofapanelofantiuhumanimmunodeficiencyvirus type1monoclonalantibodies.journalofvirology78:13232u Lynch&RM,&Wong&P,&Tran&L,&ODell&S,&Nason&MC,&Li&Y,&Wu&X,&Mascola&JR HIVU1fitnesscostassociatedwithescapefromtheVRC01classofCD4bindingsite neutralizingantibodies.journalofvirology89:4201u Holtkamp&DJ,&Kliebenstein&JB,&Zimmerman&JJ,&Neumann&E,&Rotto&H,&Yoder&TK,& Wang&C,&Yeske&P,&Mowrer&CL,&Haley&C.2012.EconomicImpactofPorcine ReproductiveandRespiratorySyndromeVirusonU.S.PorkProducers.Animal IndustryReportAS& Loula&T.1991.MysteryPigUDisease.AgriUPractice12:23U Wensvoort&G,&Terpstra&C,&Pol&JMA,&ter&Laak&EA,&Bloemraad&M,&de&Kluyver&EP,& Kragten&C,&van&Buiten&L,&den&Besten&A,&Wagenaar&F,&Broekhuijsen&JM,&Moonen& PLJM,&Zetstra&T,&de&Boer&EA,&Tibben&HJ,&de&Jong&MF,&van&t&Veld&P,&Greenland& GJR,&van&Gennep&JA,&Voets&MT,&Verheijden&JHM,&Braamskamp&J.1991.Mystery swinediseaseinthenetherlands:theisolationoflelystadvirus.theveterinary quarterly13:121u130.

17 7 15. Kimman&TG,&Cornelissen&LA,&Moormann&RJ,&Rebel&JM,&StockhofeZZurwieden&N Challengesforporcinereproductiveandrespiratorysyndromevirus(PRRSV) vaccinology.vaccine27:3704u Murtaugh&MP,&Genzow&M.2011.Immunologicalsolutionsfortreatmentand preventionofporcinereproductiveandrespiratorysyndrome(prrs).vaccine 29:8192U Kappes&MA,&Faaberg&KS.2015.PRRSVstructure,replicationandrecombination: Originofphenotypeandgenotypediversity.Virology479Z480:475U Zhao&P,&Ma&C,&Dong&X,&Cui&Z.2012.Evolutionofquasispeciesdiversityforporcine reproductiveandrespiratorysyndromevirusunderantibodyselectivepressure. ScienceChina.Lifesciences55:788U Delisle&B,&Gagnon&CA,&Lambert&ME,&DAllaire&S.2012.Porcinereproductiveand respiratorysyndromevirusdiversityofeasterncanadaswineherdsinalarge sequencedatasetrevealstwohypervariableregionsunderpositiveselection. Infection,geneticsandevolution:journalofmolecularepidemiologyand evolutionarygeneticsininfectiousdiseases12:1111u Franzo&G,&Dotto&G,&Cecchinato&M,&Pasotto&D,&Martini&M,&Drigo&M Phylodynamicanalysisofporcinereproductiveandrespiratorysyndromevirus (PRRSV)inItaly:actionofselectivepressuresandinteractionsbetweendifferent clades.infection,geneticsandevolution:journalofmolecularepidemiologyand evolutionarygeneticsininfectiousdiseases31:149u Goldberg&TL,&Lowe&JF,&Milburn&SM,&Firkins&LD.2003.Quasispeciesvariationof porcinereproductiveandrespiratorysyndromevirusduringnaturalinfection. Virology317:197U Chang&CC,&Yoon&KJ,&Zimmerman&JJ,&Harmon&KM,&Dixon&PM,&Dvorak&CMT,& Murtaugh&MP.2002.EvolutionofPorcineReproductiveandRespiratorySyndrome VirusduringSequentialPassagesinPigs.Journalofvirology76:4750U Chen&N,&Trible&BR,&Kerrigan&MA,&Tian&K,&Rowland&RR.2016.ORF5ofporcine reproductiveandrespiratorysyndromevirus(prrsv)isatargetofdiversifying selectionasinfectionprogressesfromacuteinfectiontovirusrebound.infection, geneticsandevolution:journalofmolecularepidemiologyandevolutionary geneticsininfectiousdiseases40:167u Murtaugh&MP,&Xiao&Z,&Zuckermann&F.2002.ImmunologicalResponsesofSwineto PorcineReproductiveandRespiratorySyndromeVirusInfection.Viralimmunology 15:533U547.

18 8 25. Lopez&OJ,&Osorio&FA.2004.RoleofneutralizingantibodiesinPRRSVprotective immunity.veterinaryimmunologyandimmunopathology102:155u Lunney&JK,&Steibel&JP,&Reecy&JM,&Fritz&E,&Rothschild&MF,&Kerrigan&M,&Trible&B,& Rowland&RR.2011.ProbinggeneticcontrolofswineresponsestoPRRSVinfection: currentprogressoftheprrshostgeneticsconsortium.bmcproceedings5&suppl& 4:S Boddicker&N,&Waide&EH,&Rowland&RR,&Lunney&JK,&Garrick&DJ,&Reecy&JM,&Dekkers& JC.2012.EvidenceforamajorQTLassociatedwithhostresponsetoporcine reproductiveandrespiratorysyndromeviruschallenge.journalofanimalscience 90:1733U1746. & & & & & & & & & & & & & & & & &

19 9 CHAPTER&2:&LITERATURE&REVIEW& & Viral&Variation&and&its&Consequence&on&Infection& & Sincetheirdiscoveryinthelate1800 sandearly1900 s,viruseshavebeen recognizedasasignificantcauseofdiseaseinhumans,animals,andplants.highmutation ratesandshortgenerationtimeshavemadethecontrolandpreventionofmanyviral diseaseselusive.understandingviralvariationanditseffectonvirusuhostinteractionswill leadtoinsightsintoviralpathogenesisandemergenceandthedevelopmentofbetter treatmentandpreventionstrategies. The&quasispecies&structure&of&RNA&virus&populations& AcommonfeatureacrossRNAvirusesistheirexistencewithinhostsasacomplex population cloud ofrelatedbutnonuidenticalvariantsknownasquasispecies(1u3). Quasispeciespopulationsrepresenta mutantcloud ofbothpotentiallybeneficialand potentiallydeleteriousvariantscenteredaroundasequenceaverage,orconsensus sequence(3u5).whilesomernavirusesundergomajorgeneticchanges(geneticor antigenicshift)viarecombination,asinhumanimmunodeficiencyvirustype1(hivu1;6), ortheexchangeofentiregenomicsegments,asininfluenzaavirus(7)themain mechanisminthecreationofmutantcloudsistheaccumulationofrandommutations (geneticorantigenicdrift)(3).thisisduetothehigherrorrateandlackofproofureading mechanismsofviralrnapolymerases(3,8).viralrnapolymerasemutationratesare estimatedtobebetween10 U6 to10 U4 substitutionspernucleotidepercellinfection,or

20 10 approximatelyonemutationpergenomeineachreplicationcycle(9,10).despitethe apparentinfinitepotentialforvariationwithinviralgenomes,thereisaninherent evolutionarytradeuoffforviralquasispecies.theaccumulationoftoomanymutationscan bedetrimentaltovirusfitnessbecauseoftheproductionofnonuviablegenomes,and converselytoolittlegeneticvariationdecreasesthereservoirofpotentiallybeneficial variantsinthemutantcloud,limitingthevirus capabilitytoadaptundervarying circumstancesandenvironments.selectivepressureshelptoshapethecompositionofthe viralquasispeciespopulation.negative,orpurifying,selectionactstoeliminatedeleterious ornegativephenotypictraitsfromthequasispeciespopulation,whereaspositiveselection actsonsetsofvariantswithbeneficialphenotypestoincreasetheirfrequencyinthe mutantcloud. Viralquasispeciespopulationsarenotmerelyanartifactoftheaccumulationof variantsduringreplicationviaerrorupronemechanisms,butareaninteractivegroupof variantsthatarebeneficialtornavirusfitnessandcanhavecomplementaryinteractions. Infact,severalstudieshavefoundthattheuseofhighUfidelityRNApolymeraseinviral replicationresultsindecreasedfitnessoftheviralpopulation(11,12).inastudyofa poliovirusvariantwithahighufidelityrnapolymerase,theauthorsfoundthattheresulting homogenousviruspopulationwaslessadaptabletochangingenvironmentsandless virulentinmicethanitswildtypecounterpart(11).however,artificiallyexpandingthe quasispeciesofthehighufidelityvariantviachemicalmutagenesisrescuedpathogenicity (11).Inaddition,thesamestudyconductedsingleandcoUinfectionsofapoliovirusvariant thatwasunabletoinfectthecentralnervoussystemandawildtype,neurotropic poliovirus.inthepresenceofwildtypepoliovirus,thenonuneurotropicvariantwascapable

21 11 ofinfectingthecentralnervoussystem,demonstratingcomplementationbetweenviral quasispecies(11).similarly,ahighufidelityvariantofchikungunyavirus(chikv)had reducedviremiainbothamosquitovectorandmousehostcomparedtowildtypechikv, indicatingthatadecreaseinquasispeciesheterogeneitynegativelyimpactedviralfitness (12).& & & Antigenic&variation&and&immune&escape& ThelargeamountofvariationinRNAvirusesmakeseradicationandcontrolofthese pathogensextremelydifficult.inmammals,asuccessfulimmuneresponsetoavirus, elicitedbyimmunizationorinfection,leadstotheproductionofbucellsandtucellsthat recognizespecificportionsofthevirus,calledepitopes.asubsetofthesebucellsandtucells formmemorycells,andthismemorycomponentoftheadaptiveimmuneresponsecan thenbecalledonuponsubsequentexposuretothesamepathogen,whichleadstoaquick andeffectiveimmuneresponsetopreventtheestablishmentofaproductiveinfection.of particularimportanceinpreventingsubsequentinfectionbythesamevirusisneutralizing antibodysecretedfrommemorybucells(13u15).thesememorybucellscirculateinlow numbersthroughoutthebodyafterclearanceoftheinitialinfection.uponsubsequent infectionwiththesamevirus,thememorybucellsrecognizethepathogenviathebindingof theirbucellreceptorwiththespecificvirusepitope,andelicittherapidactivationand productionofmorebucellsinordertosecreteneutralizingantibodytopreventthe infectionfromestablishinginthehost,orcleartheinfectionquickly(16).thisprocessis effectiveuponexposurewiththesamevirusthatexpressesthesameepitopethatthebucell

22 12 responserecognizedduringthefirstinfection,andcontributestothelifeulongprotection generatedbyvaccinesagainstsmallpoxvirus,poliovirus,andmeaslesvirus(13). TheimmuneresponsebymemoryBUcellsisoftennoteffectiveagainstgenetically distinctstrainsofthesamevirusduetodifferencesorchangesintheviralepitopes recognizedbyneutralizingantibody,aprocessknownasantigenicvariation.antigenic variationcancontributetothespreadandcontinualcirculationofvirusesviagenerating diversityamongandbetweenstrainsofthesamevirus.becausevirusescancontinually acquirenewmutationsandchangeastheyreplicateinhosts,eachnewhostmaybe infectedbyaslightlydifferentversionofthevirus.thiscanbeproblematicforhosts,even whentheyhavepreviouslymountedasuccessfulimmuneresponsetothevirus.ifvariation occursinviralepitopes,thememorybucellscreatedfrompriorexposuretothevirusmay notrecognizethenewinfection,andthereforecannotmountarapidbucellresponsetothe virus.antigenicvariationisamajorreasonforthelackofeffectiveinfluenza,hivu1,and footuandumouthdiseasevirusvaccines(7).theidentificationof broadlyneutralizing antibodies (bnab)inhivu1andinfluenzaavirusthatarecapableofcrossureactivityand preventinginfectionbyawidevarietyofgeneticallyvariantviruseswithinavirusfamily hasgivensomehopetotheideaofdevelopingsingle,effectivevaccinesagainstdiverse viruses;howeverthesebnabshavebeendifficulttoreliablyelicitviaimmunization(14, 17). Antigenicvariationnotonlyoccursbythetransmissionofnewviralvariants betweenhosts,butalsobytheselectionand/orreplicationofviralvariantswithin individualhosts.whenantigenicvariationoccursinasubsetofvariantsinaquasispecies populationwithinasinglehost,thecollectionofvariantsthatareresistanttotheimmune

23 13 responsethatwaseffectiveagainstthemajorityofthepopulationarecalledescape mutants.theseescapemutantscanthenreplicateandestablishanew,productiveinfection inthehostandseedanew,orexpanded,quasispeciespopulationdistinctfromtheinitial populationthatwascontrolledbythehostimmuneresponse.escapevariantsareroutinely seenwithinhivu1uinfectedpatients,whichcontributetothepersistenceoftheviruswithin theseindividuals(18). Antigenicvariationandescapefromneutralizingantibodycontributestothespread ofmanyviruses,includingtheimportanthumanpathogensinfluenzaa,hivu1,hepatitisb virus,hepatitiscvirus,aswellasagriculturallyimportantvirusessuchasavianandswine influenzaandfootuandumouthdiseasevirus(19).antigenicvariationisamajorcauseof thecontinualcirculationandannualepidemicsofinfluenzavirusinhumans,swine,and pathogenicstrainsinbirds.theimmunitygeneratedfrompreviousexposuretoinfluenza strains,viavaccineorinfection,istypicallynoteffectiveatcontrollingthenextyear s epidemicstrain(7,17,20,21).influenzaa,anegativesensernavirus,isparticularlyadept atantigenicvariationandimmuneescapebecauseitcangeneratediversitythroughboth antigenicshiftandantigenicdrift.thesegmentedgenomicstructureofinfluenzaaallows fortheexchangeofanyofitseightgenesegmentsbetweendifferentstrainsduringcou infectionofasinglecell,atypeofantigenicshiftknownasreassortment(7).theresulting geneticallymixedviruses,orreassortants,canacquirenewphenotypictraitsnotseenin eitheroftheparentalstrains.novelreassortantshaveledtoseveralinfluenzaepidemics andpandemics,includingtheoutbreakofhighlypathogenicavianinfluenzaincommercial chickenflocksincanadaandtheunitedstatesin2015(22),the2009h1nipandemicin humans(23),andthecurrentlycirculatingendemicstrainsinswine(24).however,large

24 14 changesingeneticdiversityarenotstrictlynecessaryforantigenicvariation.an antigenicallydriftedh3n2humaninfluenzastrainduringthe2014u2015seasonallowed thisstraintoescapeantibodyelicitedbythegeneticallysimilarh3n2vaccine,resultingin poorperformanceofthevaccine(25). ViruseswithnonUsegmentedgenomesmayundergoantigenicshiftvia recombination,butmanyprimarilyutilizeantigenicdriftfortheproductionofantigenic variation,immuneescape,andpersistence(6,7).itiswellestablishedforhivu1that antigenicvariationwithinthequasispeciespopulationofasinglepatientleadstocontinual escapefromthehost sneutralizingantibody.patientseraarecapableofneutralizingthe autologoushivu1quasispeciespopulationfromprevioustimesininfection,butare incapableofneutralizingconcurrentvirus(18,26u28).hivu1employsastaggeringnumber ofstrategiestoevadeandescapethehost simmuneresponse,includingmaskingof neutralizingepitopesontheenvelopeprotein(env)throughglycanshielding,and conformationalandstructuralchangesthatalterantibodybinding(14).thepositivesense RNAvirushepatitisCvirus(HCV)iscapableofpersistingforlongperiodsoftimewithina hostdue,inpart,tocontinualantigenicvariation.theresultingnabescapemutants, similartothoseseeninhivu1,utilizemultiplegeneticpathwaysofescape,leadingto ineffectivevaccinesagainsthcv(29,30).theagriculturallyimportantfootuandumouth diseasevirus,apositivesensernavirusofclovenuhoofedlivestockhasalsobeenshownto haveaconsiderableamountofantigenicvariation,andmonoclonalneutralizingantibody escapevariantshavebeendescribedinvitro(31,32). DespitetheenormousamountofantigenicvariationwithinRNAviruses,crossU reactivebroadlyneutralizingantibodiestoseveralrnaviruseshavebeenidentified,

25 15 notablyagainstinfluenzavirusandhivu1(14).influenzabnabswereidentifiedasearlyas 1993(17,33).ThesebNAbsprimarilytargetthehighlyconservedstemregionof influenza shemagglutinin(ha)envelopeprotein(stemureactiveantibodies)andblock crucialstepsinfusionoftheviralenvelopewiththehostmembrane(17,34).importantly, thesebnabscanbeproducedinhighamountsafterseasonalinfluenzaimmunizationin somepeople(34).however,thisisnotauniversaloccurrenceandfewofthestemureactive memorybcellsaremaintainedforlongperiodsafterimmunization.bettermechanismsof elicitingbnabmemorycellsareneedediflongulastingimmunitytoinfluenzavirusthrough auniversalvaccineistobeachieved(17).similarly,inhivu1,bnabhavebeenidentified andtargetmultipleregionsofenv,includingthereceptorandcoureceptorbindingsitesand themembraneuproximalexternalregion(14,35u37).passivetransferstudieswithhivu1 broadlyneutralizingantibodieshavedemonstratedthatinvivoprotectionispossible, thoughhighserumconcentrationsaretypicallyrequired(14,38u40).however,bnabwere isolatedfromlongutermhivuinfectedindividualsanddidnotprotectagainstvirus replicationintheseindividuals,likelyduetoongoingantigenicvariationandimmune escape(14).therefore,whilebnabtohivu1maypotentiallyhavetherapeuticapplications, theireffectivenessinpreventinghivu1infectionisunclear.interestingly,broadlycrossu reactiveantibodieshavealsobeendescribedforthearbovirusdenguevirus(denv),but theywerenotneutralizingandinfactactuallypromotedviralreplicationand pathogenicity,aphenomenonknownasantibodyudependentenhancement(ade)(14,41). WhilethediscoveryofbroadlyneutralizingantibodiesagainstinfluenzaandHIVU1 provideshopeforthedevelopmentofeffective,universalvaccinesagainsttheseandother geneticallydiverseviralpathogens,themyriadstrategiesofantigenicvariationand

26 16 immuneescapeemployedbythesevirusescontinuetoposesubstantialchallengesfortheir controlandelimination. Antiviral&drug&resistance& Wheneffectivevaccinesforviraldiseasesarelacking,oftenthebestcourseofaction istreatmentoftheinfectionwithantiviraldrugsorimmunotherapy.antiviralshavebeen extremelyeffectiveinthetreatmentofhivu1andhepatitiscvirus(hcv)infections.antiu retroviraltherapy(art)hasturnedinfectionwithhivu1intheunitedstatesfromadeath sentencetoamanageablechronicinfection(42,43).antiuretroviraldrugs(ards)have beendevelopedagainstvirtuallyeverystepofhivreplicationandareextremelyeffective indecreasingpatientviralloadandincreasinglifespan(43,44).similarsuccesshasbeen foundusingantiviralsforthetreatmentofhcv.foryears,chronichcvinfectionswere typicallytreatedviaimmunotherapywithinterferonualpha(ifnuα),however20u50%of patientsdidnotrespondtothetreatmentduetoquasispeciespopulationsconsistingof IFNUαresistantvariants(45).Morerecently,thetreatmentofchronicHCVhasfound successthroughdirectuactingantivirals(daa)(45u47).thesedrugswereahuge breakthroughintreatmentofhcv,andhavereportedcureratesof90u100%(45). However,similartoantigenicvariationandimmuneescape,geneticvariationin virusescanleadtoresistancetoantiviraldrugsthatwerepreviouslyeffectiveatcontrolling theinfection,leadingtothereplicationofantiviralescapevariantsandtheemergenceof druguresistantstrains.ardresistanceisasignificantprobleminthetreatmentofhivu1 infections.duetothehighmutationrateofhivu1anditscontinualreplicationwithinits host,drugresistantescapemutantscommonlyarise,bothinpatientswhoarelongterm

27 17 ARTusers,andinnewlyinfectedpeopleviathetransmissionofdrugresistantvariants(48, 49).Becauseofthis,ARTgenerallyconsistsofacombinationofARDstocombatdrug resistantescapemutants(50).however,multiudrugresistantescapemutantshavebeen described,andwhilenewardsarecontinuallybeingdeveloped,thecontinuedtrendof ARDsselectingfordrugresistancemutantsisofconcernforlongUtermHIVtreatment(48, 49).Likewise,thetreatmentofchronicHCVinfectionsishamperedbytheoccurrenceof drugresistantescapemutants.despitethesuccessofthedirectuactingantivirals,subsetsof HCVstrainsareresistanttoDAA,andnewDAAresistantescapemutantshavealsoarisen (45,47).Whileantiviralsareakeycomponentincombatingpersistentviralinfections,the continualoccurrenceofdrugresistantescapemutantswithinquasispeciespopulations underliestheneedforthedevelopmentofnewantivirals,aswellasalternativetherapies forthetreatmentofpersistentviralinfections. & Emergence&of&new&viral&diseases Inadditiontocontributingtoantigenicvariation,immuneescape,anddrug resistance,viralquasispeciescancontainsubsetsofthepopulationwithalteredtropism andhostrange,whichcanleadtotheemergenceofnewviraldiseasesthroughcrossu speciestransmissionevents.severalhighuprofileviraldiseaseoutbreakshaveoccurred sincetheturnofthecentury,mostnotablythesevereacuterespiratorysyndrome coronavirus(sarsucov)outbreakin2003,andtheebolavirus(ebov)epidemicthat ravagedthewestafricancountriesofsierraleone,guinea,andliberiain2014.several arthropoduborneviruses(arboviruses),includingchikungunyavirusandmostrecently zikavirus,haveemergedandarecapableofcausingsignificantandseverediseasein

28 18 humans.additionally,thecoronavirusporcineepidemicdiarrheavirus(pedv)hasrecently emergedinswineandcausedsignificanteconomiclossestotheporkindustryworldwide (51).ThemajorityofthesevirusesemergedviacrossUspeciestransmissionfacilitatedby geneticchangesinthevirus,allowingtheestablishmentofinfectioninanewhost. SARSUCoVemergedin2002inGuangdongprovinceinChinaandinfectedthousands andkilledhundredsofpeopleacrosstheglobethroughout2003(52,53).sarsucovwasa novelcoronavirusatthetimeofitsdiscovery,butsequenceanalysisofsarsucovisolates fromtheoutbreakwithsarsulikecoronaviruses(slucov)fromcivetsandbatsrevealed thataslucovlikelyjumpedfromabatreservoirtocivetsasanintermediatehost,where thevirusunderwentadditionalmutationstofacilitatethejumptohumans(52,54u57),the jumpfromcivetstohumanswaslikelymediatedbymutationsinthesarsucovreceptor bindingdomainintheenvelopespikeprotein,whichfacilitatesbindingtothehuman receptorangiotensinuconvertingenzyme2(aceu2)(58).arecentlyemergedcousinto SARSUCoV,MiddleEastrespiratorysyndromecoronavirus(MERSUCoV),isbelievedtohave similarlymadethejumpfrombatstohumans,possiblythroughcamelsasanintermediate host,(59u61).unlikesarsucov,mersucovtodateonlyhaslimitedhumanutouhuman spread,asitapparentlyisnotwelladaptedtothehumanhost(59,60).thecloselyrelated batcoronavirusesthatmersucovisbelievedtobederivedfromutilizetheevolutionarily conserveddipeptidylpeptidase4(dpp4)receptor,andsomeevidencesuggeststhat mutationsinthespikeproteinofmersucovallowedforbetterbindingwithhumandpp4 andfacilitatedthecrossuspeciestransmission(62).similarly,acoronavirusofswine, porcineepidemicdiarrheavirus(pedv),emergedintheunitedkingdomin1971,chinain the1980s,andtheunitedstatesin2013,likelyviatransmissionofamutatedstrainfrom

29 19 batstopigs(63).sustainedpigutoupigtransmissionandcontinualgeneticvariationinpedv hasledtoperiodicoutbreaksofhighlypathogenicpedvinchinaandtheunitedstates (63). Therecent2014EBOVoutbreakinWestAfricathatcausedmorethan28,000 infectionsandover11,000deathsisbelievedtohaveoriginatedfromasinglezoonotic transmissioneventofamutatedvariantfromabattoa2uyearoldboy(64).theebov variantwasreadilytransmissiblefrompersonutouperson,resultingintheepidemic(64). ThespecificmutationsthatfacilitatedthiscrossUspeciestransmissioneventarecurrently notknown,but173aminoacidsubstitutionsacrossallebovorfswereidentified betweenthe2014ebovoutbreakstrainandpreviouslyidentifiedebovsequences(65). Severalarboviruseshaverecentlyemergedorreemerged,causingsubstantial diseaseinhumans,includingchikungunyavirus(chkv)andzikavirus(zikv)(66,67). TheseviruseswerefirstidentifiedintheearlyUmid1900 sandcausedsmallclustersof diseaseinafricaandasia,butrecentlyhavereemergedtocauseincreasingnumbersof infectionsacrosstheglobe,resultinginarthriticulikedisease(chkv)andpotentially microcephalyininfantsandguillainubarrésyndromeinadults(zikv)(66,67).thisspread isbelievedtobedue,atleastinpart,toevolutionaryadaptationstotheirmosquitovectors (CHKV)orhumanhost(ZIKV),resultinginvariantsthataremoreeasilytransmissibleto humans(68u72).inchkv,asinglemutationintheenvelopegenehasbeenimplicatedin thespreadofchkvbyexpandingthevectorspecificityfromaedesaegyptitoaedes albopictusmosquitospecies,resultinginincreasedviralloadinthevectorand transmissiontohumans(68).whileresearchonthereemergingzikvisstillinitsinfancy,

30 20 someevidencesuggeststhatvariationincodonusageinanonstructuralproteinofzikv hasresultedinvariantsbetteradaptedforreplicationinhumans(71). & Summary&of&viral&variation& & ThehigherrorrateandquasispeciespopulationstructureofRNAvirusescanhave manysignificantimplicationsforthepathogenesisandtreatmentoftheseinfections.a diversequasispeciespopulationcontainspotentiallybeneficialvariantsthatcanemerge underdifferentselectivepressures.thishighdegreeofgeneticvariationwithinrna virusescanleadtoantigenicvariationandimmuneescape,drugresistance,andthe emergenceofnewviraldiseases.together,theseconsequencesofviralvariationpose manychallengestotheprevention,treatment,andcontrolofrnaviruspathogens. Porcine&Reproductive&and&Respiratory&Syndrome&Virus& & Porcinereproductiveandrespiratorysyndromevirus(PRRSV)isa15kbpositiveU strandedrnavirus.prrsv,whichemergedsimultaneouslyastwodivergentstrainsinthe intheunitedstates(type2prrsv)andeurope(type1prrsv)inthelate1980 scauses respiratorysymptomsingrowingpigsandspontaneousabortionsinpregnantsows(73u 76).PRRSViseasilytransmissiblethroughtherespiratoryroute,aswellassexual transmission(77).theeconomiclossesduetoprrsvareestimatedtoberesponsiblefor $670millioninlossesannuallyfortheUSporkindustry(78).Severalvaccineshavebeen developedagainstprrsv;howevertheireffectivenessishamperedbythehighamountof virusvariationandgeneticdiversity(79u81).althoughgeneticdiversityofprrsvhasbeen

31 21 documentedandcharacterizedingreatdetail(82),theevolutionaryandmolecular mechanismsthatdrivethecontinualcirculationofprrsvvariantswithinandbetween animalsarenotwellunderstood.suchunderstandingiscriticalforthedevelopmentofsafe andeffectivevaccinesthatconferbroadandlongulastingprotectionagainstprrsv. PRRSV&genome&and&virion&structure& & PRRSVisanenveloped,15kbpositivestrandRNAvirusintheArteriviridaefamilyin theordernidovirales.thegenomeofprrsvconsistsofatleastelevenopenreadingframes (ORFs)(83,84).The5 twouthirdsofthegenomeencodesthepolyproteinsorf1aand ORF1ab(producedfromaU1ribosomalframeshift)thatareproteolyticallyprocessedto formatleast12knownnonstructuralproteins(nsp),butupto16havebeenpredicted(85, 86).TheORF1aand1abpolyproteinsaresynthesizedandsubsequentlycleavedintothe individualnspsviaselfucleavageofnsp1andnsp2,withthensp4proteasemediatingthe remainingknowncleavages(85u87).oncecleaved,variousnspsmediatevirusreplication andvirusuhostinteractions,includingsuppressionoftheinnateimmuneresponse(79,88). Importantly,nsp9istheviralRNAUdependentRNApolymerase(RdRp),andnsp2,nsp3, andnsp5containtransmembranedomainsthathavebeenimplicatedinhostmembrane remodelingfortheformationofvirusreplicationvesicles(86,89,90).& ThelastthirdofthegenomecontainseightoverlappingORFs(ORF2U7,2a,and5a) thatencodethevirusstructuralproteins(91u94).orf5andorf6encodethemajor envelopeproteins,theglycosylatedgp5andm,respectively,whichinteracttoforma heterodimeronthevirionsurfacethatisbelievedimportantforattachment(95u98).orf2u 4encodestheminorenvelopeglycoproteinsGP2,GP3,andGP4,respectively,whichforma

32 22 heterotrimerthatinteractswiththecd163receptor(99u103).additionalminorenvelope componentsareencodedbyorf2aandorf5a,whicharenestedwithinorf2andorf5 andencodetheeproteinand5aprotein,respectively(93),(94).someevidencesuggests thatemayinteractwiththegp2/3/4trimerandfunctionasanionchannel(94,102,104, 105).Importantly,alloftheenvelopeproteinsareabsolutelyrequiredforproductive PRRSVinfection(94,100).ThefinalPRRSVstructuralproteinisthenucleocapsid(N), whichisencodedbyorf7(91,106).ncontainsanuclearlocalizationsignal,andprevious studieshaveshownthatitentersthehostnucleolus,suggestingthatinadditiontoserving astheprrsvnucleocapsid,nmaybeinvolvedinregulatinghosttranscription(107,108). NegativestainandcryoUelectronmicroscopyhaverevealedthatPRRSVhasaspherical, smoothvirionthatisapproximately54nmindiameter(108u110).withintheinteriorofthe virionisahollowcorecomposedofndimerslikelyinahelicalorchainulinktype arrangementassociatedwiththe(+)rnagenome(108,110). PRRSV&replication&and&molecular&pathogenesis& Likeallviruses,thePRRSVreplicationcycleconsistsofentryintothecell,protein andgenomesynthesis,andassemblyandreleaseofnewvirions.entryofavirusintoits hostcellistypicallyfacilitatedbyaseriesofcomplexinteractionsbetweenthevirusand thehost,andmanyoftheseinteractionsarenotknownforprrsv.toinitiatetheentry process,thefirstthingthatneedstohappenisaninitialinteractionbetweenthevirusand thehostcell.thisinitialattachmentstepisoftenmediatedthroughtheinteractionofthe viruswithcellular attachmentfactors,whicharehostcellproteinsthatcanbindtoviral proteinstobringthevirionintocloseproximitywiththecell,butdonotactivelypromote

33 23 entryofthevirusintothehostcell.theseinitialattachmentinteractionsarenotknownfor PRRSV,althoughthereissomeevidencethatinteractionsofCD169withGP5,andheparin sulfatewithmmaymediateattachment(95,97).followingattachment,prrsvbindstoits receptor,cd163,throughinteractionswithgp2andgp4(102,103).avirusreceptorisa cellularproteinthatactivelypromotesvirusentryintothecelluponbindingtothevirus, andistheprimarydeterminantofcelltropismforavirus.uponbindingofgp2andgp4 withcellularcd163,theprrsvvirionistakenupintothecellviaendocytosis(98). Additionally,itwasrecentlyshownthatthecellularfactorMYH9bindingtoGP5atapostU attachmentstepinentryisalsoarequiredinteractionforprrsvinfection(111).likeall envelopedviruses,prrsvmustfuseitsenvelopewiththehostcellmembrane,inthecase ofprrsvwiththeendosomalmembrane,inordertoreleaseitsgenomeintothecell, howevertheprrsvfusionproteinthatmediatesthisinteractioniscurrentlyunknown. Uponreleaseofthegenomeintothecytoplasm,PRRSVcompletesitsreplicationcycle throughthesynthesisofviralproteinsandgenomes,whichareassembledintonewvirions facilitatedbygp5andm(100),andarereleasedfromthecellviaexocytosis(86). PRRSVprimarilyinfectscellsofthemacrophagelineage,withapreferencefor porcinealveolarmacrophages(pams)(112).prrsvreplicatesinvitroinprimarycultures ofpamsandperipheralubloodmonocytederivedmacrophages(mdms),howevertheonly PRRSVpermissivecelllinesarethoseoftheMAU104lineageofAfricanGreenMonkey kidneycells,primarilyasubsetofhighlypermissivemarcu145cells(113).foryears,the hostcellreceptorforprrsvremainedamysteryduetoconflictingresultsfrominvitro studies.cd169wasimplicatedbyseveralstudiesasarequiredreceptorinpamsbutthis wascontroversial,asmarcu145cellsdonotexpresscd169(97,114u116).anadditional

34 24 cellularreceptor,cd163,whichisexpressedinpamsandmarcu145cells,wasimplicated astheprrsvreceptorthroughstudiesthatshowedthatexpressionofcd163couldmake nonupermissivecellspermissivetoprrsv(117).however,otherstudiesfoundthat expressionofcd163alonewasnotsufficientforprrsvpermissivity(115,116).aseriesof experimentsutilizinggeneueditedpigswithknockoutsofeithercd169orcd163found thatpigswithoutcd169werejustassusceptibletoprrsvinfectionaswildtypepigs; however,cd163knockoutpigswerecompletelyresistanttoprrsvinfection,decisively showingthatcd163,andnotcd169,isarequiredprrsvreceptor(103,118).therecent discoveryofmyh9asarequiredcellularfactorforprrsvinfectionmayexplainwhysome studiescouldnotproducepermissivecellsthroughtheexpressionofcd163alone(111). AttachmentofthePRRSVviriontothehostcellislikelymediatedbyinteractionsof themajorenvelopeproteindimergp5/mwithcellularattachmentfactors.thisis circumstantiallysupportedbythefactthatthegp5/mdimerisveryhighlyexpressedon thevirionsurface,withfeweroftheminorgp2/3/4complexespresent(108,110,119). WhileCD169isnotarequiredPRRSVreceptor,GP5hasbeenshowntointeractwith CD169andthisinteractionmayfacilitateviralattachment(95,97).However,thisisclearly notarequiredinteractionascd169knockoutpigsweresusceptibletoprrsv,andthe permissivemarcu145cellslackcd169(118,120).inadditiontocd169,mhasbeenfound tointeractwithheparinsulfateandfacilitateattachmentinpamsandmarcu145cells(97, 98,121).PriortotheCD163knockoutpigexperiments,CD151hadbeenimplicatedasa possibleprrsvreceptor,althoughnointeractionbetweencd151andanyprrsvprotein hasbeenfound(120).afterattachment,prrsvbindingtothereceptorfacilitatesreceptoru mediatedendocytosis(122).gp2andgp4interactwiththeprrsvreceptorcd163(102),