allen d. leman swine conference - university of minnesota · 2012 allen d. leman swine conference...

Allen D. Leman Swine Conference

Volume 39 2012 Published by: Veterinary Continuing Education Sponsors We thank the following sponsors: Platinum Bayer Animal Health Pfizer Animal Health Gold Novartis Animal Health Silver Boehringer Ingelheim Vetmedica, Inc. National Pork Board Newport Laboratories Bronze Merck Animal Health Copper AgStar Financial Services Elanco Animal Health GlobalVetLINK IDEXX Novus International, Inc. PIC USA USDA PRRS CAP University of Minnesota Institutional Partners College of Veterinary Medicine University of Minnesota Extension College of Food, Agriculture and Natural Resources Sciences

2012 Allen D. Leman Swine Conference 49

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Use and interpretation of sequencing in PRRSV control programsMichael P. Murtaugh, PhDDepartment of Veterinary and Biomedical Sciences, University of Minnesota

Take-home messages• PRRSVsequencingisaroutineserviceofdiagnos-ticlabsservingtheswineindustryinCanadaandtheU.S.

• Comparisonofsequencesbyalignmentrevealsthedegreeofsimilarity.

• Phylogeneticanalysis(dendrogramsortrees)withreferencesequencesshowsrelatednesstoknownvirusfamilies.

• Interpretationofsimilarities–arethevirusesrelatedornot–requiresasmuchadditionalinformationascanbeobtainedsinceratesofgeneticchangecanbehighlyvariable.

• 97%or98%sequencesimilarityisausefulguideindicatingrelatednessoftwovirusisolates,butge-neticsimilarityisanunreliablepredictorofimmu-nologicalsimilarityindicativeofcross-protectiveimmunity.

IntroductionDiagnosticPRRSVsequencingbeganinthemid-1990’stoaidproducersandveterinariansinthecontrolofPRRS.ThebestdiagnosticmethodofPRRSVidentificationatthetimewasRFLPtyping.ThismethodwasbasedonrestrictionenzymecutsitesinORF5thatgavevariouspatternsforeachofthreeenzymes.ItwasmostusefulfordifferentiatingtheIngelvacMLV(knownthenasResPRRSandResPRRSRepro)vaccinefromfieldviruses,sinceIngelvacMLVhadaunique2-5-2pattern,andfieldviruseshadother3-digitidentifiers.However,withinashortperiodoftimeitbecameapparentthatgeneticallyunrelatedviruseshadthesamecutpattern.Forexample,twocommoncutpatternswere1-4-2and1-4-4,butgeneticallydifferentvirusesappearedwiththesamecutpatterns.Figure1showsadendrogramfromthe1990’sinwhichgeneticallydiversevirusessharedthesamecutpatterns.Also,viruseswerebeingisolatedfromthefieldthatwerenot2-5-2butbyothercriteriaseemedtoberelated toIngelvacMLVvaccine.Clearly,abetterclassificationmethodwasneededtounderstandwhatwashappeninginthefield.

DiagnosticsequencingwasintroducedasanalternativetoRFLP typing to improve the ability to differentiatePRRSVfieldisolates,identifyvaccinereisolates,andac-commodatetheproliferationofvaccines(e.g.Suvaxyn,PRIMEPACPRRS,IngelvacATP,FosteraPRRS)andlivevirusinoculumsthatcouldnotbedifferentiatedbyRFLPtypingfromcommonfieldisolates.

ORF5,thegeneencodingthemajorenvelopeglycoprotein,isthestandardsequencingtargetbecauseitisusedforRFLPtyping,anditshowsextensivegeneticdiversity.Italsowasassumedtobeanimportanttargetforimmuneprotection,thoughitsimmunologicalsignificanceisnowuncertain.

The basic idea behind sequencingDiagnosticPRRSVsequencingintheyear2012isstraight-forwardintheUSandCanada.SerumororalfluidsampleswithmoderatetohighamountsofPRRSV(Ctvaluesofabout 27 or less, dependingon the diagnostic lab) aretreated to isolateRNA, and submitted to an academicorcommercialfacilitytosequenceORF5.Thisprocessgenerally takesoneto threedays.Therawdataresultsaresenttothediagnosticlab,checkedforquality, thenprocessed and trimmed to identify the specificORF5region.Thereporttypicallyincludessequencesimilaritytostandardvaccinestrains(e.g.IngelvacMLV,IngelvacATP,FosteraPRRS,Suvaxyn),thededucedaminoacidsequence,and theRFLPpattern.Somereports includeacomparisontoastandardreferencepanelofwildtypePRRSVisolates,orcomparisontoacompanyorclinicPRRSVsequencedatabase.

Analysis of PRRSV sequencesSequencesimilarity,oridentity,isdeterminedbyaligningtwoormoresequencesusingacomputerprogram.AnexamplealignmentoffourORF5sequencesisshowninFigure2.Threefieldvirussequencesarequitesimilartoeachother,varyingonlyby1.2to2.1percentinpairwisecomparisons(Table1).Alternatively,theyare97.9%to98.8%similar.Akeyquestionforproducersandveterinar-iansiswhetherthesedifferencesrepresentnormalvaria-tioninapopulationofvirusesinabarn,orareconsistentwithindependentvirusesthatarenotcloselyrelated.In

50 2012 Allen D. Leman Swine Conference

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Figure 1: RFLP patterns of genetically diverse PRRSV isolates. The dendrogram (black lines) contains 70 ORF5 sequences comprising 5 major PRRSV families. Forty-three (61%) of the sequences are in 6 RFLP types as indicated, including “untypeable.” All types except for 252 are present in multiple families. Dashed lines are included to assist alignment of boxes with the dendrogram.


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Use and interpretation of sequencing in PRRSV control programs

Figure 2: Base sequence alignment of 3 field isolates and Ingelvac MLV vaccine.

Table 1: Pairwise comparison table of PRRSV ORF5 sequences in Figure 2. Top diagonal is the pairwise percent sequence similarity. Bottom diagonal is the percent sequence difference.

Isolate 11-048146 12-018127 12-018776 Ingelvac MLV11-048146 100 98.7 98.8 85.112-018127 1.3 100 97.9 85.312-018776 1.2 2.1 100 84.7Ingelvac MLV 14.9 14.7 15.3 100

otherwords,are twoviruses relatedorunrelated.Thisquestionisaddressedbelow.Thefourthisolate,IngelvacMLVvaccine, isobviouslydifferent,specificallybeing14.7to15.3%different,andshowingbasemutationsfrombeginningtoend(Figure2).

Pairwise comparisons arewidely used tomake deci-sions about relatedness of PRRSV isolates, but theydonotprovideanygeneticinformationaboutthevirustype.SomePRRSVfamilies,suchas1-8-4,1-18-2,and1-4-4viruses,commonlyshowhighvirulence,airbornetransmission,andapropensitytoovercomepre-existingimmunity.Geneticrelatednessofisolatesisdeterminedfromsequencealignmentsthatcontainknownreferencestrains,andconstructionofdendrograms.Linearorradialdendrograms(Figure3)showthesamerelationshipsindifferentwaysthatmaybemoreeasilydigestedbyview-ers.Inourexample,thethreefieldisolatesarerevealedtobeacloselyrelatedgroupthatisanoffshootofthe1-18-2

familyofvirusesthatappearedintheU.S.around2008.Importantly,thequalityofinformationinandresolvingpowerofadendrogramisonlyasgoodasthereferencestrains thatareused.Thedendrogramsshownhere in-cluderepresentativesofmajorPRRSVfamilies,someofwhichhaveextensivegeneticvariation.The1-8-4familyhas tremendous genetic diversity.Thus, if only a fewrepresentativesequencesareused,itiseasytomistakenlyconcludethatanewisolateisonlydistantlyrelatedandcouldthusbeanewgenotype.Asecondlimitationintheexample shown is the absence ofEuropean-typevirusrepresentatives.Their presence distorts the shape of adendrogrambecausetheyaresodifference,makingithardtodiscriminatevariationamongtype2NorthAmericanisolates. Identifying thepresenceofa type1Europeanisolateiseasilydonebyinspectionofanalignmentsincethereareobviousgapswithmissingbases,and50to60%oftheexpectedbasesaredifferent.


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Figure 3: Phylogenetic analysis of three PRRSV field strains. Dendrogram (A) and radial dendrogram (B) of field strains (gray outlines) in a set of 43 type 2 North American reference sequences. The Ingelvac MLV-related and Ingelvac ATP-related families are identified, in addition to the 1-8-4 family. Note that the two dendrograms are identical representations of the same phylogenetic analysis.

SequenceanalysiscomputerprogramsalsocantranslatethebasesequenceofORF5intotheaminoacidsequenceoftheenvelopeglycoprotein,GP5.InPRRSV,theaminoacid sequence differences in proteins are often higherthanthebasedifferencesinthegenes.AnalysisofproteinsequencevariationcanprovideinsightsintomechanismsthatdrivePRRSVevolution,andcontribute to thebio-logical properties of the virus.However, an enormousgapexistsinPRRSVknowledgebetweengenesequencevariationandbiologicaldifferencesinvirulence,transmis-sibility,andcross-protectiveimmunity.SincethereisnosolidevidencelinkingaminoacidsequenceofGP5withspecificbiologicaltraits,PRRSVsequenceanalysisshouldbebasedongeneticvariationinthebases.

Interpretation of PRRSV sequencesThe sequenceofORF5 is about 600bases.While thisamountisonly4%ofthefull-lengthPRRSVgenome,itstillcontainsabout40timemoreresolvingpowertodif-ferentiateviralisolatesthandoesRFLP,whichisbasedonspecificbasepatternsforthreerestrictionenzymecutsitesinORF5.Withthehindsightof18yearsanalyzingPRRSVORF5sequencevariation,itisclearthatthe600baseregionofORF5isanexquisitelysensitiveindicatorof genetic variation and change inPRRSV. In one ex-ampleinacommercialswineoperation,IngelvacMLVwasadministeredtopigs,andreisolatedoveraperiodof6months.During this time, 20ORF5 sequenceswere


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Figure 3: Phylogenetic analysis of three PRRSV field strains. Dendrogram (A) and radial dendrogram (B) of field strains (gray outlines) in a set of 43 type 2 North American reference sequences. The Ingelvac MLV-related and Ingelvac ATP-related families are identified, in addition to the 1-8-4 family. Note that the two dendrograms are identical representations of the same phylogenetic analysis.

obtained,whichvariedbetweenzeroand0.8%(i.e.,zeroto5basedifferences)fromtheinitialvaccinesequence(MurtaughandHarding,2003).Additionalisolateswereobtainedoveraperiodof7yearsthatshowedincreasingvariationupto3.8%differencefromIngelvacMLV.Theoverallrateofbasechangewas0.48%to1.32%peryearforallisolatesinthisstudy.

InanexperimentalstudyofPRRSVtransmissioncarriedoutbysequentiallyinfectingpigsoveraoneyearperiodoftime,basesequencedifferencesvariedby0.7%fromtheinitialinoculum(Changetal.,2002).Takentogether,thefieldexperienceandexperimentalstudysuggestthatPRRSVchangesatarateofabout0.5%to1%peryearinthefield.Otheranecdotalobservationsareconsistentwiththisestimatedrange.Forexample,ORF5geneticdiversityamongtype2PRRSVisolatescollectedfrom1989to1993

wasabout9%.Atpresentitisabout25%,anincreaseof16%in19years.Itisimportanttorememberthatthesefiguresareaveragesbasedonalimitedsetofobservations,whereasmanyfactorsaffecttherateofgeneticchange.

Geneticchangeisdeterminedlargelybymutations.Thegenomemutationrateisprimarilyafunctionofthefidelityofthepolymeraseenzymethatcopiesthegenome.Thisproteinishighlyconservedandshowslittlegeneticvaria-tion(Changetal.,2002).Becauseitshowslittlevariation,itwillintroducemutationsinallPRRSVisolatesataboutthesamerateeachtimeagenomeiscopied.Therefore,geneticvariationwillbeinfluencedprimarilybythenum-beroftimesavirusreplicatesinagivenperiodoftime.PRRSV rate of growth and frequencyof transmissionaretheprimarydeterminantsofgeneticvariation.Asaresult,variationintherateofgeneticchangeisdetermined


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primarilybynon-viralfactorssuchasdifferentlevelsofimmunity.Thelevelofspecificanti-PRRSVimmunityinpigshasahugeimpactonviralgrowthandtransmission,exertingastronginhibitorypressurethatdiminishedthenumber of genome copies.Lower viral loads result inreducedtransmissionrates,furtherreducingoverallviralreplicationanddecreasingtherateofchange.Nonspecificinnateimmunityalsoexertsasubstantialanti-viraleffectthatvariesgreatlyamongindividualpigs.Age-dependentvariationinPRRSVresistanceiswelldocumented,butevenamongpigsofthesameageextensivevariationinviralloadoccurs(Klingeetal.,2009).Likewise,trans-missionrateishighlydependentonviralload(Choetal.,2007).Thesamevirusunderdifferenthostconditions,orvirusesthatgrowatdifferentratesinequivalentpigs,areexpectedtoshowdifferentratesofgeneticchange.Thus,allofthebiologicalfactorsthataffectviralgrowthwithinpigsandtransmissionamongpigscanresultinhigherorlower rates of genetic changeoutside of the suggestedrangeof0.5%to1%peryear.

Diagnostic issues further complicate the interpretationofPRRSVsequenceinformation,especiallythecentralquestionofwhethertwosequencesarecloselyrelatedorindependent.Sporadicsamplingwithinabarnorsystemmayresultinanincompletepictureofoveralldiversityin the population of indigenous viruses, and sporadicsamplingovertimemayresultinmissinglinksthatcon-nectvirusesthatotherwisemightappeartobeunrelated.Givenalloftheseuncertainties,itremainsthatPRRSVisolatesareroutinelydeterminedasrelatedornotbasedonapercentsimilaritycut-offof97%or98%.Unpublisheddatafromseveralextensivemonitoringprojectssuggestthatthesetwocut-offsgiveequivalentresults.

Itisobviousthatblindacceptanceofa2%or3%geneticdifferencebetweenisolates,withoutincorporationofad-ditionalknowledge, can lead toerroneousconclusions.ThealignmentexamplefromFigure2showsthreecloselyrelatedviruses,butstrictapplicationofa98%similaritycut-offwouldleadtoanillogicalconclusion.Likewise,inthecasecitedbyMurtaughandHarding,2003,theisolatethatis3.8%differentfromIngelvacMLVafter7yearsisnotadifferentvirus.Intensivemonitoringovertime,knowledgeofotherPRRSVgenotypesintheregion,andheardhealthstatusinformationexcludesthepossibilityofafieldvirusintroduction.

Is97%or98%toostringent?Theimplementationofa98%cut-offdatesto1998andwasbasedonsomeofthesamedatapresentedhere(Collins,1998;MurtaughandFaaberg,2001).Aprimarypurposeatthattimewastodetermineiffieldisolateswererelatedtovaccinesanditwasgener-ally assumed that the time framewas relatively short;i.e.,oneortwoyears.Hence,98%wasausefulcut-off.

Later,whenitbecameapparentthatbothvaccine-derivedandfieldvirusinoculation-basedimmuneprotectionwasvariable,geneticrelatednesswasexaminedasapredictorofimmuneprotection.Simplyput,ifhomologousimmuneprotectionwas good and heterologous immunitywasnotgood,thenwhatlevelofgeneticrelatednessdefinedhomologousandheterologous?OnehundredpercentwastoostringentsincePRRSVsequencechangesalmostev-erytimeitgrowsinapig(Changetal.,2002).The98%cut-offwasgenerallyused,ormodifiedby±1%,sinceitwasalreadyestablishedand sincenobetter alternativeforpredictionofimmuneprotection,suchasglycotypeorMJtype,couldbeestablished(Murtaughetal.,2010).Thus,97%or98%sequencesimilarityisausefulguideindicatingrelatednessoftwovirusisolates,butgeneticsimilarity is an unreliable predictor of immunologicalsimilarityindicativeofcross-protectiveimmunity.

Atthistime,wehavealimitedunderstandingofPRRSVgeneticvariationwithinswine-growingregions.Acom-prehensivepictureofviralvariationatthebeginningofcontrolandeliminationprojects iscritical foreffectivemonitoringprogressandeffectivenessofimplementationprocedures,andforidentificationofnewintroductionstofarmsandtotheregion.Intensivesamplingandsequenc-ing iswithin reach due to technical improvements inviralRNAisolation,PCRamplification,andsequencingmethods.Thoroughsamplingwilleliminatesubstantialdiagnosticuncertaintyandprovidebetterinsightstobio-logicalfactorsthatinfluencegeneticvariation.

Lastly,itispossiblethatORF5mightnotbethebestpor-tionofthePRRSVgenometoevaluategeneticvariation.Thequestion is primarily academic at this point sincethere issomuchgenetic informationforORF5,andsolittleforeveryotherregion.Also,thereisnoevidencethatanyotherfragmentwouldbesuperiorforassessingisolaterelatedness.However,itisclearnowthatgeneticvaria-tioninORF5neitherpredictsnorexplainsimmunologicalprotectionoroutbreaks in immuneherds.Theongoingdifficulty in obtaining reliable, durable immunologicalprotection,usingeitherattenuatedvaccinesorlivevirusinoculation,combinedwiththeinabilitytoidentifypre-dictorsofsuccess,isamajorstumblingblockincontrolofPRRSV.AssumingthatsomesequencefeatureinthePRRSVgenomeisassociatedwith immuneprotection,wholegenomesequencinghasbeeninitiatedinvariouslaboratoriesonaresearchbasistosearchforgeneticmark-ers.Thisworkisinadevelopmentalstageatthistime.

References1.Chang,C.C.,K.-J.Yoon,J.J.Zimmerman,K.M.Harmon,P.M.Dixon,C.M.T.Dvorak,andM.P.Murtaugh.2002.Evolutionofporcinereproductiveandrespiratorysyndrome(PRRS)virusduringsequentialpassagesinpigs.J.Virol.76:4750–4763.


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2.Cho,J.G.,J.Deen,andS.A.Dee.2007.InfluenceofisolatepathogenicityontheaerosoltransmissionofPorcinereproduc-tiveandrespiratorysyndromevirus.Can.J.Vet.Res.71:23–27.3.Collins,J.1998.InterpretingPRRSVsequencingdata.AllenD.LemanSwineConf.Proceed.25:1–4.4.Klinge,K.L.,E.M.Vaughn,M.B.Roof,E.M.Bautista,andM.P.Murtaugh.2009.Age-dependentresistancetoPorcinere-productiveandrespiratorysyndromevirusreplicationinswine.Virol.J.6:177–187.5.Murtaugh,M.P.andK.S.Faaberg.2001.HowtointerpretandusePRRSVsequenceinformation.AllenD.LemanSwineConf.Proc.28:60–66.

6.Murtaugh,M.P.andJ.Harding.2003.PRRSVgenomics:applicationtoafieldinvestigation.AllenD.LemanSwineConf.Proc.30:41–43.7.Murtaugh,M.P.,T.Stadejek,J.E.Abrahante,T.T.Lam,andF.C.Leung.2010.Theever-expandingdiversityofporcinerepro-ductiveandrespiratorysyndromevirus.VirusRes.154:18–30.

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