diagnostic phylogenetics reveals a new porcine circovirus 2 cluster

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VirusResearch217(2016)32–37

ContentslistsavailableatScienceDirect

Virus

Research

jo u r n al h om ep age :w w w . e l s e v i e r . c o m / l o c a t e / v i r u s r e s

Diagnostic

phylogenetics

reveals

a

new

Porcine

circovirus

2 cluster

Brendan

Davies

a

_,

_Xiong

_Wang

a

_,

_Cheryl

_M.T.

_Dvorak

a

_,

_Douglas

_Marthaler

b

_,

Michael

P. Murtaugh

a,∗

a_Departments_of_Veterinary_and_Biomedical_Sciences,_University_of_Minnesota,_St._Paul,_MN_55108,_United_States b_Departments_of_Veterinary_Population_Medicine,_University_of_Minnesota,_St._Paul,_MN_55108,_United_States

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18December2015

Receivedinrevisedform3February2016 Accepted3February2016

Availableonline3March2016

Keywords:

Porcinecircovirus2(PCV2)wasprevalentinswineintheUnitedStatesbeforePCV2-associateddisease (PCVAD)appearedin2006.Limitednucleotidesequencingofopenreadingframe2(ORF2)encoding cap-sid,theonlystructuralprotein,revealedthepresenceoftwogenotypes,PCV2aandPCV2b.Later,PCV2c andmutantPCV2b,orPCV2d,werealsodescribed.However,extensivePCV2ORF2sequencedatabases inveterinarydiagnosticlaboratorieshavenotbeenanalyzedsystematicallytodeterminethegenetic diversityoffieldisolates.Here,weinterrogated>1100PCV2ORF2nucleotidesequencestoassess popu-lationdiversityandgeneticvariation.WedetectedanovelPCV2genotypethatissubstantiallydifferent, primarilyinORF2,fromallknownPCV2.Notably,ORF2containsauniquecarboxylterminalaminoacid insertionresultingina238aminoacidORF2.AllotherPCV2ORF2proteinsare233or234aainlength. PhylogeneticanalysisindicatesthatitismoreancientthanotherPCV2genotypes.Thefindings demon-stratethevalueofanalyzingroutinediagnosticlaboratorysequencedatabasesinpopulationgenetic analysesofanimalpathogens.

1. Introduction

Porcinecircovirus2(PCV2)isasmall,circular,single-stranded DNAvirusofswine.PCV2wasdiscoveredinassociationwith out-breaksofapostweaning,multisystemicwastingsyndrome(PMWS) inthe1990’sinEuropeandCanada(Allanetal.,1998;Ellisetal., 1998). Retrospective analysis of archived diagnostic specimens showeditwaspresentinmultiplecountriesinEurope,Asiaand NorthAmericainpreviousdecades(Dupontetal.,2008;Patterson andOpriessnig,2010).AnationalsurveyoftheU.S.swineherdprior todiseaseoutbreaksshowedthatPCV2infectionwaspresentin nearlyallherdsataprevalencegreaterthan80%ofpigsbefore diseasesassociatedwithPCV2wereobserved(Haleyetal.,2011; Puvanendiranetal.,2011).

DiagnosticsequencingofPCV2openreadingframe2(ORF2), encodingtheviralcapsidprotein,waswidelyadoptedinNorth Americaandotherswinegrowingregionstoassistin understand-ingtheetiologyofdiseases,includingPMWS,porcinedermatitis andnephropathysyndrome,porcinerespiratorydiseasecomplex, andreproductivediseases,associatedwiththepresenceofPCV2

∗_{Corresponding}_author.

E-mailaddress:murta001@umn.edu(M.P.Murtaugh).

(Segales,2012;Segalesetal.,2005).Inthefollowingyears, exten-sivephylogeneticanalysisshowedthatPCV2consistedofseveral geneticclusters,withoutaclearconsensusrelatinggenotypic vari-ation tobiological characteristicsor todiseasecausation(Chae, 2015;Franzoetal.,2015;Wangetal.,2009;Xiaoetal.,2015).

NumerousstudieshavedescribedPCV2fieldisolatesandtheir phylogeneticrelationshipsinswinegrowingregions.However,a systematicanalysisofunpublishedORF2sequencescollectedin veterinary diagnostic laboratorieshas not been conducted. We examinedarepositoryof>1100PCV2ORF2sequencesdatingback to2001intheMinnesota,USA,VeterinaryDiagnosticLaboratory (MVDL).Aclusterofnovelsequenceswasidentifiedthat,by phy-logeneticanalysis,isancestraltoallknownPCV2.Wholegenome sequencingconfirmedthatthesevirusesareuniqueandformanew geneticgroupwhichwerefertoasPCV2e.

2. Materialsandmethods

TheMVDLPCV2ORF2databaseinAugust2015contained1289 sequences generated by Sanger sequencing and linked by case numbertodateofisolation,geographiclocation,anddescriptive reports.Thefilesweremergedandorganizedbygeographicregion andyearofisolationthensubjectedtodataqualityanalysis. Wild-typefieldoriginwasdeterminedbyaccessingMVDLcasefilesfor

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Fig.1. Maximumlikelihood(ML)phylogeneticanalysisof729PCV2ORF2sequencesintheMinnesotaVeterinaryDiagnosticLaboratorydatabase.Substitutionpatternand rateswereestimatedundertheTamura-Neimodel(+G)(TamuraandNei,1993),andadiscreteGammadistributionwasusedtomodelevolutionaryratedifferencesamong sites(5categories[+G]).ForestimatingMLvalues,atreetopologywasautomaticallycomputed.EvolutionaryanalyseswereconductedinMEGA6(Tamuraetal.,2013).

Fig.2.Pairwisesimilarityanalysisof729MinnesotaVeterinaryDiagnosticLaboratoryPCV2ORF2sequences.Histogrambinsarein1%increments.Genotypesareshown relativetoPCV2b.AnalysiswasperformedinMEGA6(Tamuraetal.,2004,2013).Thetotalnumberofcomparisonswas531,448.

missingorsuspectinformationandtoremovesequencesgenerated inresearchstudies.Sequencesthatcouldnotbeverifiedasfield isolateswerediscarded.Afastafilewasgeneratedwiththe result-inglistof905sequences,renamedtoshowvirusname(PCV2),

geographiclocation(country,stateorprovince),auniqueidentifier, andyearofisolation.

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34 B.Daviesetal./VirusResearch217(2016)32–37

Fig.3.AminoacidsequencealignmentofrepresentativePCV1andPCV2genotypeclustersshowingregionsofsimilarityanddissimilarity.

Fig.4.VariationinPCV2attheORF23′_gene_terminus_(A)_and_the_carboxyl_terminus_of_the_protein_(B)._{Representative}_strains_are_ordered_by_amino_acid_length_of_the_protein, andbygenotypeasindicated.

sequences are ≥702 bases. Sequences with an ORF less than 650were deleted,and there were nonebetween 650and 702 bases.Sequenceswith>5ambiguousbasesweredeleted,andfor sequenceswith≤5ambiguities,originaltracefileswereexamined manually.Sequenceswithoneormoreambiguitiesthatcouldnot beresolvedweredeleted.Theresulting729validatedsequences weresubjectedtophylogeneticanalysis.

Nucleotide sequences were aligned in MUSCLEwith default parametersandclusteredwithGeneiousTreeBuilderusingdefault parameters.AmaximumlikelihoodtreewasconstructedinMEGA 6.06 using GTR and default parameters without bootstrapping. Independentlandmarkgenotypestoorientthetreewereadded fromGenbankasdescribed(Franzoetal.,2015).

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Fig.5.RecombinationanalysiscomparingPCV2ewholegenomestoallPCV2genomesinGenbank.AnalysiswasperformedusingtheRecombinationAnalysisTool(RAT) (Etheringtonetal.,2005).

Fig.6. FrequencypatternofPCV2ORF2sequenceacquisitionintheMinnesotaVeterinaryDiagnosticLaboratoryfrom2001to2015.

novelgenomesidentifiedinthisstudy.Recombinationanalysiswas performedusingRecombinationAnalysisTool(RAT)(Etherington etal.,2005).

PCV2 ORF2 sequences were deposited in Genbank with accession numbers KT867794-KT868522. Full length genome sequences PCV2/USA/NE-001/2015 and PCV2/USA/NE-002/2015 weredepositedasKT870146andKT870147,respectively.

3. Results

Thegeneticstructure of729validatedPCV2ORF2sequences fromfieldcasessubmittedtotheMVDLisshowninFig.1.Notable featuresincludethelimitedgeneticvariationinPCV2b,especially thepresenceof126identical,independentlyacquiredsequences intheyears2005–2013,andasecondsetof88identicalsequences from2005to2012;amorevariablePCV2agenotypewithacluster of33identicalsequencesfrom2006to2012;aconservedcluster ofmPCV2b(=PCV2d)isolatedin2012–2015;andanovel,distant clusterof10sequencesfrom4midwesternU.S.statesandMexico, collectedfrom2006to2015,proposedhereasPCV2e(Fig.1). Pair-wisesequencecomparisonofthe729sequencesalsoshowedthat thenovelPCV2eclusterwasunique(Fig.2,Table1).Aminoacid sequencealignmentsofrepresentativeisolates,asshowninFig.

3,andnucleotidesequencecomparisons(notshown)alsoshowed thatPCV2e,aswellasotherPCV2genotypes,werestrikingdifferent fromPCV1.

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36 B.Daviesetal./VirusResearch217(2016)32–37

Table1

PairwisecomparisonofORF2nucleotidesequencesofPCV1andPCV2a-e.

Genbank

Genotype PCV1 PCV1 PCV2e PCV2c PCV2a PCV2b PCV2d PCV2d

AAlength 233 233 238 234 233 233 234 233

YearofIsolation 2009 1990 2015 1990 2006 2013 2012 2013

GU371908 98.3 65.6 66.3 65.9 67.1 65.2 66.0

KJ408798 98.3 66.6 67.3 66.6 68.0 66.2 67.0

PCV2/USA/NE-002/2015

65.6 66.6 84.4 79.5 81.6 82.8 83.0

EU148505 66.3 67.3 84.4 85.7 89.9 89.1 88.9

PCV2/USA/MN-163/2006

65.9 66.6 79.5 85.7 91.6 88.9 89.9

PCV2/USA/IA-053/2013

67.1 68.0 81.6 89.9 91.6 93.3 94.3

PCV2/USA/MN-003/2012

65.2 66.2 82.8 89.1 88.9 93.3 98.7

PCV2/USA/IA-072/2013

66.0 67.0 83.0 88.9 89.9 94.3 98.7

Table2

PCV2egeneandgenomenucleotidesequenceidentitiestonearestneighbors.

Isolate Genbank Yearisolated Country (%)identitya

A.ORF2

PCV2/USA/NE-002/2015 KT870147 2015 USA 100 DK1980PMWSfree EU148503 1980 Denmark 87 DK1987PMWSfree EU148504 1987 Denmark 87 DK1990PMWSfree EU148505 1990 Denmark 87

India-GN-07 GU808525 2007 India 87

EU-RO-WB2010-794 JN382183 2011 Romania 87

B.ORF1

PCV2/USA/NE-001/2015 KT870146 2015 USA 100

SPA1 AF201308 1999 Spain 99

Yantai-1 KP313251 2014 China 99

P2425NT JX099786 2088 Vietnam 99

(blank) AY713470 2004 Germany 99

YL5 HQ202972 2010 Taiwan 99

C.Wholegenome

PCV2/USA/NE-001/2015 KT870146 2015 USA 100

P2425NT JX099786 2008 Vietnam 93

TZ60607 FJ158607 2006 China 93

HaiNan AY556476 2004 China 93

10BJ-2 HaiNan 2010 China 93

LZ DQ363860 2006 China 93

a_Percent_identity_was_taken_from_BLASTn_analysis_results_performed₇_Sept.₂₀₁₅

ontheNationalCenterforBiologicalInformation(NCBI)BLASTwebsite.

WholegenomesequenceanalysisoftwoPCV2eisolates con-firmed the unique identity of this group of viruses. BLASTn analysis of the non-redundant Genbank database identified 19 genomeswith93%identity.ComparisonofonePCV2eisolatetofive representativegenomes,witharangeofisolationdatesand geo-graphicallocations,showsthatthegeneticvariationislocalized predominatelyin ORF2(Table2).Inaddition,BLAST analysisof ORF1,encodingthereplicase gene,showedthatPCV2e isa cir-covirus and is not the product of a recombination event that combinedORF2withgeneticmaterialfromanothersource(Table2 andFig.5).Inaddition,wholegenomesimilarityanalysisofPCV2 showedthatPCV2ewasnotcreatedbyrecombinationwithinPCV2 familymembers.Interestingly,nearlyallPCV2genomesexceptfor PCV2eflowedtogetherasasinglegroup,indicatingthat recombi-nationdidnotgiverisetoPCV2eandsuggeststhatrecombination isunlikelytocontributesignificantlytoevolutionofPCV2(Fig.5).

4. Discussion

PhylogeneticanalysisofaPCV2ORF2sequencedatabaseina veterinarydiagnosticlaboratorythat servesthemidwestern US swineindustryrevealedthepresenceofa novelPCV2genotype thatwasfirstobservedin2006,andanabundanceofPCV2b iso-lates withlimited genetic diversity even though it waswidely distributedthroughouttheU.S.nationalherdin2006,suggestinga recentspreadofthegenotypepriortotheappearanceofvaccines (Puvanendiranetal.,2011).Widespreaddistributionofa geneti-callyconservedvirusintheabsenceofclinicaldiseasecouldhave beenaccomplishedbyspreadthroughinfectedsemenorbreeding stock.PCV2agenotypesalsowerecommonandwidelydistributed inU.S.swinebutshowsubstantiallymoregeneticdiversity,ashas beendescribedpreviously(Franzoetal.,2015;Xiaoetal.,2015). BothPCV2aandPCV2bsequenceshavebeenobtainedeveryyear since 2006at theMVDL,regardless ofthe samplingfrequency, whichpeakedin2006and2007,thenincreasedagainin2012and 2013(Fig.6).

PresenceofadistantPCV2genotypeintheU.S.atthe begin-ningofPCVAD outbreaksin 2006and sporadicisolationtothe presenttimewasunexpected.Themarkeddivergencefromother PCV2genotypesindicatesanancientoriginwhosesourceisyetto berevealed.Additionalanalysisofdiagnosticlaboratorydatabases containing extensive inventoriesof viral and microbial genetic information may reveal further insights. The value of screen-ingexistingdiagnosticdatabasesfor geneticdiversity inanimal pathogensisevidentinthecaseofPCV2,apathogenthatdoesnot reproduciblycausediseaseinexperimentalstudies,andispresent inswineherdswithoutobviousdisease(Allanetal.,2007;Guoetal., 2012;Opriessnigetal.,2010,2008;Puvanendiranetal.,2011;Shen etal.,2010).ThePCV2eisolatesidentifiedherewererecoveredfrom diagnosticsubmissionassociatedwithPCVADandwouldhavenot beenrecognizedasnovelwithoutsequencing.Diagnostic sequenc-ingoftypicalPCVADcasesindependentlyatIowaStateUniversity showsthatPCV2eisolatesaredetectedwithroutinediagnosticPCR thatalsodetectsotherisolates(Harmonetal.,2015).

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thatPCV2aand PCV2bORF2encodes233aacapsidexclusively, whilePCV2ccontainsonlythe234aaformandPCV2dgenotypes contain233or234aacapsids.Asimplemodeltoaccountforthese changesisthatPCV2eistheprototype,withasingle12nt dele-tiongivingrisetoaprogenitorof2a–d;andthatPCV2candPCV2d acquiredasecond,3ntdeletion.Theuniquecarboxylterminusof PCV2ecapsiddoesnotappeartoaffectthepredicted3-dimensional proteinstructurecomparedto233or234aalengthcapsids mod-eledusingRaptorX(Kallbergetal.,2012).

Acknowledgements

TheworkwasfundedinpartbysupportfromBoehringer Ingel-heimVetmedica(BIV)andbytheUSDANationalInstituteofFood andAgriculture,multistateprojectMIN-63-112.XWwassupported byaMnDRIVEGlobalFoodVenturesfellowship.BDisan under-graduatestudentatUniversityofMinnesota,Morris.MPMreceives consultingincomefromBIV.Therelationshiphasbeenreviewed andmanagedbytheUniversityofMinnesotainaccordancewith itsconflictofinterestpolicies.

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