Characterization of New Isolates of Apricot vein clearing-associated virus and of a New Prunus

15 Pages·2015·0.51 MB·English

RESEARCHARTICLE
Characterization of New Isolates of Apricot
vein clearing-associated virus and of a New
Prunus-Infecting Virus: Evidence for
Recombination as a Driving Force in
Betaflexiviridae Evolution
ArmelleMarais1,2*,ChantalFaure1,2,EldarMustafayev3,ThierryCandresse1,2
1 INRA,UMR1332BFP,Villenaved’Ornon,France,2 UniversitédeBordeaux,UMR1332BFP,Villenave
d’Ornon,France,3 GeneticResourceInstituteoftheAzerbaijanNationalAcademyofSciences,Baku,
Azerbaijan
* [email protected]
Abstract
DoublestrandedRNAsfromPrunussamplesgatheredfromvarioussurveyswereanalyzed
byadeep-sequencingapproach.Contigannotationsrevealedthepresenceofapotential
OPENACCESS
newviralspeciesinanAzerbaijanialmondtree(Prunusamygdalus)anditsgenomese-
Citation:MaraisA,FaureC,MustafayevE,
quencewascompleted.Itsgenomicorganizationissimilartothatoftherecentlydescribed
CandresseT(2015)CharacterizationofNewIsolates
ofApricotveinclearing-associatedvirusandofaNew Apricotveinclearingassociatedvirus(AVCaV)forwhichtwonewisolateswerealsochar-
Prunus-InfectingVirus:EvidenceforRecombination acterized,inasimilarfashion,fromtwoJapaneseplums(Prunussalicina)fromaFrench
asaDrivingForceinBetaflexiviridaeEvolution.PLoS
germplasmcollection.Theaminoacididentityvaluesbetweenthefourproteinsencodedby
ONE10(6):e0129469.doi:10.1371/journal.
thegenomeofthenewvirushaveidentitylevelswiththoseofAVCaVwhichfallclearlyout-
pone.0129469
sidethespeciesdemarcationcriteria.Thenewvirusshouldthereforebeconsideredasa
AcademicEditor:UlrichMelcher,OklahomaState
newspeciesforwhichthenameofCaucasusprunusvirus(CPrV)hasbeenproposed.Phy-
University,UNITEDSTATES
logeneticrelationshipsandnucleotidecomparisonssuggestedthattogetherwithAVCaV,
Received:February12,2015
CPrVcoulddefineanewgenus(proposedname:Prunevirus)inthefamilyBetaflexiviridae.
Accepted:May8,2015
Amoleculartesttargetingbothmembersofthenewgenuswasdeveloped,allowingthede-
Published:June18,2015 tectionofadditionalAVCaVisolates,andthereforeextendingtheknowngeographicaldistri-
Copyright:©2015Maraisetal.Thisisanopen butionandthehostrangeofAVCaV.Moreover,thephylogenetictreesreconstructedwith
accessarticledistributedunderthetermsofthe theaminoacidsequencesofreplicase,movementandcoatproteinsofrepresentativeBeta-
CreativeCommonsAttributionLicense,whichpermits
flexiviridaememberssuggestthatCitrusleafblotchvirus(CLBV,typememberofthegenus
unrestricteduse,distribution,andreproductioninany
Citrivirus)mayhaveevolvedfromarecombinationeventinvolvingaPrunevirus,further
medium,providedtheoriginalauthorandsourceare
credited. highlightingtheimportanceofrecombinationasadrivingforceinBetaflexiviridaeevolution.
ThesequencesreportedinthepresentmanuscripthavebeendepositedintheGenBank
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformationfiles. databaseunderaccessionnumbersKM507061-KM504070.
Funding:Theauthorshavenosupportorfundingto
report.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0129469 June18,2015 1/15
AVCaVandNewPrunus-InfectingBetaflexiviridae
Introduction
ThefamilyBetaflexiviridaeiscomposedofplantviruseswithparticlesofflexuousmorphology.
Currently,sevengeneraarerecognizedinthisfamily,foratotalofcloseto90species,including
unassignedvirusesinthefamily(http://www.ictvonline.org/virusTaxonomy.asp).Duringthe
lastfiveyears,anumberofviralspeciesbelongingtothefamilyBetaflexiviridaehavebeenchar-
acterizedbyeitherclassicalapproaches(e.g.[1–9])orusinghigh-throughputnextgeneration
sequencing(NGS)technologies[10–12].Inthelatercase,differentstrategieshavebeenused,
targetingdifferentnucleicacidtemplates(single-strandedordouble-strandedRNAs,smallin-
terferingRNAs,messengerRNAs,partiallyorcompletelypurifiedviralparticles)orthese-
quencingtechnology(Illuminaor454pyrosequencing[13–17]).Fruittreematerialshavebeen
subjectedtoasystematicNGSscreeningofviralinfections,inparticulartoelucidatediseasesof
stillunknownetiology[18].Inthiscontext,thefailuretodetectviralsequenceotherthanLittle
cherryvirus1(LChV1)insourcherrysourcesoftheShirofungenstuntdiseaseledtothesug-
gestionthatLChV1mayberesponsibleforthissyndrome[19].Establishingtheassociationbe-
tweenviralinfectionandsymptomatologyissometimescomplicatedbymixedviralinfections,
whichareveryfrequentlyobservedinfruittreesandinothervegetativelypropagatedplants.
Forexample,anovelTepovirusinfectingPrunusspecies,tentativelynamedPrunusvirusT,
wasrecentlycharacterizedfollowingapyrosequencinganalysisofdsRNAsrecoveredfrom
plumandcherrytrees,butitwasnotpossibletoassociatethisnewagentwithspecificsymp-
tomsbecauseofitspresenceinco-infectionwithcommonfruittreeviruses[20].Illuminase-
quencingofdsRNAsobtainedfromanapricottreewithveinclearingsymptomsrecently
allowedtheidentificationofanovelunclassifiedBetaflexiviridaemembernamedApricotvein
clearingassociatedvirus(AVCaV);howeveritsco-infectionwithPlumbarknecrosisstempit-
tingassociatedvirus(PBNSPaV)similarlyhamperedtheestablishmentofacausalrelationship
betweenAVCaVandtheveinclearingsymptoms[21].
Betaflexiviridaemembershavegenomeswhichencodeareplicasewithcharacteristicsofthe
alphavirus-likesuperfamily[22–24].Twogenera,Carlavirus,Foveavirus,andsomeunassigned
memberspossessasetofthreeopenreadingframes(ORFs)collectivelyknownasthetriple
geneblock(TGB),involvedincell-to-cellmovement.Theremaininggenera,Citrivirus,Capil-
lovirus,Trichovirus,Vitivirus,andtherecentlyacceptedgenusTepovirushaveasinglemove-
mentprotein(MP)belongingtothe"30K-like"superfamily[24–25].InthegeneraVitivirus
andCarlavirus,andinsomemembersofthegenusTrichovirus,anadditionalORFispresent3’
ofthecoatprotein(CP),andencodesanRNA-bindingproteinwithazinc-ribbonmotif,iden-
tifiedasasuppressorofsilencinginGrapevinevirusA[26].Therecentlydescribedspecies
AVCaV,proposedasanunassignedspeciesinthefamily,hasasimilargenomeorganization
[21].ThisnovelviralspecieshasonlybeenreportedsofarfromItaly,andapreliminarysurvey
suggestedalimitedprevalence,withonlyonevarietyofapricotandthreeoutof78Prunus
samplesfoundtobeinfected[21].
Inthepresentstudy,thecompletegenomesequenceofanovelviralspeciesfromanAzer-
baijanialmondtree,forwhichthenameofCaucasusprunusvirus(CPrV)isproposed,was
determinatedbyadeep-sequencingapproach.Thesamestrategyprovidedthegenomese-
quenceoftwoadditionalisolatesofAVCaV,extendingourknowledgeonthemoleculardiver-
sity,geographicaldistributionandhostrangeofthisrecentlydiscoveredvirus.Phylogenetic
analysesallowdefinitionofatentativenewgenus,Prunevirus,composedofCPrVandAVCaV
asthetypemember,andconfirmtheroleofrecombinationasadrivingforceinBetaflexiviri-
daeevolution.
PLOSONE|DOI:10.1371/journal.pone.0129469 June18,2015 2/15
AVCaVandNewPrunus-InfectingBetaflexiviridae
MaterialsandMethods
Plantsamplesandvirusisolates
Thefieldstudiesdidnotinvolveendangeredorprotectedspeciesandnospecificpermissions
wererequiredforthevariouslocations.
ThetwoJapaneseplums(Prunussalicina)Pairand13025aswellasthedomesticplum(Pru-
nusdomestica)381-07-4wereobtainedfromthecollectionheldbytheCtifl(Lanxade,France)
andweregraftedonGF305peachseedlingsunderlevel3containmentgreenhouseconditions.
Thealmond(Prunusamygdalus)Aze204wascollectedinArinc,inthemountainsoftheNakh-
chivanareaofAzerbaijan.TheJapaneseapricot(Prunusmume)S4andtheapricot(Prunus
armeniaca)S15sampleswerecollectedduringsurveysinChina.Thepeach(Prunuspersica)
Iran1wascollectedinIranduringasurveyin2005.AnumberofPrunussamplesbelongingto
variousspecieswerecollectedduringsurveysinAzerbaijan(P.domestica,P.amygdalus,P.per-
sica,P.armeniaca,andP.avium),inChina(P.mume,P.armeniaca,P.sibirica,andP.persica),in
Kazakhstan(P.armeniaca),inItaly(P.armeniaca,P.avium,andP.cerasus),inFrance(P.domes-
ticaandP.salicina),andintheCzechRepublic(P.cerasus,P.avium,P.armeniaca,andP.domes-
tica)andwereusedtoassesstheprevalenceofCPrVandAVCaV.SomeMalusspp.,Mespilus
germanicaandCydoniaoblongasamplesfromAzerbaijanwerealsotestedforvirusinfection.
Determinationofcompletegenomesequencesbypyrosequencing
Double-strandedRNAs(dsRNAs)wereextractedfromfreshleavesofGF305graftedwiththe
13025andPairsources,andfromdriedleavesoftheAze204almond.Afterwhole-genomeam-
plification,theamplifiedfragmentswereanalyzedby454pyrosequencing,followingthestrate-
gydescribedinCandresseetal.[19].Afterdemultiplexing,thereadswereassembledusingthe
CLCGenomicsWorkbench6.5(http://www.clcbio.com)andannotatedbyBlastXandBlastN
comparisonwithGenBank,usinga10−3e-valuecut-off.Mappingofcontigsonthegenomeof
AVCaV(forthe13025andPairsources)orofCitrusleafblotchvirus(CLBV)(fortheAze204
source)usedasreferencesallowedthescaffoldingandorderingofcontigsforeachviralisolate.
Inordertocompletethegenomicsequences,thegapsbetweenthecontigsgeneratedfromthe
pyrosequencingdataaswellasregionsforwhichsequenceconfirmationwassoughtweream-
plifiedfromtotalnucleicacids(TNA)extractedfromthe13025,PairandAze204sourceswith
primersdesignedfromthesequenceofthecontigs(S1Table)intwo-stepRT-PCRprocedures,
asdescribedbyMaraisetal.[27].The5'endofeachviralgenomeweredeterminedusinga
RandomAmplificationofcDNAEnds(RACE)strategyandinternalprimersdesignedfrom
thecontigs(S1Table),followingthemanufacturer'sinstructions(TakaraBioEurope/Clon-
tech©,Saint-Germain-en-Laye,France).Whenneeded,anestedamplificationwasperformed
toincreasetheamplificationsignal.The3’genomicregionswereamplifiedusingforwardin-
ternalprimersdesignedfromthecontigsandtheLD-primeprimerinlong-distancePCRam-
plificationreactionsfollowingtheprotocoldescribedinYoussefetal.[2](S1Table).All
amplificationproductsweresequencedonbothstrands(GATCBiotechAG,Mulhouse,
France),eitherdirectlyorafteracloningstepintothepGEM-TEasyvector(Promega,Char-
bonnières-LesBains,France).Thesequencesobtainedwerefinallyassembledwiththeinitial
contigstogeneratethecompletegenomicviralsequences.
DeterminationofthepartialgenomicsequenceoftheAVCaVIran1
isolate
TNAwereextractedfromP.persicaIran1leavesaccordingtotheprotocol2describedbyFois-
sacetal.[28]andsubmittedtothePolyvalentDegeneratedOligonucleotide(PDO)nested
PLOSONE|DOI:10.1371/journal.pone.0129469 June18,2015 3/15
AVCaVandNewPrunus-InfectingBetaflexiviridae
RT-PCRasdescribedbyFoissacetal.[28],targetingtheactivesiteoftheviralpolymerase.A
primer(IF1:5'GTGTGTTGAGTCTGATTACGAAG3')wasdesignedfromthenucleotidese-
quenceofthePDOfragmentandused,togetherwithLD-polyTandLD-primeprimers(S1
Table)toamplifytheregionbetweenthePDOfragmentandthepolyAtailatthe3'endofthe
genomeusingLong-Distance(LD)PCR.Theamplifiedfragmentof2.8kbpwasclonedinto
thepGEM-TEasyvector(Promega)andsequencedonbothstrands(GATCBiotechAG).
TotalnucleicacidsextractionanddetectionofAVCaVandthenewCPrV
byRT-PCR
TNAwereextractedfromdriedorfreshleafsamplesasdescribedabove.Specificdetectionof
AVCaVwasperformedfollowingthetwo-stepRT-PCRassaydescribedbyElbeainoetal.[21].
Thisassayallowstheamplificationofashortfragment(ca.330nt)ofthereplicasegeneusing
primersVC37657sandVC28239a.Twoprimerpairsweredesignedinthepresentstudyto
allowthesimultaneousdetectionofAVCaVandCPrVisolates.Oneprimerpair(NB-F1i:5'
ATGYTIGTIMGIAARYTIGARATHCARGA3'andNB-R1:5'GAACTKACYAAAACTGG
CAARGTCTCTGA3')allowstheamplificationofa274-ntlongfragmentinthenucleicacid
bindingproteingene(NB).Theotherone(Pol-F1i:5'CARYTITGYACIAARTAYGARAAR
CARTAYGT3'andPol-R1:5'CCRATWGCSTTTGCTGGGGATGAYATGTG3')wasde-
signedtoamplifyafragmentof499ntinthepolymerasegene.Atwo-stepRT-PCRassaywas
applied.TNA(5μl)weresubmittedtoareversetranscriptionfollowingtheprotocoldescribed
byMaraisetal.[29].ThecDNA(5μl)wasthenamplifiedina50-μlvolumecontaining10mM
Tris-HCl(pH8.8),1.5mMMgCl ,50mMKCl,250μMdNTPs,primersat1μMeach,and1U
2
ofDyNAzymeIIDNApolymerase(Finnzymes/FisherScientific,Illkirch,France)and40cycles
wereapplied,eachof95°Cfor30sec,48°C(NB-F1i/R1)or42°C(Pol-F1i/R1)for30sec,and
72°Cfor30sec,followedbyafinalextensionstepof10minat72°C.Theamplifiedfragments
werevisualizedunderUVlightafternon-denaturatingelectrophoresisonagarosegeland
ethidiumbromidestaining.Theampliconsweresequenced(GATCBiotechAG)eitherdirect-
ly,orafteracloningstepintopGEM-TEasyvector(Promega).
Sequenceandphylogeneticanalyses
Manipulationsof454pyrosequencingsequencedatawereperformedasdescribedbyCan-
dresseetal.[19]usingtheCLCGenomicsWorkbench6.5.Phylogeneticandmolecularanaly-
seswereconductedusingMEGAversion6[30].Phylogenetictreeswerereconstructedusing
theneighbor-joiningtechniquewithstrictnucleotideoraminoaciddistancesandrandomized
bootstrappingfortheevaluationofbranchingvalidity.Geneticdistances(p-distancescalculat-
edonnucleotideoraminoacididentity)werecalculatedusingMEGAversion6.
Results
PyrosequencingofdsRNAsextractedfromthePairand13025
JapaneseplumsourcesandfromtheAze204almondsample
Afterdemultiplexingandqualitytrimmingof454pyrosequencingdata,atotalof29,538reads
wereobtainedforthePairsource,16,750readsforthe13025source,and18,418readsforthe
Aze204sample.Thesesequenceswerethentreatedaspreviouslydescribed[19].Blastcompari-
sonsfollowingdenovoassemblyofcontigsshowedthatinthePairsourcethreeviruseswere
presentinco-infection,namelyPBNSPaV(twoisolates,collectivelyrepresenting60.9%ofthe
reads,[29]),Prunusnecroticringspotvirus(PNRSV,1.3%ofthereads),andanisolateof
PLOSONE|DOI:10.1371/journal.pone.0129469 June18,2015 4/15
AVCaVandNewPrunus-InfectingBetaflexiviridae
AVCaV,representing10.8%ofthereads[29].Forthelattervirus,alongcontigof8,120nucle-
otides(nt)wasobtained,spanningtheAVCaVgenomefromnt205tont8,325.
Inthe13025Japaneseplumsample,Blastcomparisonsallowedidentificationoftwoviruses:
PBNSPaV(twodistinctisolates,43%ofthereads)andanisolateofAVCaV,representing2%
ofthereads.SevencontigsidentifiedasbelongingtoAVCaVweremanuallyassembledby
mappingagainstthereferenceAVCaVgenome,thuscreatingascaffoldof6,141ntmissingthe
5'and3'genomeendsandcontainingsixinternalgaps.ThegenomicsequencesofthePairand
13025AVCaVisolateswerecompletedbytargetedsequencingasdescribedintheMaterials
andMethodssection.TheassembledcompletegenomesequencesweredepositedintheGen-
BankdatabaseunderaccessionnumbersKM507062andKM507063,respectively.
FortheAze204almondsample,onlyafewcontigscouldbeidentifiedashavingapossible
viraloriginbyBlastNandBlastXanalyses.Fivecontigsrepresenting0.09%ofthereadsandto-
taling1,853ntwerethusshowntohaveweaksequencesimilaritiestovariousmembersoffam-
ilyBetaflexiviridaesuchasAVCaV,CLBV,Applechloroticleafspotvirus(ACLSV),orAsian
prunusvirus1(APV1),dependingonthecontigconsidered.Assumingthatallcontigsbe-
longedtothesameagent,ascaffoldwasconstructedbyalignmentwiththeCLBVsequence,
whichspannedtheCLBVgenomefrompositions403to6,820,withfourinternalgaps.Primers
weredesignedfromthecontigsequencesandamplificationsusingAze204sourceTNAwere
performedinordertocompleteandvalidatethesequenceofthistentativescaffold.Asinprevi-
ousefforts[19,20,29],essentiallynodifferenceswereobservedbetweentheinitiallyassembled
scaffoldandtheresequencedregions,thusvalidatingboththeinitialcontigsandthetentative
scaffolding.TheassembledsequencewasdepositedunderaccessionnumberKM507061inthe
GenBankdatabase.
PartialsequencingoftheAVCaVisolatepresentintheIran1Prunus
persicasource
TNAextractsfromtheIran1P.persicasourceweresubmittedtoaPDOnestedRT-PCRwhich
permitsthedetectionofTrichovirus,Foveavirus,andCapillovirusmembersaswellasofsome
unassignedBetaflexiviridaemembers[28].InBlastanalyses,the310-ntlongfragmentobtained
showedstrongnucleotideidentity(92%)withthecorrespondingfragmentofAVCaV[21].
UsingaforwardprimerlocatedinthePDOfragmentandLD-polyTprimertargetingthe
polyAtailofthegenome,a2,883-ntlongfragmentwasamplified,clonedandsequencedon
bothstrandsbyprimer-walking.Theassembledsequenceof3,037ntwasdepositedunderAc-
cessionnumberKM507070intheGenBankdatabase.
ComparisonofAVCaVisolatesgenomicsequences
CompletegenomesequencesofthreeAVCaVisolatesandonepartial(3,037nt3'terminal)are
nowavailable.BothcompleteAVCaVsequencesdeterminedhereare8,358ntlong,significant-
lylongerthanthereferenceisolate(7,315nt).Theiroverallgenomicorganizationandgene
orderareconservedasallencodefourproteinsfrom5'to3'(Fig1):areplication-associated
protein(Pol),amovementprotein(MP),acoatprotein(CP)andanucleicacidbindingprotein
(NB).TheCPistheonlyproteinconservedinsize(221aminoacids[aa])betweenthefour
AVCaVisolates(S2Table).ThePolofthe13025andPairisolatescontainsanadditional342
aainsertedataaposition405ofthePolreferenceisolate,sothatthesizeoftheirproteinis
2,021aainsteadof1,679forthepreviouslysequencedisolate[21].Thefivedomainsmethyl-
transferase(MeT),AlkB,endopeptidase,helicaseandRNA-dependentRNApolymeraseare
conserved,withthelargeindelpositionedbetweentheMeTandAlkBdomains,inthehyper-
variablepartoftheBetaflexiviridaePolprotein.Interestingly,inthe13025andPairisolates,
PLOSONE|DOI:10.1371/journal.pone.0129469 June18,2015 5/15
AVCaVandNewPrunus-InfectingBetaflexiviridae
Fig1.SchematicrepresentationofthegenomesofApricotveinclearingassociatedvirus(AVCaV,
referenceisolateandPairisolate),Caucasusprunusvirus(Aze204agent)andCitrusleafblotchvirus
(CLBV).Thesizeinaminoacidsoftheencodedproteinsisindicatedbetweenbrackets.Thefourgenomes
arealignedonthebasisoftheC-terofthepolymerase.ThehatchedboxesrepresentpartsofthePolandMP
geneswhichareeitherabsentornotexpressed,respectively,intheAVCaVreferenceisolatebutpresentand
expressedintheIran1,Pairand13025isolates.Theblackarrowsrepresenttheamplifiedfragmentsusing
thetwoprimerpairsPol-F1/R1andNB-F1/R1.MP,Movementprotein;CP,Coatprotein;NB,Nucleicacid
bindingprotein.
doi:10.1371/journal.pone.0129469.g001
theindelisborderedbytheduplicatedheptanucleotidesequenceGCAACTT.TheMPofthe
referenceisolateisalsosignificantlyshorter(292aa)duetoaframeshiftmutationleadingto
prematureMPORFterminationascomparedtotheotherthreeisolates,whichhaveaMPof
460aa.ThreeofthefourAVCaVisolatesencodeaNBof139aa,butthatoftheIran1isolateis
23aalongerduetoapointmutation(TtoC)atnucleotide419intheNBgene,whichsup-
pressesthefirststopcodonattheendoftheORF.ThelargeindelinthePolgeneandthemuta-
tionsaffectingthelengthofthevariousORFswereallconfirmedbytargetedsequencingofthe
relevantisolates.The5'noncodingregion(NCR)ofthePairand13025isolatesis78ntlong,
fiventlongerthanthatofthereferenceisolate,duetoindels.The3'NCRisalsovariableissize,
being152ntlonginthePairand13025isolates(13ntlongeratthe3'endthaninthereference
isolate)butonly84ntlongintheIran1isolate,duetotheextensionoftheNBgenetothesec-
ondinframestopcodon.Overall,andwhennottakingintoaccountthelargeindelinthePol
gene,thethreecompletegenomesshowaveryhighlevelofnucleotideidentity(96%-96.8%).
AsshowninS2Table,thesameapplieswhenconsideringthedifferentgenomeregions(and
thefourth,partiallysequencedisolate),withtheexceptionofthe5'NCR,whichishighlydiver-
gentinthereferenceisolateandshowsonlyabout58%identitywiththatoftheothertwo
isolates.
PLOSONE|DOI:10.1371/journal.pone.0129469 June18,2015 6/15
AVCaVandNewPrunus-InfectingBetaflexiviridae
Fig2.Phylogeneticanalysisofthepolymeraseaminoacidsequencesofrepresentativespeciesin
theAlphaflexiviridaeandBetaflexiviridaefamilies.TheabbreviationsofvirusnamesaregiveninS3
Table.Thefamiliestowhicheachvirusbelongsareindicatedattheright.TheisolatesofAVCaV,CLBVand
CPrVAze204areindicatedinbold.Thetreeswereconstructedbytheneighbour-joiningmethodfromstrict
aminoacididentitydistancesandthestatisticalsignificanceofbrancheswasevaluatedbybootstrapanalysis
(1,000replicates).Onlybootstrapvalueshigherthan70%areindicated.Thescalebarsrepresent10%amino
aciddivergence.
doi:10.1371/journal.pone.0129469.g002
GenomeorganizationofAze204agentanddeterminationofits
phylogeneticaffinities
ThegenomeofthevirusfoundintheAze204sourcehasasizeof8,255ntexcludingthepolyA
tail.ThegenomestructureissimilartothatofsomemembersoffamilyBetaflexiviridae,name-
lyCherrymottleleafvirusandPeachmosaicvirus(bothmembersofthegenusTrichovirus),
andAVCaV,withrelativelyshortNCRs(65ntat5'and152ntat3')andfourencodedproteins:
areplication-associatedprotein(Pol,1,986aa),a30K-typemovementprotein(MP,438aa),a
coatprotein(CP,222aa),andanucleicacidbindingprotein(NB,150aa)(Fig1).Inaddition
tothesimilargenomicstructure,thesizesoftheencodedproteinsaresimilartothoseof
AVCaV,especiallywhenconsideringthetwoAVCaVisolatescharacterizedinthepresent
work(seeabove).Blastanalysesfailedtorevealanycloselyrelatedagent,AVCaVbeingde-
tectedastheclosestbutstillquitedistantrelative(resultsnotshown).Phylogeneticanalysisof
thesequencesoftheencodedproteinsshowthatAze204agentandAVCaVclustertogether
withhighlysignificantbootstrapvalues(100%)whateverproteinisconsidered(Figs2–4and
S1Fig).Thesameclusteringisobtainedusingwholegenomesequences(datanotshown).
ComparisonwithsequencedBetaflexiviridaemembersshowedthatwhateverORFis
PLOSONE|DOI:10.1371/journal.pone.0129469 June18,2015 7/15
AVCaVandNewPrunus-InfectingBetaflexiviridae
Fig3.Phylogeneticanalysisofthemovementproteinaminoacidsequencesofrepresentative
speciesinthefamilyBetaflexiviridae.TheabbreviationsofvirusnamesaregiveninS3Table.Theisolates
ofAVCaV,CLBVandCPrVAze204areindicatedinbold.Thetreeswereconstructedbytheneighbour-
joiningmethodfromstrictaminoacididentitydistancesandthestatisticalsignificanceofbrancheswas
evaluatedbybootstrapanalysis(1,000replicates).Onlybootstrapvalueshigherthan70%areindicated.The
scalebarsrepresent10%aminoaciddivergence.
doi:10.1371/journal.pone.0129469.g003
considered,onlydistantrelationshipsandidentitylevelsareobserved,withthesoleexception
oftheNBORF,whichshows73%aminoacididentitywiththatofAVCaV(Table1).Asob-
servedintheBlastanalyses,theclosestrelativeofthenewagentisconsistentlyAVCaV,which
showsonly47.1–51.6%ntidentity(36.5–44%aaidentity)fortheCP,MPandPolgenes
(Table1).PhylogeneticanalysesatthewholegenomelevelandforthePolandMPgenesalso
revealaffinitiesbetweenAVCaV,theAze204agentandCLBV,thetypememberofthegenus
Citrivirus[31].ThePolgeneofCLBVshows49%nucleotideidentity(40.7%aminoacididenti-
ty)withthoseofAVCaVandoftheAze204agent,andevenhighervaluesareobservedwhen
comparingtheMPs(Table1).However,theCLBVCPhasnodetectablephylogeneticaffinities
withthoseofAVCaVandoftheAze204agent(Fig4)andverylownucleotide(30.4%)or
aminoacid(11.2%)identity(Table1).Infact,theCLBVCPhasaffinitieswiththoseofBeta-
flexiviridaememberswithatriplegeneblock(TGBinFig4).
Developmentofabroad-spectrumdetectiontestforAVCaVandthenew
Aze204agent
ThecomparisonofthethreecompletegenomesequencesofAVCaV(Pair,13025andtherefer-
enceisolate)inadditiontothepartialsequenceofIran1isolateandthecompletesequenceof
thenewagentfromtheAze204sourceallowedtheidentificationofseveralconservedmotifsin
thededucedproteins.Twoconservedregionswereselected,oneinthePolandtheotherinthe
NB.Lowdegeneracyinosine-containingprimerstargetingthesetworegionsweredesigned.As
presentedinS2–S3Figs,thetwoviralspecies(AVCaVandtheAze204agent)wereefficiently
andreproduciblydetectedinJapaneseplums(lanes4and7)andinalmond(lane2),yielding
theexpected274-or499-bpPCRproducts.ThedetectiontesttargetingtheNBgeneconsis-
tentlygavestrongeramplificationsignalsthantheonetargetingthePolgene.
PLOSONE|DOI:10.1371/journal.pone.0129469 June18,2015 8/15
AVCaVandNewPrunus-InfectingBetaflexiviridae
Fig4.Phylogeneticanalysisofthecoatproteinaminoacidsequencesofrepresentativespeciesin
thefamilyBetaflexiviridae.TheabbreviationsofvirusnamesaregiveninS3Table.Thetypeofthe
movementprotein(30K-typeorTripleGeneBlock,TGB)towhicheachvirusbelongsareindicatedatthe
right.TheisolatesofAVCaV,CLBVandCPrVAze204areindicatedinbold.Thetreeswereconstructedby
theneighbour-joiningmethodfromstrictaminoacididentitydistancesandthestatisticalsignificanceof
brancheswasevaluatedbybootstrapanalysis(1,000replicates).Onlybootstrapvalueshigherthan70%are
indicated.Thescalebarsrepresent10%aminoaciddivergence.
doi:10.1371/journal.pone.0129469.g004
Table1. Percentageofidentityofgenesandencodedproteinsofthenewagentidentifiedinthe
Aze204PrunussourcewiththecorrespondinggenesandproteinsofBetaflexiviridaememberswitha
30K-typemovementprotein.
Virusspecies Pol MP CP NB
AVCaV 51.6(44) 50.2(42.1) 47.1(36.5) 68.3(73)
CLBV 49(40.7) 56.7(53) 30.4(11.2) naa
PVT 42.9(29.2) 29.9(13.4) 38.7(21.6) naa
ASGV 40.2(26.5) 30.9(13.5) 36.7(25.9) naa
GVA 40.1(27) 30.2(9.2) 38.3(24.9) 34.4(11.1)
ChMLV 40.9(26.4) 33.4(13.2) 41.1(30.4) 32.4(13.6)
ACLSV 40.8(26.8) 31.1(10.2) 39.1(24.6) naa
Theaminoacididentitypercentagesareindicatedinitalicsbetweenbrackets.Pol,Polymerase;MP,
Movementprotein;CP,Coatprotein;NB,Nucleicacidbindingprotein.Theabbreviationsofvirusnames
aregiveninS3Table.
anotapplicable
doi:10.1371/journal.pone.0129469.t001
PLOSONE|DOI:10.1371/journal.pone.0129469 June18,2015 9/15
AVCaVandNewPrunus-InfectingBetaflexiviridae
ToassesstheincidenceofbothvirusesinvariousPrunusaccessions,412samplesgathered
duringseveralsurveysweresubmittedtothepolyvalentRT-PCRassays.Noadditionalsample
wasfoundtobeinfectedbythenewvirus.Incontrast,threeadditionalsampleswerefoundto
beinfectedwithAVCaV(S2–S3Figslanes3,5,and6),enlargingthenaturalhostrangeofthis
virustoP.mume(sampleS4),andtoP.domestica(sample381-07-4),anditsgeographicaldis-
tributiontoChina(samplesS4andS15)(S4Table).Sequencingoftheamplifiedfragments
confirmedthespecificityofthedetectionassays.Theunrootedneighbor-joiningtreescon-
structedusingthepartialsequencesoftheNBgeneorofthePolgeneshowedthatallidentified
AVCaVisolatesappeartoformarelativelytightcluster,withtheexceptionoftheIran1isolate,
whichismoredivergent(datanotshown).Theintragroupnucleotidediversityintheclusterof
closelyrelatedisolatesisinthesamerangeforbothgenomicregions(1.5%and1.4%nucleotide
divergenceintheNBandPolregions,respectively),whereasthenucleotidedivergencebetween
theIran1isolateandthemajorgroupreaches5.4%intheNBregionand7.4%inthePol
region.
Discussion
Inthepresentstudy,thehigh-throughputsequencingofdoublestrandedRNAsextractedfrom
variousfruittreesamplesallowedthedetectionandtheefficientsequencingofthecompletege-
nomeoftwoisolatesofthenewlydescribedAVCaVandtheidentificationofanovelvirusbe-
longingtothefamilyBetaflexiviridae.TheseresultsfurtherillustratethepoweroftheNGS
(NextGenerationSequencing)basedapproachestoidentifyandcharacterizeknownornovel
fruittreeviruses[12,19–21,29,32,33].
ThecomparisonofthetwocompleteandofthepartialAVCaVgenomicsequencesdeter-
minedherewiththetypeisolatefromItaly(NC023295,[21])showtheItalianisolatetohave
severaluniquefeatures,includingthelarge,1,029ntdeletioninthehypervariableregionofthe
Polgeneanda168aa-shorterMPcausedbyaframeshiftmutation.Theimpactofthesemuta-
tionsonthebiologyorevenontheinfectivityoftheItalianisolateremaintobeevaluated.The
existenceofdefectiveRNAs(D-RNAs)hasbeenreportedbeforeinthefamilyBetaflexiviridae
[34]butthesemoleculeshaddeletionsof1.2–1.5kb,spanningseveral3’genomegenes.The
factthatthesedeletionswereborderedbyshort2to4bpduplications[34]mayconstitutea
paralleltotheobservationthattheItalianAVCaVisolatedeletionisborderedbytheduplicated
heptanucleotideGCAACTT.Nevertheless,allisolatesshowhighgenomeidentityvaluesde-
spitetheirverydifferentorigins,suggestinganarrowgeneticdiversityofAVCaV,whichwill
needtobeconfirmedbyfurtherstudies.AVCaVwasfirstdiscoveredinanapricottreefrom
Italyandapreliminarysurveyindicatedalimitedspreadofthevirusinnature[21].Thepres-
entworkallowedestimationofaca.1.4%incidenceofAVCaVinalargePrunuscollectionand
toenlargeitsknowngeographicaldistributiontothreefurthercountries(France,China,and
Iran),anditsnaturalhostrangetoseveralPrunusspecies:P.salicina,P.persica,P.mume,and
P.domestica.However,asintheworkofElbeainoetal.[21],duetomixedviralinfectionsinall
thesevenidentifiedAVCaV-positivetrees,itisstilldifficulttoassociatespecificsymptoms
withAVCaVinfection.Allidentifiedhostplantsweresimultaneousinfectedbycommonfruit
treeviruses(S4Table),suchasPlumpoxvirus(Potyvirus),CherryvirusA(Capillovirus),
PBNSPaV,orPNRSV,aswellasbyaputativenewTrichovirusspeciesrelatedtoPcMVand
ChMLV[29],sothatitisnotpossibletoassociatethesymptomsobservedontheleavesofthe
varioustrees,suchasmarbling,chlorosis,orreddeningwithAVCaVpresence.
Thegenomicorganization,sequencecomparisonsandphylogeneticanalysesshowthatthe
viralagentidentifiedintheAzerbaijanialmondplantAze204,hasitsclosestaffinitieswith
AVCaV.However,thenucleotideorencodedaminoacidsequenceidentitiesfortheirCPand
PLOSONE|DOI:10.1371/journal.pone.0129469 June18,2015 10/15