Polymorphic patterns of the merozoite surface protein-3β in Korean isolates of Plasmodium vivax

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Polymorphic patterns of the merozoite surface protein-3β in Korean isolates of Plasmodium vivax
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Kang, Jung-Mi
Ju, Hye-Lim
Cho, Pyo Yun
Moon, Sung-Ung
Ahn, Seong Kyu
Sohn, Woon-Mok
Lee, Hyeong-Woo
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Background: The merozoite surface protein-3β of Plasmodium vivax (PvMSP-3β) is one of the candidate antigens for blood stage malaria vaccine development. The polymorphisms in PvMSP-3β have been reported in certain P. vivax isolates. However, the diversity of PvMSP-3β throughout its global distribution has not been well understood. In this study, the genetic diversity and the effects of natural selection in PvMSP-3β among P. vivax Korean isolates were analysed. Methods: Blood samples were collected from 95 patients with vivax malaria in Korea. The region flanking full-length PvMSP-3β was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvMSP-3β sequence of each isolate was determined and the polymorphic characteristics and effects of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs. Results: Five different subtypes of PvMSP-3β were identified based on single nucleotide polymorphisms (SNPs), insertions, and deletions. Although a high level of sequence diversity was observed in the PvMSP-3β gene, the coiled-coil tertiary structure of the PvMSP-3β protein was well conserved in all of the sequences. The PvMSP-3β of Korean isolates is under natural selection. DNA polymerase slippage and intragenic recombination likely contributed to PvMSP-3β diversity in Korean P. vivax isolates. Conclusions: The PvMSP-3β of Korean P. vivax isolates displayed polymorphisms, with SNPs, insertions and deletions scattered throughout of the gene. These results of parasite heterogeneity are relevant to the development of a PvMSP-3β based vaccine against P. vivax and the implementation of malaria control programmes in Korea. Keywords: Plasmodium vivax, Merozoite surface protein-3β, Genetic polymorphism, Natural selection, Korea
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Kang et al. Malaria Journal 2014, 13:104 http://www.malariajournal.com/content/13/1/104; Pages 1-9
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doi:10.1186/1475-2875-13-104 Cite this article as: Kang et al.: Polymorphic patterns of the merozoite surface protein-3β in Korean isolates of Plasmodium vivax. Malaria Journal 2014 13:104.

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RESEARCHOpenAccessPolymorphicpatternsofthemerozoitesurface protein-3 inKoreanisolatesof PlasmodiumvivaxJung-MiKang1,Hye-LimJu1,PyoYunCho2,Sung-UngMoon3,SeongKyuAhn2,Woon-MokSohn1, Hyeong-WooLee4,Tong-SooKim2*andByoung-KukNa1*AbstractBackground: Themerozoitesurfaceprotein-3 of Plasmodiumvivax (PvMSP-3 )isoneofthecandidateantigens forbloodstagemalariavaccinedevelopment.ThepolymorphismsinPvMSP-3 havebeenreportedincertain P.vivax isolates.However,thediversityofPvMSP-3 throughoutitsglobaldistributionhasnotbeenwellunderstood. Inthisstudy,thegeneticdiversityandtheeffectsofnaturalselectioninPvMSP-3 among P.vivax Koreanisolates wereanalysed. Methods: Bloodsampleswerecollectedfrom95patientswithvivaxmalariainKorea.Theregionflankingfull-length PvMSP-3 wasamplifiedbypolymerasechainreactionandclonedintoaTAcloningvector.ThePvMSP-3 sequence ofeachisolatewasdeterminedandthepolymorphiccharacteristicsandeffectsofnaturalselectionwereanalysed usingtheDNASTAR,MEGA4,andDnaSPprograms. Results: FivedifferentsubtypesofPvMSP-3 wereidentifiedbasedonsinglenucleotidepolymorphisms(SNPs),insertions, anddeletions.Althoughahighlevelofseque ncediversitywasobservedinthePvMSP-3 gene,thecoiled-coiltertiary structureofthePvMSP-3 proteinwaswellconservedinallofthesequences.ThePvMSP-3 ofKoreanisolatesisunder naturalselection.DNApolymeraseslippageandintragenicrecombinationlikelycontributedtoPvMSP-3 diversityin Korean P.vivax isolates. Conclusions: ThePvMSP-3 ofKorean P.vivax isolatesdisplayedpolymorphisms,withSNPs,insertionsanddeletions scatteredthroughoutofthegene.Theseresultsofparasiteh eterogeneityarerelevanttothedevelopmentofaPvMSP-3 basedvaccineagainst P.vivax andtheimplementationofmalariacontrolprogrammesinKorea. Keywords: Plasmodiumvivax ,Merozoitesurfaceprotein-3 ,Geneticpolymorphism,Naturalselection,KoreaBackgroundPlasmodiumvivax isthemostwidelydistributedmalaria parasiteinworldwide,causing70 – 80millioncasesannually,andisamajorpublichealthproblemcontributingtotheglobalburdenofmalariamorbidityinregions outsideofAfrica[1]. Plasmodiumvivax ismoredifficult tocontrolandeliminatethan Plasmodiumfalciparum mainlybecauseofitstendencytorelapse.Moreover, vivaxmalariahasre-emergedinmanytemperateregions inrecentyears,includingSouthKorea,whereithad beenlargelyeradicatedduringtheglobalmalariacontrol campaigns. Understandingthepopulationstructuresofthemalariaparasitesisanimportantfactorindrug-resistance surveillanceandestimatingtheperformanceofvaccines underdevelopmentinaparticularparasitepopulation [2].Althoughthepopulationstructuresof P.falciparum isolateshavebeenexaminedonaglobalscale,thoseof P.vivax remainlargelylessunderstood.Withaglobal distributionthatrangesfr omtropicaltotemperate regions, P.vivax isolateshaveexhibitedvariationin biologicalcharacteristics,suchasrelapsepatternsand transmissibility,whichcanbe usedtodistinguishgeographicalisolatesandevensubspecies[3].Moreover,extensive geneticdiversityhasalsobeenidentifiedwithinalocal *Correspondence: tongsookim@inha.ac.kr ; bkna@gnu.ac.kr2DepartmentofParasitologyandInhaResearchInstituteforMedicalSciences, InhaUniversitySchoolofMedicine,Incheon400-712,RepublicofKorea1DepartmentofParasitologyandTropicalMedicine,andInstituteofHealth Sciences,GyeongsangNationalUniversitySchoolofMedicine,Jinju660-751, RepublicofKorea Fulllistofauthorinformationisavailableattheendofthearticle 2014Kangetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated.Kang etal.MalariaJournal 2014, 13 :104 http://www.malariajournal.com/content/13/1/104

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P.vivax population.Therefore,understandingthenatureof geneticpolymorphismsof P.vivax populationsinendemic areasmightprovideinformati onregardingparasiteheterogeneitythatisrelevanttomalariacontrolefforts. The P.vivax merozoitesurfaceprotein-3(PvMSP-3) familyconsistsofseveralrelatedproteins,PvMSP-3 PvMSP-3 andPvMSP-3 .Althoughthelevelofshared aminoacidsequenceidentityamongtheproteinsislimited,allofthemcontainacentralalanine-richdomain thatformsacoiled-coiltertiarystructure[4-6].Limited sequencepolymorphismhasbeenobservedintheMSP-3 of P.falciparum (PfMSP-3)[7,8],butthePvMSP-3 is likelytobehighlypolymorphicandisreportedtobea reliablegeneticmarkerforpopulationanalysis[9-13]. PvMSP-3 alsoshowedpolymorphicpatternsamongseveralgeographicallydifferent P.vivax isolates[14,15],but itspolymorphicpatterninworldwideisolatesispoorly understoodcomparedtoothervaccinecandidateantigens. AsPvMSP-3 hasbeenregardedasapotentialvaccine target[16],amorethoroughunderstandingofgenetic diversityofthegeneisrequiredsincegeneticpolymorphisminthecandidateantige ncanhampertheefficacyof avaccine. Followingitsre-emergenceinKoreain1993,vivax malariahaspersisted,withvaryingnumbersofindigenous casesannuallyinthecountry.Severalrecentstudieshave suggestedthatrapidgeneticvariationhasoccurredinthe Korean P.vivax populationinrecentyears[17-21],which supportssettlementoflocaltransmissionofvivaxmalaria inKorea.Inthisstudy,thepolymorphicnatureofPvMSP3 inKorean P.vivax isolateswasanalysed.ThePvMSP3 geneisradicallypolymorphicinKorean P.vivax population,withmultiplegenesizesandsinglenucleotide polymorphisms(SNPs)whicharescatteredthroughout thegene.MethodsBloodsamplesAtotalof95bloodsamplesusedinthisstudywerecollectedfromKoreanpatientsinfectedwith P.vivax in Koreabetween2007and2012(2007 – 2010, n =15foreach year;2011, n =18;2012, n =17).Allthepatientsinhabited inmalariaendemicareas,Ilsan,KimpoorYonchon,and havenotbeenabroadatleastinrecent2yearswhentheir bloodsampleswerecollected.The P.vivax infectionswere identifiedbymicroscopicexaminationofthinandthick bloodsmears,andconfirmedbypolymerasechainreaction (PCR)[10,20].About5mlofbloodwascollectedfrom eachindividual.Thebloodwasseparatedintopacked cellsandplasmaandthenstoredat 80Cuntiluse.Informedconsentwasobtainedfromallofthepatients beforebloodcollection.Thestudyprotocolwasapproved bytheEthicsCommitteeoftheInhaUniversitySchool ofMedicine.GenomicDNAextractionandamplificationofPvMSP-3 GenomicDNAwasextractedfrom200 lofwholeblood sampleusingtheQIAampDNABloodKit(Qiagen, Hilden,Germany).Thefull-lengthregionencodingPv MSP-3 wasamplifiedusingtworoundsofPCR.The primersweredesignedbasedonPvMSP-3 sequences ofSalI(XM_001613146)andBelem(AF099662)strains depositedinGenBank.Theprimersusedforthefirst roundofPCRwere5 -TTCGCAACACTCGCCTTATT TCGCTCAACG-3 and5 -CCCCCAATTCGTCACCAA TTTGTTTAGCAT-3 .Theprimersusedforthenested PCRwere5 -TTTCGCTCAACGCGCGCATCTAAAA TG-3 and5 -TTAGCATATTTTCTTCCGCCTCCTTT A-3 .Thefollowingthermalcyclingconditionswereused forbothamplifications:94Cfor5min;30cyclesof94C for1min,52Cfor1minand72Cfor3min,andafinal extensionat72Cfor10min.ThePCRproductwas analysedona1.2%agarosegel,purifiedfromthegel, andligatedintotheT&Acloningvector(RealBiotech Corporation,BanqiaoCity,Taiwan).Eachligationmixturewastransformedinto Escherichiacoli DH5 competentcellsandpositivecloneswiththeappropriate insertwereselectedbycolonyPCR.Thenucleotidesequencesoftheclonedinsertwereanalysedbyautomatic DNAsequencingusingthevectorprimers,M13forward andM13reverseprimers.Sequencinganalyseswithtwo additionalspecificinternalprimers(5 -AGCAAAAACA GAAGCAGAAACAGCACA-3 and5 -GGAAATTTTC AGCTTCCGTTTTTGCTT-3 )werealsoperformedto obtainthesequenceofcentralregionofthePvMSP-3 Atleasttwoclonesfromeachisolateweresequencedto ensureaccuracy,andsomeisolatesunderwentthree-fold sequencecoveragetoconfirmtheexistenceofrarepolymorphisms.Thenucleotidesequencesreportedinthis studyhavebeendepositedintheGenBankdatabaseunder theaccessionnumbersJX667768-JX667772.SequenceandphylogeneticanalysisofPvMSP-3 Thenucleotideanddeducedaminoacidsequencesof PvMSP-3 wereanalysedusingEditSeqandSeqMan intheDNASTARpackage(DNASTAR,Madison,WI, USA).Thephylogenetictreewasconstructedusingthe neighbour-joiningmethodinMEGA4computationalprogram[22].Bootstrapproportionswereusedtoassessthe robustnessofthetreewith1,000bootstrapreplicates.The coiled-coilmotifsineachofthesequenceswerepredicted usingthePaircoil2structuralanalysisprogram[23].SequencepolymorphismanalysisDNAsequencepolymorphismanalysiswasperformed onthe95PvMSP-3 sequences.Thenumberofsegregatingsites(S),haplotypes(H),haplotypediversity(Hd), nucleotidediversity( ),andtheaveragenumberofpairwisenucleotidedifferenceswithinthepopulation( K )wereKang etal.MalariaJournal 2014, 13 :104 Page2of9 http://www.malariajournal.com/content/13/1/104

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estimatedusingtheDnaSPver.5.10.00[24].The was calculatedtoestimatethestep-wisediversitythroughout theentirePvMSP-3 basedonaslidingwindowof100 baseswithastepsizeof25bp.Theratesofsynonymous (dS)andnon-synonymous(dN)substitutionswereestimatedandwerecomparedusingtheZ-test(P<0.05)in MEGA4program[22]usingNeiandGojobori  smethod [25]withtheJukesandCantorcorrection.Toevaluatethe neutraltheoryofevolution,theTajima  sDtest[26]was performedwiththeDnaSPver.5.10.00[24].FuandLi  sD andFstatistics[27]werealsoanalysedusingtheDnaSP ver.5.10.00[24].RecombinationparametersandlinkagedisequilibriumTherecombinationparameter(R),whichincludedthe effectivepopulationsizeandprobabilityofrecombinationbetweenadjacentnucleotidespergeneration,and theminimumnumberofrecombinationevents(Rm) weredeterminedusingtheDnaSPver.5.10.00[24].The linkagedisequilibrium(LD)betweenthedifferentpolymorphicsiteswascomputedbasedontheR2index.ResultsanddiscussionThefull-lengthregionencodingtoPvMSP-3 wasamplifiedfromthegenomicDNAfromeachof95 P.vivax Koreanisolates.Theapproximatesizesofamplified productswerevariedrangingfrom2.1to2.5kb.The nucleotidesequenceanalysisofthesequencesrevealed SNPsscatteredthroughoutofthegene,aswellassmall orlargeinsertionsanddeletionsthatresultedinvariations inthesizebetweenthesequences.Basedontheresultsof thesequenceanalysis,the95PvMSP-3 sequencesof Koreanisolateswereclassifiedasfivedifferentsubtypes (subtypes1,2,3,4,or5)(Figure1).TheN-terminaland C-terminaldomainsofPvMSP-3 arefairlypolymorphic, containingSNPsandsmallinsertions.Regardingthetwo largesegmentsofinsertAandinsertB,whichwerefound inSalIsequencebutnotinBelemsequence[14],only twosubtypes(subtypes4and5)hada120aminoacidlengthinsertA,butnota200aminoacid-lengthinsertB. Theremainingthreesubtypes(subtypes1,2and3)had neitherinsertAnorB.Subtypes2,3,4,and5contained smallinsertsintheN-terminalportion,whereassubtype1 hada2aminoacid-lengthsmallinsertinC-terminal Figure1 SequencepolymorphismofthePvMSP-3 inKorean Plasmodiumvivax isolates. Sequenceanalysisof95 P.vivax Koreanisolates revealedfivedistinctsubtypesofPvMSP-3 .Thelarge-scaleinsertAandinsertBareboxedwiththebluelineordottedredline,respectively.The fivesmallinsertions(S1-S5)intheN-terminalandC-terminaldomainsareindicatedwithboldlinesonthesequences.Thetotalnumberofsequences foreachsubtypearelistedintherightpanel.ThePvMSP-3 sequencesoftheSalvadorI(SalI),BelemandChesswerecomparedtothoseofKorean P.vivax isolates. Kang etal.MalariaJournal 2014, 13 :104 Page3of9 http://www.malariajournal.com/content/13/1/104

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region.Subtype3wasthemostprevalent(n=40),followedbysubtype4(n=23).Nosignificantdifference wasobservedintheannualprevalenceofthesubtypes (Figure2).Thephylogeneticanalysisrevealedthatthe KoreanPvMSP-3 subtypeswereclusteredintofivedifferentclades,whichdiffertopresentlyreportedPvMSP-3 sequences(Figure3).Interestingly,theKoreansubtypes werewidelydistributedamongdifferentisolatesfromdistinctgeographicregions.Thisresultdiffersfromthepreviouslyreportedresult,whichwasthatPvMSP-3 alleles tendedtoclusterbasedongeographiclocationoftheisolates[14].But,consideringthatsequencedataforPvMSP3 fromworldwideisolatesarescant,furthercomplicated studiesof P.vivax isolatesfromdifferentgeographical areasarerequiredtocharacterizepatternsinPvMSP-3 polymorphismsonaglobalscale. AlthoughthebiologicalfunctionofPvMSP-3 isnot fullyunderstood,thealanine-richcentralcore,animportantstructuralfeaturefoundinitsaminoacidsequence, suggeststhattheproteinformsacoiled-coiltertiarystructure[4,5,14].Basedonitsexpressiononthesurface ofmerozoites,PvMSP-3 ispredictedtobeassociated withothermerozoitesurfacemolecules,possiblythrough protein-proteininteractionsinvolvingthecoiled-coilstructure[4-6],inasimilarmannertoPfMSP-3[7].Theseoverallcoiled-coilstructureswerewellconservedinallKorean PvMSP-3 subtypes,eventhoughcentralcoiled-coildomainsweremorepolymorphicthantheN-andC-terminal regions,whicharerelativelyconserved(Figure4).These resultsareconsistentwiththoseofpreviousstudiesthat theoveralltertiarystructureofPvMSP-3 iswellconserved in P.vivax isolatesdespitesequencevariationamongthe isolates[14]. DNAsequenceanalyseswereperformedtoexaminethe nucleotidediversityandgeneticdifferentiationatPvMSP3 amongKorean P.vivax isolates.Nucleotidediversity wasnotconsistentlydistributedwithineachdomainor throughouttheentirePvMSP-3 geneofKorean P.vivax isolates.Theaveragenumberofpair-wisenucleotidedifferences( K )fortheN-terminaldomainofPvMSP-3 was 73.296(Table1).Theoverallhaplotypediversity(Hd)and nucleotidediversity( )forN-terminaldomainwasestimatedtobe0.7730.028and0.072710.00240,respectively(Table1).TheC-terminaldomainofPvMSP-3 was moreconservedthantheN-terminaldomain.Theoverall Hdand fortheC-terminaldomainofPvMSP-3 was 0.7330.028and0.030400.00179,respectively.Meanwhile,theinsertAwashighlyconservedinsubtypes4and 5,whichrepresentedasinglehaplotype.Toexamine whethernaturalselectionhadcontributedtoPvMSP-3 diversitywithintheKorean P.vivax population,theratio oftheaveragenon-synonymousandsynonymousmutationrates(dN/dS)inthePvMSP-3 sequenceswasanalysed.ThedN/dSratioiswidelyusedtoevaluatethe effectsofnaturalselectionongenesequences.Anexcess ofdNrelativetodS(dN/dS 1)indicatespositiveselection,whereasalackofdNrelativetodS(dN/dS<1) suggestsnegativeorpurifyingselectionimposedby functionalconstraints[25,28].TheestimateddN/dSfor theN-terminaldomainwas1.286,indicatingthatpositive naturalselectionmaybeoccurringintheN-terminaldomainofPvMSP-3 ofKorean P.vivax isolates(Table1).ThedN/dSvalueoftheC-terminaldomainwaslessthan 0 5 10 15 20 20072008 2009 201020112012 Subtype 1 Subtype 2 Subtype 3 Subtype 4 Subtype 5 Number of isolates Figure2 AnnualdistributionofPvMSP-3 subtypesduringthe studyperiod. The95PvMSP-3 sequencesfromKoreanisolates wereanalysedbyyearofcollection. Br1B (AY454080) EcuB (AY454090) Belem (AF099662) Br1T (AY454081) Subtype 2 (JX667769) Br56 (AY454085) Br67T (AY454087) Br68B (AY454086) Subtype 1 (JX667768) Chess (AY454083) Subtype 4 (JX667771) SK0814 (GU476525) Subtype 5 (JX667772) Br781B (AY454088) EcuT (AY454091) Ong (AY454094) Subtype 3 (JX667770) Sal I (XM_001613146) Br781T (AY454089) NK (AY454093) Thai (AY454098) Bangl (AY454084) Br69 (AY454082) India (AY454092) SL (AY454096) SL57 (AY454097) 100 83 100 100 100 78 100 99 99 62 76 100 33 40 29 74 99 56 91 93 97 100 0.05 Figure3 PhylogeneticanalysisofthePvMSP-3 inKorean Plasmodiumvivax isolates. Thephylogenetictreesforthefive PvMSP-3 Koreansubtypeswereconstructedusinganeighbour-joining methodwiththeMEGA4program.Thenumbersonthebranches indicatethebootstrapproportions(1,000replicates). Kang etal.MalariaJournal 2014, 13 :104 Page4of9 http://www.malariajournal.com/content/13/1/104

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1(0.455),whichsuggestingpurifyingselectionpressure mayactonthedomain.Inordertomorecloselyexplore theeffectsofnaturalselectiononeachdomainofPvMSP3 ,Tajima  sDtestwasapplied.TheTajima  sDvaluesfor N-terminaldomainandC-terminaldomainofPvMSP-3 were3.15439(P<0.01)and2.27420(P<0.05),respectively, indicatingbothdomainshadbeeninfluencedbypositive naturalselection(Table1).Analysisoftheslidingwindow plot(windowlength100bp,stepsize25bp)showedthat mostofthenucleotidediversitiesfoundintheN-terminal domainoccurredprimarilyinthemiddleandC-terminal endsoftheN-terminaldomain(Figure5A).Meanwhile, inthecaseofC-terminaldomain,thediversitieswere concentratedintheN-terminalend,decreasingtoward theC-terminalendofthedomain(Figure5A).These findingsareconsistentwiththoseofapreviousreportof unequallydistributednucleotidediversitywithinPvMSP3 [14]. Basedontheoccurrenceofsmallindelsinthesequenceandthepresenceofdegeneraterepeatoneither sideoftheindels,DNApolymeraseslippagehasbeensuggestedasapossiblemechanismgeneratingdiversityof PvMSP-3 [14].SimilarsmallindelsandflankingdegeneraterepeatswerealsoidentifiedinPvMSP-3 sequencesof Korean P.vivax isolates,whichsuggestedslipped-strand mispairingduringDNAreplicationmayhavecontributed toPvMSP-3 diversity.The11aminoacid-lengthrepeated elementsthatwereidentifiedinthelarge-scaleinsertion [14]werealsoconservedintheendoftheN-terminal domainandtheendofinsertAofPvMSP-3 ofKorean P.vivax isolates.Thediversityof Plasmodium antigens, includingPvMSP-3 ,isalsoknowntobegeneratedby intragenicrecombinationevents,andismaintainedby balancingselection[9,10,20,28].Similarly,ithasbeen postulatedthatrecombinationisanothermajorfactor generatingallelicdiversityofPvMSP-3 [14].TounderstandtheroleoftherecombinationeventinPvMSP-3 inKorean P.vivax isolates,therecombinationparametersandthelinkagedisequilibriuminthegenewere analysed.FortheN-terminaldomainofPvMSP-3 ,the minimumnumberofrecombinationeventsbetweenadjacentpolymorphicsites(Rm)was13,whereastheRbetweenadjacentsites(Ra)andpergene(Rb)was0.0036 and3.6,respectively.InthecaseofC-terminaldomain ofPvMSP-3 ,theRmwas4withaRaof0.0029anda Rbof3.1.Thesehighrecombinationparametervalues suggestedthatrecombinationmayhaveoccurredbetweensites,contributingtogeneticdiversityinPvMSP3 gene.Thelinkagedisequilibriumindex,R2,wasalso declinedacrosstheanalysedregion,whichfurtherindicatesthatintragenicrecombinationmighthavecontributedtothediversityobservedinboththeN-terminal andC-terminaldomainsofPvMSP-3 inKorean P.vivax isolates(Figure5B). Subtype 1Subtype 2 Subtype 3 A A A BSubtype 4Subtype 5Sal IProbability of a coiled coil Probability of a coiled coilAmino acid residues Amino acid residues Amino acid residues Figure4 Conservationofthecoiled-coiltertiarystructure. ThefivesubtypesofPvMSP-3 sequencesofKorean P.vivax showedahigh probabilityofformingcoiled-coils.Theportionscorrespondingtoinsert A andinsert B aremarkedwiththeblueordottedredlines,respectively. Kang etal.MalariaJournal 2014, 13 :104 Page5of9 http://www.malariajournal.com/content/13/1/104

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Table1DNAsequencepolymorphismsineachdomainofPvMSP-3 amongKoreanisolatesFragmentSegregating sites(S) Singleton variablesites Parsimony informativesites Totalno.of mutations K HHdSD SDdN-dSTajima ’ sDFuandLi ’ sDFuandLi ’ sF N-terminaldomain187018719473.29650.7730.0280.072710.002401.2863.15439(P<0.01)2.53522(P<0.02)3.36192(P<0.02) InsertA000001000000 C-terminaldomain9609610032.92050.7330.0280.030400.001790.4552.27420(P<0.05)2.39279(P<0.02)2.81226(P<0.02)K ,averagenumberofpairwisenucleotidedifferences;H,numberofhaplotypes;Hd,haplotypediversity; ,observedaveragepairwisenucleotidediversity;dN,rateofnon-synonymousmutations;dS,rateof synonymousmutations.Kang etal.MalariaJournal 2014, 13 :104 Page6of9 http://www.malariajournal.com/content/13/1/104

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Thegeneticdiversityofmalariaparasitesiscloselyassociatedwiththelevelsofendemicityandintensityoftransmission[29,30].The P.vivax populationinhyperendemic areas,suchasPapuaNewGuinea,ishighlydiverseand showsmultiplicityofinfections[31,32].However,complex geneticstructureshavealsobeenidentifiedin P.vivax populationsinhypo-endemicareas,suchasChina,Myanmar andThailand[10,33-38].Severalrecentstudieshavesuggestedthatgeneticdiversitywithinthe P.vivax population inKoreahasincreasedinrecentyears[17-21].Considering thelowendemicityandtransmissionintensityinKorea,it remainsunclearhowgeneticdiversityof P.vivax inKorea israpidlydisseminatedandmaintainedinrecentyears. Anincreasedinfluxofinternationaltravellersandforeign workersmighthavecontributedtotheincreasingallelic variationintheKorean P.vivax populationthroughthe introductionofmalariaparasitesfromotherendemic areas[17].ItisalsopossiblethattheKorean P.vivax populationhasevolvedunderevolutionarypressure,most probablyrelatedtohostimmuneresponse.Furtherstudies ofgeneticdiversityintheKorean P.vivax populationin Koreaarewarrantedtoclarifythepatternsofgeneticvariationandthebiologicalrelevanceofincreasing P.vivax diversity.ConclusionsRadicalgeneticpolymorphismwasidentifiedinthePv MSP-3 geneofKorean P.vivax isolates.Thesizeofthe PvMSP-3 varied,asaresultoflargeandsmallinsertions/ deletions,andnumerousSNPswereobservedthroughout thegene.Despitethehighlevelofsequencediversity,the coiled-coiltertiarystructureoftheproteinwaswellconservedinalloftheKorean P.vivax isolates,suggestingthat theproteinisunderfunctionalconstraints.Thegenetic diversityofthePvMSP-3 ofKorean P.vivax isolates hasbeeninfluencedbynaturalselection,particularlyin theN-terminalandC-terminaldomains.Polymerase slippageduringDNAreplicationandintragenicrecombinationmighthavecontributedtothegeneticdiversity ofPvMSP-3 intheKorean P.vivax population.These findingsofparasiteheterogeneityarerelevanttothedevelopmentofaPvMSP-3 -basedvaccineagainst P.vivax AB Figure5 NaturalselectionandrecombinationeventinPvMSP-3 .(A) Theslidingwindowplotofnucleotidediversitypersite( )was constructedtocomparethelevelofgeneticdiversityintheN-terminalandC-terminaldomainsofPvMSP-3 .The valueswerecalculatedusing theDnaSPwithawindowlengthof100bpandstepsizeof25bp. (B) Thelinkagedisequilibrium(LD)plotshowednon-randomassociations betweenthenucleotidevariantsin95Korean P.vivax isolatesatdifferentpolymorphicsites.TheR2valueswereplottedagainstnucleotidedistance usingatwo-tailedFisher ’ sexacttestforstatisticalsignificance. Kang etal.MalariaJournal 2014, 13 :104 Page7of9 http://www.malariajournal.com/content/13/1/104

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andtheimplementationofmalarialcontrolprogrammes inKorea.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions JMKandHLJperformedalltheexperimentsandanalysedthesequence data.PYC,SKAandSUMcollectedbloodsamplesorperformedsequence andphylogeneticanalyses.BKNandTSKdesignedthestudyandsupervised thestudyprocess.JMKandBKNwrotethepaper.TSK,HWLandWMS assistedinwritingandeditingthemanuscript.Allauthorsreadand approvedthefinalmanuscript. Acknowledgements ThisworkwassupportedbytheNationalResearchFoundationofKorea (NRF)grantfundedbytheKoreagovernment(MEST)(2011 – 0028135). Authordetails1DepartmentofParasitologyandTropicalMedicine,andInstituteofHealth Sciences,GyeongsangNationalUniversitySchoolofMedicine,Jinju660-751, RepublicofKorea.2DepartmentofParasitologyandInhaResearchInstitutefor MedicalSciences,InhaUniversitySchoolofMedicine,Incheon400-712, RepublicofKorea.3DepartmentofInternalMedicine,SeoulNationalUniversity BundangHospital,Seongnam463-707,RepublicofKorea.4Departmentof Pathology,Immunology,andLaboratoryMedicine,CollegeofMedicine, UniversityofFlorida,J-566,1275CenterDrive,Gainesville,FL32610,USA. Received:2January2014Accepted:7March2014 Published:17March2014 References1.MendisK,SinaBJ,MarchesiniP,CarterR: Theneglectedburdenof Plasmodiumvivax malaria. AmJTropMedHyg 2001, 64: 97 – 106. 2.CuiL,EscalanteAA,ImwongM,SnounouG: Thegeneticdiversityof Plasmodiumvivax populations. 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