Molecular diversity of human parvovirus B19 during two outbreaks of erythema infectiosum in Brazil

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Molecular

diversity

of

human

parvovirus

B19

during

two

outbreaks

of

erythema

infectiosum

in

Brazil

Rita

de

Cássia

Nasser

Cubel

Garcia

_,

_Renata

_Freire

_Alves

_Pereira

_,

Kátia

Martins

Lopes

de

Azevedo

_,

_Tatiana

_Xavier

_de

_Castro

_,

_Francisco

_C.A.

_Mello

_,

Sérgio

Setubal

_,

_Marilda

_M.

_Siqueira

_,

_David

_Brown

_,

_Solange

_Artimos

_de

_Oliveira

a_{UniversidadeFederalFluminense,InstitutoBiomédico,DepartamentodeMicrobiologiaeParasitologia,Niterói,RJ,Brazil} b_{UniversidadeFederalFluminense,FaculdadedeMedicina,ProgramadePósGraduac¸ãoemCiênciasMédicas,Niterói,RJ,Brazil} c_{UniversidadeFederalFluminense,FaculdadedeMedicina,DisciplinadeDoenc¸asInfecciosaseParasitárias,Niterói,RJ,Brazil} d_{Fiocruz,InstitutoOswaldoCruz,LaboratóriodeVírusRespiratórioseSarampo,RiodeJaneiro,RJ,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9June2016 Accepted5November2016 Availableonline30November2016

Keywords: B19Vgenotypes Outbreak Erythemainfectiosum Brazil

a

b

s

t

r

a

c

t

Thisstudywasconductedtoprovideinformationonthegeneticdiversityofhuman parv-ovirusB19(B19V)circulatinginthemunicipalityofNiterói,RiodeJaneiro,SoutheastBrazil during1996–2006,aperiodwithtwodistinctoutbreaksofB19Vinfection:1999–2000and 2004–2005.Atotalof27serafrompatientswitherythemainfectiosumandfiveserafrom HIV-infectedpatientsthattestedpositiveforB19VDNAduringthestudyperiodwere ana-lyzed.TogenotypeB19Vstrains,asemi-nestedPCRforpartialamplificationofthecapsid genewasperformedandsequenceanalysisrevealedthat31sequencesbelongedto subgeno-type1a(G1a)ofthemaingenotype1andonesequencewascharacterizedassubgenotype3b (G3b).ThephylogenetictreesupportedthedivisionoftheG1aintotwowell-definedclades with1.3%ofdivergence.ThelowdiversityoftheG1astrainsmaybeexplainedbythefact thatallpatientshadacuteB19Vinfectionand30/32serawerecollectedduringtwodistinct outbreaks.TheG3bstrainwasfromanHIV-infectedpatientwhoseroconvertedtoanti-B19 IgGantibodiesinSeptember/2005.ThisisthefirstreportofG3binthestateofRiodeJaneiro. ©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

The human parvovirus B19 (B19V), a member of the

Erythroparvovirusgenus(Parvoviridaefamily),isasmall, non-enveloped icosahedral virus composed of two structural proteins VP1 (83kDa) and VP2 (58kDa) surrounding a lin-earsingle-strandedDNAgenomeofapproximately5.6Kb.1,2

∗ _{Correspondingauthor.}

E-mailaddress:ritacubel@id.uff.br(R.C.CubelGarcia).

Currently,threedistinct genotypesofB19Vhavebeen iden-tified:(i)genotype1,withsubtypes1a(theprototypicvirus) and 1b;(ii)genotype2(A6 andLaListrains);and (iii) geno-type3,withsubtypes3a(V9strain)and3b(D91.1strain).2–4 Genotype1isthemostprevalentworldwide,genotype2has

http://dx.doi.org/10.1016/j.bjid.2016.11.002

1413-8670/©2016SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

been detectedinpatients born before1973 from European countries,theUnitedStatesandBrazil,andgenotype3 pre-dominatesinpartsofWestAfrica.2,5

The most common clinical presentation of B19V infec-tion in immunocompetent individuals is the erythema infectiosum(EI)orfifthdisease,aself-limitedchildhood exan-themacharacterizedbya“slapped-cheek”rash.Adultswith EI, particularly women, frequently present clinically with arthropathy.ItisassumedthatEIandthejointsymptomsare theresultofdepositionofimmunecomplexes.2,6,7

Previousstudies conducted by our group demonstrated thatB19Vinfectionisacommoneventinschoolchildrenaged fivetonineyearsoldandthatEIoutbreaksseemtooccurevery 4–5years.6,7_{AllthreegenotypesofB19Vhavebeenreportedin} patientspresentingwithacuteand/orpersistentB19V infec-tioninBrazil.5,8–12_{Nonetheless,thereisalackofinformation} regardingthegenotypesofB19Vcirculatingduringoutbreaks ofEIinourcountry.

Thisstudywasconductedtoprovideinformationonthe geneticdiversity ofB19V circulating duringa period span-ningtwooutbreaksofEIinthe municipalityofNiterói,Rio deJaneirostate.Inaddition, B19VstrainsfromHIV-positive patientsinfectedduringthestudyperiodwerealso character-ized.

Theserumsamplesanalyzedinthisstudywerecollected duringa10-year-period (1996–2006)from 131patients with exanthematicdisease,28ofthemwitharthropathy,attending theInfectiousDiseasesDepartment,AntonioPedroUniversity Hospital,FederalFluminenseUniversityinNiterói,RJ.These serahad previouslytestedpositiveforanti-B19VIgMusing acommercial enzymeimmunoassay(Biotrin International, Dublin,Ireland).6,7_{Of131patients,79werefemale.Theirages} rangedfrom9monthsto54years.Inaddition,serafromfive HIV-infectedadultpatientswhoattended thegeneral med-icaloutpatientclinicforroutinecareand werefoundtobe positiveforB19VDNAduringthestudyperiodwerealso exam-ined.Thesecaseswereretrievedfromastudytoestimatethe frequencyofB19VseroconversioninacohortofHIV-infected patients.13_{Writteninformedconsentwasobtainedfromall} participantsand the project was approvedbythe Hospital ReviewBoard(CMM/HUAP134/05).

For molecular detection of B19V and sequencing, viral DNA was extracted from serum samples using a QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). Genotyp-ing of B19V strains was performed with a semi-nested PCRusingtheprimerpairsP12/P16(4127–4689)andP13/P16 (4214–4689) for partial amplification of the VP1/VP2 cap-sid gene.5 _{The 476-bp amplicons were purified using the} illustra GFXTM _{PCR DNA and Gel BandPurification kit (GE,}

Healthcare®, Buckinghamshire, UK) and then subjected to directsequencing using the BigDye terminatorv. 1.1 cycle sequencing kit (Applied Biosystems, CA, USA) and an ABI Prism® 3730DNA analyzer (Applied Biosystems,CA, USA). Bothstrandsofeachampliconweresequencedatleasttwice. Thenucleotidesequences obtained duringthis study were depositedintheGenBankdatabaseunderaccessionnumbers KP115293–KP115325.

For sequence analysis and phylogeny, sequences were alignedusingtheBioEditSequenceAlignmentEditorv7.2.5. Phylogenetic trees were constructed by Bayesian Markov

Chain Monte Carlo (MCMC) statistical framework imple-mentedintheBEASTv1.7.414_{andmaximumlikelihood(ML)} availableintheMEGAv.6.0software.15_{Theevolutionarymodel} Hasegawa-Kishino-Yanoplusgamma(HKY+␥)wasthe sub-stitutionmodelusedasthebestfittedbyModeltest3.7.16

Todeterminetheselectionpressureactingatvariable pos-itionsweusedtheFixedeffectslikelihood(FEL),Randomeffect likelihood(REL)andSinglelikelihoodancestorcounting(SLAC) oftheDatamonkeysoftware.17

B19Vinfectionwascharacterizedbyvariablecombinations of fever,flu-like symptoms, arthropathy, and gastrointesti-nal symptoms. All cases presented with an erythematous maculopapularrash.“Slappedcheek”appearanceand reticu-larorlace-likerashwere seeninonly30%ofthechildren. Noadultpresentedthistypicalrash.Acutearthropathywas reportedmorefrequentlyinadults,mainlyinwomen,andin generalitwassymmetrical,affectingpreferentiallythesmall jointsofthehands,feet,kneesandwrists.Inmostofthecases polyarthropathy completelyresolved withintwoweeks.No clinicalmanifestationsofB19Vinfectionweredetectedinthe medicalrecordsofthefiveHIV-infectedadultpatientsduring theseroconversionperiod.

A476bpfragmentoftheB19Vcapsidgenewasamplifiedby PCRfrom67/131(51.1%)serafrompatientswithEI.The major-ityofthepositivesampleswerecollectedduringthe1999–2000 (n=40)and2004–2005(n=23).

Nucleotidesequencesof27/67amplifiedB19VDNAhada satisfactoryqualityforanalysis.Allthe27sequencesdetected inEIpatientsbelongedtosubgenotype1a(G1a)(Fig.1).

AmongstthefiveB19Vsequencesobtainedfromthe HIV-infected patients, four (RJ109/2002, RJ011/2005, RJ026/2005, RJ350/2006)weregroupedintosubgenotypeG1aandthefifth sequence(RJ071/2005)into3b(G3b).Ofthesefiveserum sam-ples,threewerecollectedduringthe2004–2005outbreak.

Thephylogenetic treesupportedthedivisionofthe G1a in two well-defined clades (posterior probability=1). The sequences from EIpatients were distributedalong the two cladeswhile3/4sequencesfromHIV-infectedpatientswere keptonthesameclade(Fig.1).

TheaveragenucleotidedistanceobservedbetweentheG1a sequencesofthisstudywithotherG1aandG1bwas1.6%and 5.6%,respectively,andwithG3bwas14.5%.Within subgeno-type 1a, the viral sequences were more closely related to eachother(d=1.0%)thantoG1aisolatesfromBrazilorother countries (d=1.2–1.8%).The nucleotidedivergencebetween thetwoG1acladeswas1.3%(aminoaciddivergenceof0.1%) andthegeneticdistancewithinthecladeswasverylow (p-distance=0.9%and0.6%).

Comparisonofthe 427bpfragmentwithG1a sequences retrieved from GenBankrevealed four nucleotide substitut-ions at positions 4260 (A→T), 4267 (A→C), 4262 (A→T), and 4552(T→C)that wereidentified onlyinstrains ofthis study during 1999–2000 outbreak (RJ674/1999, RJ676/1999, RJ677/1999, RJ680/1999, RJ683/1999, RJ690/1999, RJ695/1999, RJ/742/1999,RJ776/1999,RJ772/1999,RJ903/2000).Anotherfour changesatpositions4349(C→T), 4414(T→C),4507(A→G) and4570(C→T)werealsofoundinBrazilianstrainsfromPará andSãoPaulo.Amongtheeightnucleotidechangesallbutone weresynonymous(position4260),afindingeasilyexplained since6/8changesoccurredinthe3rdcodonposition.

TheexaminationoftheselectivepressuresbytheFEL,REL andSLACalgorithmsofDatamonkeypackagedemonstrated thattheregionissubjecttostrongnegativeselection.No pos-itivelyselectsitewasfound.

One sequence (RJ071/2005) from a serum of an adult female HIV-infectedpatient receiving antiretroviraltherapy whoseroconvertedtoanti-B19IgGantibodies inSeptember 2005showedmorethan14.5% ofdivergencewith subgeno-type1ainthe427bpVP1/VP2region,12.2%withgenotype2 and1.8%withsubgenotype3b.Thephylogenetictreebased on these data showed that this sequence clustered in the samebranchas othersbelongingtogenotype 3b.Itshared highernucleotidehomology (99.8%)withother Brazilian3b sequencesobtainedfromapatientwithsystemiclupus ery-thematous from Pará (EF0892229/1999) and another with anemiaafteraliving-donorkidneytransplantfromSãoPaulo (AY647977/2004)(Fig.2).5,9

ThisanalysisofB19Vgenotypescirculatingfrom1996to 2006was performed sincetherewas alackofinformation regardingthegenotypesofB19Vcirculatingduringoutbreaks ofEIinBrazil.Duringthestudyperiodtwodistinctoutbreaks ofB19Vinfection occurred:1999–2000and 2004–2005.6,7 _We demonstratedthepredominanceofsubgenotype1a.Thisis consistentwithpreviousstudiesperformedinBrazil(North, Midwest,South,andSoutheastBrazil)inwhichthemajorityof thesequencesfromseraofpatientswithacuteB19Vinfection belongedtosubgenotype1a.5,9,10,12_{Sequencesfrom} subgeno-type1aofthisstudyhadaveragegeneticdiversityof1.0%and thediversityfrom3bisolateswasaround14.5%.Theseresults areconsistentwithpreviousstudiesinwhichthesameregion ofVP1/VP2genewasanalyzed.3,4,5

Thefirstevidencethattwogroupsofsubgenotype1a coex-ist around the worldarosefrom the phylogenetic analysis ofstrains fromDutch blood donorsand patientswithfifth

Fig.1–PhylogeneticanalysisofB19VVP1/VP2partialsequences(427bp),obtainedfrompatientswitherythema infectiosum(blue)andHIV-infectedpatients(red)inNiterói,RJ,Brazil,from1996to2006.Thereferencesequencesare showedwiththeirGenBankaccessionnumbersinblack.TheBayesianMaximumCladeCredibilitytreewasbasedonthe relaxedmolecularclockandtheMCMCanalysiswasrunfor1×109_{generationstoachievetheconvergenceofparameters.}

Fig.2–Maximumlikelihoodphylogenetictreeof427bpfragmentoftheVP1/VP2capsidgeneofB19V(Genotype3b)based onHasegawa-Kishino-Yanodaplusgamma(HKY+␥)evolutionmodel.Bootstrapsvalues(>50%)basedon2000replicatesare shownabovethebranches.ThereferencesequencesareshowedwiththeirGenBankaccessionnumbers.

disease.18_{Thesubdivisionofsubgenotype1aintotwogroups} wasalsoproposedbyFreitasetal.5_{inastudythatanalyzedthe} same427bpfragmentofVP1/VP2regionfrompatientswith severalclinicalmanifestationsofB19Vinfectioninthe Ama-zonregion(NorthBrazil).Recently,Slavovetal.12_examininga 446bpfragmentcorrespondingtotheNS-1geneofB19Vfrom patientswithhemoglobinopathies andblood donorsinthe StateofSãoPaulo(SoutheastBrazil)alsoreportedthedivision ofsubgenotype1aintotwoclades.

Inagreementwithpreviousreports,theseresultsshowtwo well-definedcladesintosubgenotype1a.Thegeneticdistance betweenthetwocladeswasverylow(d=0.013)whichis con-sistentwithotherstudies.5,18

The low diversity and occurrence of non-synonymous substitutionsofthestrainsG1aofthisstudymaybeexplained by:(i)allserawerefrompatientswithacuteinfection6,7,13_;(ii) themajority ofthesera were collected duringtwodistinct outbreaks; and (iii) almost all the nucleotide substitutions occurred inthe 3rdcodon positionwhich may notlead to aminoacidchange.

Nucleotidechangesmightsuggesttemporalorgeographic clustering but neither the genotypic grouping nor the co-circulationofmultiplelineagesofG1acouldbeassociatedwith aparticularoutbreak.Itisimportanttonotethattheamplified

fragmentcorrespondtothevariablecarboxy-terminalregion ofVP1/VP2capsidgene,aregionthatcontainsseveral neutral-izingepitopes.1

Unlike acuteinfection,B19V isolatesfrom patientswith persistentinfectionexhibitahigherdegreeofvariability,both atDNAandproteinlevel,duetoalowbutcontinuousvirus replicationover prolongedperiodsoftimewhichmay con-tributetoaccumulatedmutations.1

The association of high nucleotide diversity with low amino acid variability observedin B19V isconsistent with theapparentlackofdifferenceinclinicalmanifestationsand antigenicreactivityinserologicaltests.1,3

InBrazil,all threeB19V genotypeshavebeen identified. Whilstgenotypes1and3havebeendetectedinserum sam-plesfrompatientswithEIaswellinseraandbonemarrow of patients withB19-related hematological symptoms,5,8–12 genotype2hasonlybeendetectedinthebonemarrowofolder patients(meanage62.5years)withcytopeniasofunknown origin.11,19

Surprisingly,asequenceobtainedfromaserumofan HIV-infectedpatientduring2005oubreak20_{wascharacterizedas} subgenotype 3b.The origin ofthis subgenotype isdifficult todeterminesincethe serumwascollected duringroutine follow-up examination of the patient under antiretroviral

therapy. No travel information was available on medical records.Furthermore,thisisthefirstreportofgenotype3b inthestateofRiodeJaneiro,SoutheastBrazil.

Thepredominanceofgenotype1(B19V),thelowfrequency ofgenotype 3and theabsence ofgenotype 2inour study are similar to those previously found in other regions of Brazil.5,8,9,11,12,19 _{As the PCR assay used amplifies all three} genotypesofB19V,thelackofdetectionindicatestheabsence ofgenotype2inthesepatients.Genotype 2DNAhasbeen foundmostlyintissuesofindividuals bornbefore1960.2,19 Mostsequencesanalyzedinthisstudywerefromacute infec-tionsinpatientslessthan40yearsold.

Thecurrentstudyrepresentsafurthercontributiontothe molecularcharacterizationofB19VinBrazilsinceitpresents ananalysisofthegenotypescirculatingduringtwodistinct outbreaksoferythemainfectiosuminourcountry.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Thisstudywas supportedbyConselhoNacionalde Desen-volvimento Científico e Tecnológico (CNPq), Universidade FederalFluminense–Pró-ReitoriadePesquisa,Pós-Graduac¸ão e Inovac¸ão (UFF-PROPPI), Fundac¸ão de Amparo à Pesquisa Carlos Chagas Filho (FAPERJ) and Instituto Oswaldo Cruz – Fundac¸ãoOswaldoCruz(IOC-FIOCRUZ).Theauthorswishto thankProf.FelipePiedadeGonc¸alvesNevesandAndré Vic-tor Barbosa for technical support on behalf of Plataforma de Sequenciamento de DNA, Laboratório Multiusuários de MicrobiologiaeParasitologia(LMMP)ofUniversidadeFederal Fluminense.

r

e

f

e

r

e

n

c

e

s

1. GallinellaG,VenturoliS,ManaresiE,MusianiM,ZerbiniM. B19virusgenomediversity:epidemiologicalandclinical correlations.JClinVirol.2003;28:1–13.

2. Servant-DelmasA,LefrèreJJ,MorinetF,PilletS.Advancesin humanB19erythrovirusbiology.JVirol.2010;84:9658–65. 3. ParsyanA,SzmaragdC,AllainJP,CandottiD.Identification

andgeneticdiversityoftwohumanparvovirusB19genotype 3subtypes.JGenVirol.2007;68:428–31.

4. ToanNL,DuechitingA,KremsnerPG,etal.Phylogenetic analysisofhumanparvovirusB19,indicatingtwosubgroups ofgenotype1inVietnamesepatients.JGenVirol.

2006;87:1941–9.

5. FreitasRB,MeloFL,OliveiraDS,etal.Molecular

characterizationofhumanerythrovirusB19strainsobtained

frompatientswithseveralclinicalpresentationsinthe AmazonregionofBrazil.JClinVirol.2008;43:60–5.

6.OliveiraSA,SiqueiraMM,CamachoLA,etal.Theaetiologyof maculopapularrashdiseasesinNiterói,StateofRiode Janeiro,Brazil:implicationsformeaslessurveillance. EpidemiolInfect.2001;127:509–16.

7.OliveiraSA,CamachoLA,PereiraAC,etal.Clinicaland epidemiologicalaspectsofhumanparvovirusB19infectionin anurbanareainBrazil(Niteróicityarea,StateofRiode Janeiro,Brazil).MemInstOswaldoCruz.2002;97:965–70. 8.CostaAC,BenditI,deOliveiraAC,KallasEG,SabinoEC,

SanabaniSS.InvestigationofhumanparvovirusB19 occurrenceandgeneticvariabilityindifferentleukaemia entities.ClinMicrobiolInfect.2013;19:E31–43.

9.KellerLW,BarbosaLM,MeloFL,etal.Phylogeneticanalysisof anear-full-lengthsequenceofanerythrovirusgenotype3 strainisolatedinBrazil.ArchVirol.2009;154:1685–7. 10.Mendonc¸aMC,FerreiraAM,SantosMG,etal.Genotypingof

humanparvovirusB19inclinicalsamplesfromBraziland Paraguayusingheteroduplexmobilityassay,single-stranded conformationpolymorphismandnucleotidesequencing. MemInstOswaldoCruz.2011;106:502–4.

11.SanabaniS,KleineNetoW,PereiraJ,SabinoEC.Sequence variabilityofhumanerythrovirusespresentinbonemarrow ofBrazilianpatientswithvariousB19-relatedhematological symptoms.JClinMicrobiol.2006;44:604–6.

12.SlavovSN,HaddadSK,Silva-PintoAC,etal.Molecularand phylogeneticanalysesofhumanParvovirusB19isolatedfrom Brazilianpatientswithsicklecelldiseaseand␤-thalassemia majorandhealthblooddonors.JMedVirol.2012;84:1652–65. 13.AzevedoKM,SetúbalS,CamachoLA,etal.ParvovirusB19

seroconversioninacohortofhumanimmunodeficiency virus-infectedpatients.MemInstOswaldoCruz. 2012;107:356–61.

14.DrummondAJ,SuchardMA,XieD,RambautA.Bayesian phylogeneticswithBEAUtiandtheBEAST1.7.MolBiolEvol. 2012;29:1969–73.

15.TamuraK,StecherG,PetersonD,FilipskiA,KumarS.MEGA6: molecularevolutionarygeneticsanalysisversion6.0.MolBiol Evol.2013;30:2725–9.

16.PosadaD.ModelTestServer:aweb-basedtoolforthe statisticalselectionofmodelsofnucleotidesubstitution online.NucleicAcidsRes.2006;34:W700–3.

17.KosakovskyPondSL,FrostSD.Datamonkey:rapiddetection ofselectivepressureonindividualsitesofcodonalignments. Bioinformatics.2005;21:2531–3.

18.Molenaar-deBackerMW,LukashovVV,vanBinnendijkRS, BootHJ,ZaaijerHL.Globalco-existenceoftwoevolutionary lineagesofparvovirusB191a,differentingenome-wide synonymouspositions.PLoSONE.2012;7:e43206.

19.GarciaSdeO,KleineNetoW,daCostaAC,etal.Parvovirus amongpatientswithcytopeniaofunknownorigininBrazil:a case–controlstudy.JClinMicrobiol.2011;49:1578–80. 20.PereiraRF,GarciaRC,AzevedoKM,SetúbalS,SiqueiraMA,

OliveiraAS.Clinicalfeaturesandlaboratoryfindingsof humanparvovirusB19inhumanimmunodeficiency virus-infectedpatients.MemInstOswaldoCruz. 2014;109:168–73.