Phylogeny of European Bat Lyssavirus 1 in Eptesicus

isabellinus Bats, Spain

Sonia Vázquez-Morón, Javier Juste, Carlos Ibáñez, José M. Berciano, and Juan E. Echevarría

TobetterunderstandtheepidemiologyofEuropean bat lyssavirus 1 (EBLV-1) in Europe, we phylogenetically characterizedLyssavirusfrom Eptesicus isabellinusbats in Spain. AnindependentclusterofEBLV-1possiblyresulted from geographic isolation and association with a different reservoirfromotherEuropeanstrains.EBLV-1phylogeny

inbatsapparentlycoincideswiththedistribution ofE. isabellinusbats;10casesofhumanexposure aftercontact withinfectedbatshavebeenreported;eachwasassociated withE. isabellinus bats.

Twosubtypeshave been proposedforEBLV-1: EBLV-1a,whichextendsfromtheNetherlandstoRussia inawest–eastaxis,andEBLV-1b, whichincludesstrains that extend south through France and the Netherlands andtheonly2published strainsfromIberia(1).We phylogenetically characterizedEBLV-1strainsassociated withE.isabellinusbats,areservoirintheIberianPeninsula thatdiffersfromE. serotinus bats.

The Study

Wesequenced12batbrainspositiveforLyssavirus antigendetectedbyimmunofluorescence andreverse transcription–PCR(RT-PCR)asdescribed(10).Allviruses wereidentifiedasEBLV-1.Forphylogeneticanalyses,the

400-bp5′variableextremeofthenucleoproteingeneofthese EBLV-1strainswasamplifiedbyspecificEBLV-1nested RT-PCRandsequencedbyusingthefollowingprimers:

iscomplexandprobablyassociatedwithhostevolutionary

SEQVAR1F5′-ACGCTTAACAACCAGATCAAAG

-3′,

history.

SEQVAR2F5′-

AAAAATGTAACACYYCTACA

-3′,

EBLVSEQVAR1R5′-

CAGTCTCAAAGATCTGTTCC

596

AT -3′, andEBLVSEQVAR2R 5′-

TAGTTCCCAGT

hegenusLyssaviruscomprises3speciesthatcaninfect batsinEurope:Europeanbatlyssavirus1(EBLV-1),

575

ATTCTGTCC -3′.

552

Europeanbatlyssavirus2,and West-Caucasianbatvirus (1,2).Mostlyssavirus-infected batshavebeenfoundin north-centralEurope(Germany,theNetherlands,Denmark, Poland,andFrance); ofthese,>95%wereserotine bats (Eptesicusserotinus)infected byEBLV-1 (3–5).EBLV-1 inotherbatspecieshasrarelybeendescribed(3,6).EBLV-

1–infectedbatsbecomeincreasingly scarcefromnorthto southinEurope,andnocasesinnorthernSpainorItalyhave beenreported.Thesametrendhasbeenconsistentlyfound withinGermany (3)exceptforanartifactthatarosefrom variedsurveillance intensityamongdifferentcountries. However,severalinfected serotinebatsinsouthern Spain havebeenreported(7).Thesebatshavebeenassigned tothespecies E.isabellinus,whichhascloselyrelated populations ontheAfricansideoftheGibraltarStrait(8). ThisspeciesisstronglydivergentfromE.serotinusbats (>16%ofcytochrome bgene)inthenorthernIberian Peninsula(9).InSpain,thedistributionofEBLV-1cases

Author affiliations: Instituto de Salud Carlos III, Majadahonda, Madrid,Spain(S.Vázquez-Morón,J.M.Berciano,J.E.Echevarría); Centro de Investigación Biomédica de Epidemiología y Salud Pública, Barcelona,Spain(S.Vázquez-Morón,J.E.Echevarría); and Consejo Superior de Investigaciones Científicas Estación Biológica de Doñana, Seville, Spain (J. Juste, C. Ibáñez)

Allrabies-positiveserotinebatscamefromsouthern

Spain (Huelva, Seville, Murcia, and Badajoz) and weremolecularlyidentified asE.isabellinus(8).An alignment wasperformed byusingClustalX(www. clustal.org) tocombine theobtained sequencesandother availableEBLV-1sequences fromGenBank, includinga Duvenhage virususedastheoutgroup (onlineAppendix Table, Before conducting further analyses, we used jModel Test( selectthebestfittingsubstitutionmodelforoursequences according tothecorrected Akaikeinformation criterion. Maximum-likelihood phylogenieswerereconstructedby usingPHYML( byusingageneralizedtime-reversiblemodelandtheγparameterestimatedintheanalyses.Maximum-parsimony analyses wereconductedbyusingPAUP*4.0b10( paup.csit.fsu.edu/)weightingtransversions15×according tothetransitions/transversion ratioestimatedinthe jModelTestanalyses.Confidenceinthetopologiesfor themaximum-likelihood andthemaximum-parsimony analyses wasestablished with1,000bootstrapreplicates. A Bayesian phylogenetic inference was obtained by using MrBayesversion 3.1( withrandomstartingtreeswithoutconstraints. Two simultaneous runsof107 generationswereconducted, eachwith4Markovchains,andthetreesweresampled

Figure1.Europeanbatlyssavirus1(EBLV-1)phylogeneticreconstructionbasedonthefirst400bpofthenucleoproteingene. Thetree wasobtainedbyBayesianinferencerunfor107 generations;treesweresampledevery100generations.Thefirst25%oftreeswere excluded from the analysis as burn-in. Black numbers indicate posterior probabilities. Bootstrap supports after 1,000 replicates for each nodearealsoshownformaximum-parsimony(greennumbers)andmaximum-likelihood(bluenumbers)analyses.Net p-distancevalues (aspercentages)betweengroupsareindicatedbyarrows.Aparsimony-basednetworkispresentedforeachmajorlineage;sizesofyellow circlesareproportionaltothenumberofindividualssharingagivenhaplotype,andreconstructedhaplotypes(medianvectors)areshown in red. DUVV, Duvenhage virus.

every100generations. Netp-distancesbetweengroups werecalculatedbyusingMEGA4( net/) (Figure1).

Thegeneticstructureandrelationships between haplotypes wereexaminedwithinthemainlineages throughaparsimony-based networkbuiltwithamedian- joining algorithm implemented in the Network 4.5.1

program(11).Toevaluateandcomparegeneticvariability andpolymorphism amonglineages,weestimatedthe numberof haplotypes,mutations,andsegregatingsites aswellashaplotype diversityandnucleotide diversityby usingDNAspversion4.5(12)forthemajorclades(Table). Finally,toinvestigatepopulationdynamicsacrosslineages, theFuFsandTajimaDstatisticswerecalculated(Table).

Table.GeneticdiversitystatisticsforEBLV-1*

PopulationnSEtaHapHdVarHdPiThetaNuckTajimaDFuFs

EBLV-1a524548260.8360.002670.006640.026562.6546–2.5693 (0.00000)

EBLV-1b253535180.9700.000380.022020-023178.8067–0.1885 (0.48000)

EBLV-1Spain139970.7950.011910.005380.007252.1538–1.0138 (0.18100)

–21.676 (0.00000)

–4.555 (0.05100)

–2.067 (0.06143)

*EBLV, Europeanbatlyssavirus;n, no.sequences;S,no.segregatingsites;Eta,no.mutations;Hap,no.haplotypes;Hd,haplotypediversity;VarHd, haplotypevariance;Pi,nucleotidediversity;ThetaNuc,estimatedpopulationmutationrateper site; k,averageno.nucleotidedifferences;andneutrality tests(Tajima D and FuFs).

Figure 2. Geographic distribution of Eptesicus serotinus bats (red), E. isabellinus bats (blue), and cases of rabies in bats (dots), Europe, 1990–2009. Obtained from Rabies Bulletin Europe (www. who-rabies-bulletin.org/).

These2statisticsareconsidered tobethemostpowerful testsfor detectingexpansionevents(13).

Conclusions

Allphylogenetic analyses,regardlessofthe reconstruction criterionused,formedamonophyletic clusteroftheEBLV-1 strainsfromSpain(onlythe Bayesianinferencereconstructionisshown).TheBayesian inference,maximum-likelihood,andmaximum-parsimony analysesidentifiedtheclusterfromSpainandEBLV-1a andEBLV-1basbeingmonophyletic(Figure1),although onlymaximum-likelihood andmaximum-parsimony analysessuggestedacloserrelationshipbetweenEBLV-

1bandthecluster fromSpain.Thegenetic differentiation oftheEBLV-1strainsfromtheIberian Peninsulamatches theirassociationwithanotherbatspecies(Figure2),which suggeststhatthehostbat’sevolutionaryhistoryplaysa majorroleinEBLV-1molecularepidemiology,ashasbeen proposed for rabiesvirus inbatsinNorth America(14).

Thelowgeneticdiversityandthe FuFsandTajimaD statistics(Table)allsuggestrapidpopulationexpansionof EBLV-1a,whichisconsistentwiththestar-likestructure ofthenetwork forthislineage(Figure1).Conversely, haplotype and nucleotide diversity descriptors (Table) havethehighestvaluesforEBLV-1b andacomplex networkstructurewithdifferentiated subnetworks.All theseelements indicatethatthislineagehasacomplex evolutionaryhistory.ThelineagefromSpainalsohaslow diversityandastar-shaped network,butneutralevolution cannotberejected onthebasisoftheFsandDstatistics. Netdistancesaresimilarwithinandbetweenlineages,

exceptforEBLV-1a, whichisslightlymoredifferentiated (Figure1).Consequently,thesuggestedEBLV-1expansion fromSpainintoEurope(15)isnotsupportedbyourresults, whichrecordthehighestvariability andmostcomplex phylogenetic structureforFranceandtheNetherlands (Figure1).Thiscomplexstructuresuggestseitheralonger evolutionaryhistoryintheseareasorarecentcontactof distinctbatlineagesinthiszone.

Theresults ofthisstudyshowthatthestrains from Spaindonotbelongtosubtype 1bbecause oftheir association withadifferentreservoir(E.isabellinusbats). Moreover, whatiscurrentlyconsidered tobeEBLV-1b seemstoincludeatleast4lineagesthataremoregenetically diverseandhaveacomplexhistory.EBLV-1a, however, haslowgeneticdiversitydespiteitsextensive geographic distribution, suggestingarelativelyrecentandsuccessful expansion ofthislineage.Theseresultscallintoquestion thecurrentclassificationofEBLV-1into2singlesubtypes. ToprovideabetterunderstandingofEBLV-1molecular epidemiologyinEurope,additionalstudiesthatconsider differentgenesshouldbeconducted andthecurrent classificationshouldberevisedaccordingly.

Acknowledgments

WethanktheGenomicsUnitoftheInstitutodeSaludCarlos IIIforanalysesofthegenomicsequences andEnriqueRoyuela Casamayorfor hisinvolvementinthedailywork.

Thisprojectwasfinancially supportedbyanagreement betweenthePublicHealthDepartmentoftheSpanishMinistryof HealthandtheInstitutodeSaludCarlosIIIforthedevelopmentof “RabiesSurveillanceinSpain”andbyprojectsSAF2006-12784- C02-01andSAF 2006-12784-C02-02oftheGeneralResearch Programmeof theSpanishMinistryof ScienceandEducation.

DrVázquez-MorónisaPhDcandidateattheInstitutode SaludCarlosIIIandComplutense UniversityofMadrid.Her mainresearchinterestsaretheepidemiologyandpublichealth implicationsof rabiesandemergingviruses inbats.

References

1. AmengualB,WhitbyJE,KingA,CoboJS,BourhyH.Evolutionof

Europeanbatlyssaviruses. J GenVirol.1997;78:2319–28.

2. BotvinkinAD,PoleschukEM,KuzminIV,BorisovaTI,Gazaryan SV, YagerP,etal.NovellyssavirusesisolatedfrombatsinRussia. EmergInfectDis. 2003;9:1623–5.

3. MüllerT,JohnsonN,FreulingCM,FooksAR,SelhorstT,VosA.

Epidemiologyofbat rabiesin Germany.ArchVirol.2007;152:273–

88. DOI: 10.1007/s00705-006-0853-5

4. Van der PoelWH,Van der HeideR,VerstratenER,TakumiK,Lina PH, Kramps JA. Europeanbatlyssaviruses, theNetherlands.Emerg InfectDis. 2005;11:1854–9.

5. Picard-MeyerE,BarratJ,TissotE,VerdotA,PatronC,BarratMJ,et al.BatrabiessurveillanceinFrance,from 1989throughMay2005. Dev Biol(Basel).2006;125:283–8.

6. Serra-CoboJ,AmengualB,AbellanC,BourhyH.Europeanbat lyssavirusinfectioninSpanishbatpopulations.EmergInfectDis.

2002;8:413–20.DOI: 10.3201/eid0804.010263

7. Vázquez-MorónS,JusteJ,IbáñezC,Ruiz-VillamorE,AvellónA, VeraM,etal.EndemiccirculationofEuropeanbatlyssavirustype

1inserotinebats,Spain.EmergInfectDis.2008;14:1263–6. DOI:

10.3201/1408.080068

8. JusteJ,BilginR,MuñozJ,IbáñezC.MitochondrialDNAsignatures at differentspatialscales:fromtheeffectsoftheStraitsofGibraltar topopulation structureinthemeridional serotinebat(Eptesicus isabellinus).Heredity.2009;103:178–87.DOI:10.1038/hdy.2009.47

9. Ibáñez C, Garcia-Mudarra JL, Ruedi M, Stadelmann B. The IberiancontributiontocrypticdiversityinEuropeanbats. Acta Chiropterologica. 2006;8:277–97. DOI: 10.3161/1733-5329 (2006)8[277:TICTCD]2.0.CO;2

10. Vázquez-MorónS,AvellónA, EchevarríaJE. RT-PCRfor detection of all seven genotypes of Lyssavirus genus. J Virol Methods.

2006;135:281–7.DOI: 10.1016/j.jviromet.2006.03.008

11. BandeltHJ,ForsterP,RohlA.Median-joiningnetworksforinferring intraspecificphylogenies.MolBiolEvol.1999;16:37–48.

12. RozasJ,Sánchez-DelBarrioJC,MesseguerX,RozasR.DnaSP, DNApolymorphismanalysesbythecoalescentandothermethods. Bioinformatics.2003;19:2496–7. DOI:10.1093/bioinformatics/ btg359

13. Ramos-OnsinsSE,RozasJ.Statisticalpropertiesofnewneutrality testsagainstpopulationgrowth. MolBiolEvol.2002;19:2092–100.

14. Hughes GJ, OrciariLA, RupprechtCE. Evolutionarytimescale ofrabiesvirusadaptationtoNorthAmericanbatsinferredfrom the substitution rate of the nucleoprotein gene. J Gen Virol.

2005;86:1467–74.DOI: 10.1099/vir.0.80710-0

15. DavisPL,HolmesEC,LarrousF,VanderPoelWH,Tjornehoj K, Alonso WJ, et al. Phylogeography, population dynamics, and molecularevolutionof Europeanbat lyssaviruses. J Virol.

2005;79:10487–97.DOI: 10.1128/JVI.79.16.10487-10497.2005

Addressforcorrespondence: SoniaVázquez-Morón, Diagnostic Microbiology Service,NationalCenterforMicrobiology, Institutode SaludCarlosIII,CarreteradeMajadahonda-PozueloKm2,Majadahonda

28220, Madrid,Spain;email: