Sizematters:WestNileVirusneutralizingantibodies inresidentandmigratorybirdsinSpain
JordiFiguerolaa,*,MiguelAngelJime´nez-Claverob,1,GuillermoLo´peza, ConsueloRubiob,Ramo´nSoriguera,ConchaGo´mez-Tejedorb,AntonioTenorioc
aEstacio´nBiolo´gicadeDon˜ana,CSIC,ApartadodeCorreos 1056,41080-Sevilla,Spain
bLaboratorio Central deVeterinaria, CarreteraAlgeteKm 8,28110-Algete,Spain
cCNM-InstitutodeSaludCarlosIII,CarreteraPozueloKm 2,28220-Majadahonda,Spain
Abstract
TherapidrangeexpansionofWestNileVirushasraisedinterestinunderstandingthepopulationdynamicsanddispersal patternsofemerginginfectiousdiseasesbywildlife.WeanalyzeddifferentecologicalandevolutionaryfactorsrelatedtoWest NileVirusneutralizingantibodyprevalencein72birdspeciessampledinsouthernSpain.Prevalenceofantibodiesreachedits maximumduringtheautumnand winterincomparisontosummermonths.Prevalenceofantibodieswasdirectlyrelatedtobody massandmigratorybehaviour.Thegreaterprevalenceofantibodiesobservedinsummermigrantscanbeexplained,among otherfactors,bythediversityoflocalitiesinvolvedintheirlifecyclesorthegeographicareasvisitedduringtheirmigrations. Greaterprevalenceinlargerspecieswasexplainedbytheirlongevitybecausetherelationshipwasalreadysignificantwhen analyzingonlyfirstyearbirds,andprobablyalsoinvolvedahighattractiontovectorsbylargerhosts.Colonialityandwinter gregarismwereunrelatedtotheprevalenceofantibodiesagainstthis highlyhostgeneralistpathogen.Evolutionaryrelationships between species wereunrelated todifferences intheprevalence ofantibodies. Ourresultssuggest largerspecies asgood candidatesforeasy,fasterandcheapermonitoringoflocal,seasonalandannualchangesinWNvirusserology.
Keywords: Dispersal;Hostecology;Migratorybirds;Monitoringprogram;Reservoirdiversity;Virusdispersal;WestNileVirusecology
1.Introduction
* Correspondingauthor.Tel.:+34954232340.
E-mailaddress: (J.Figuerola).
1 Present address: CISA-INIA, Carretera Algete-El Casar s/n,
28130-Valdeolmos,Spain.
WestNileVirus(WNV) isamemberofthe Flavivirusgenus(familyFlaviviridae),transmittedby mosquito bites.HumansinfectedbyWNVmay developavarietyofsignsrangingfrommildfever tomoresevereillnessessuchasacuteencephalitis,
poliomyelitis,meningitis,orhepatitisandis fatalina smallpercentage(1%)ofcases(Huba´lek and Halouzka,1999).WNViswidelydistributedthrough- outAfrica,Asia,Europe,Australia(Kunjin virus).It wasfirstdetectedinNewYorkin1999,andinjust6 yearshasspreadthroughout allofNorthAmerica (CDC,2005).Ithasbeensuggestedthatoneofthe causesofthisrapidexpansion isthehighmobilityof thevirus’avianreservoirs(Rappoleand Huba´lek,
2003) anditswidehostrange(already detectedin morethan285avianspecies;CDC,2005).
InEuropeandAfricaWNVinfectionisusually non-fatalforbirds(Huba´lekandHalouzka,1999);in theNewWorld,however, thevirushaskilledmany birds(Marraet al.,2004),and reducedpopulationsof moresusceptible hostsbyupto45%sinceWNV arrival(LaDeauetal.,2007).Asanexample ofthe differentepidemiologyinEuropeandNorthAmerica, whileexperimentalinfectionwithWNV ofNorth Americanbirdsusuallyresultinhighmortalities(i.e.
32.3%of87experimentally infectedbirdsof25 species,Komaretal.,2003),experimental infections doneinEuropehavereported noapparent mortality duetoWNV(9geeseexperimentally infectedby MalkinsonandBanet,2002).Thereasonsforthishigh virulenceinNorthAmerica remain largelyunknown; nevertheless, thefactthatspeciesfromtheNearctic havenever beenexposed tothevirusandthehigher pathogenicityobservedintheintroducedstrain(Brault etal.,2004)mayexplainthesedifferences.
Anumberofecological factorscanbeassociated withahigherprevalence ordiversity ofpathogens in birds:migratorybehaviour, coloniality orgregarism, habitatuse,mating systems, andimmune system capacity (MøllerandErritzoe,1996;Claytonand Moore,1997;Figuerola,1999,2000;Figuerolaand Green,2000;Tella,2002).However,toourknowl- edge,no studyhas focusedonvector-bornegeneralist pathogens.Despitethethousandsofbirdsthathave beentestedforWNV oritsantibodiesinNorth America, analysesfocusingontherelationship betweenbirdecologyandexposuretothevirusare stilllackingbuturgentlyneeded.Inthisstudywetake advantageof thedifferencesintheimpactof WNVin Europe andinNorthAmerica toanalyzethe relationship between bird ecology and phylogeny andprevalenceofWNVneutralizingantibodies. The relevanceofthisstudyistwofold,ontheonehand,the
lowhostspecificity ofWNVmakesthissystem different fromthepathogens usedinprevious studies (mainlybloodparasitesandectoparasites), andmay affecttherelevanceofdifferentecologicalfactors.On theotherhand,giventherelevanceofWNVforhuman healthandwildlifeconservation wealsoaimto identifythecharacteristics ofthespeciesthatcanbe mostusefulformonitoringinEurope.
Inthispaper,wefirst analyzetherelationship betweenhostevolutionary andecologicalcharacter- isticsandtheprevalence ofWNVneutralizing antibodiesinbirds.Second,aswereportimportant differencesintheprevalenceofantibodies according tohostcharacteristics weusedastatisticalpower analysistodiscuss therelevanceofourresultsin relationtoWNVmonitoringinEurope.
2.Materials andmethods
BetweenJanuary2003andFebruary 2005we captured1213individualsbelongingto72species(49 genera,22families,and8orders).Birdswerecaptured withoutdamageusingmist-netsandwalk-in-traps in theGuadalquivirandOdielMarshes (SWSpain). Bloodsamples weretakenwithsyringesfromthe brachial, femoral, orjugularvein,birdsweremarked withnumbered aluminum ringsandreleasedafter manipulation. Thevolumeofbloodextracted depended onthesizeofthespeciesandnever exceeded1%ofbodymass(range0.080–1ml).Blood wascollectedineppendorf tubes,allowedtoclotat ambienttemperature, andplacedintocoolersuntil centrifugationduringthesameday.Allsampleswere obtainedfromadult(fullgrown)individualstoensure thattheantibodies werenottheresultofthepassive transferofmaternalimmunity(Gibbsetal.,2005). Whenpossibleagewasdetermined (471first-year individualsand540afterfirst-year individuals) accordingtoPrateretal.(1977),Baker(1993)and Svensson(1996).
WNVstrainEg101andtheE6 cloneofVerocells usedforviruspropagationwereobtainedfromHerve´ Zeller(InstitutPasteur deLyon).TheUsutu virus (SAAR 1776isolate)wasobtainedthroughthe Centre for Ecology and Hydrology,Oxford, UK, and propagated in Vero cells (American Type CultureCollection,Manassas,VA).Virustiterswere
determined byend-pointtitrationfollowing the method usedbyReedandMuench (1938). WNV- Neutralizing antibodytitersweredeterminedbya micro-virus-neutralizationtest(micro-VNT)in96- wellplates,adapted fromapreviously described method(Jime´nez-Claveroetal.,2001).Arecentstudy showsthatamicro-VNT assayandthestandard PRNT90performcomparablyin sensitivityat detect- inganti-WNVantibodiesinbirds(Weingartletal.,
2003).Serumsampleswereinactivatedat568Cfor
30 minpriortotheanalysis.Dilutionsoftestsera (25ml)wereincubatedwith100TCID50 ofWNV strainEg101inthesamevolume(25ml)for1hat
37 8C in Eagle’smedium (EMEM)supplemented with L-glutamine,100U/ml penicillin,100mg/ml streptomycin,followedbytheaddition of50 mlofa suspension(2 x105cells/ml)ofVeroE6 cellsinthe same medium plusfetalcalfserumtoafinal concentration of5%.Themixturewasfurther incubatedfor6–7days(378C ina5%CO2 and saturatinghumidityatmosphere) untilcytopathic effect(cpe)wasobservedincontrolwellscontaining
10TCID50 ofvirus.Thescreeningofsampleswas performed at1:10and1:20dilutions oftestedsera (dilutionsconsideredbeforetheadditionofvirus,that is,inavolumeof25 ml).Onlysamplesyielding positiveneutralization(completeabsenceofcpe)at
1:20werescoredaspositives andfurther titrated by analyzing serialserum dilutions from 1:20to1:640. Neutralizingserumtiterwasconsideredasthehighest valueofthereciprocal serum dilutiongivinga completeabsenceofcpe.
Thespecificity oftheassaywasassessedintwo ways. First, byanalyzingapanelofserafroman externalqualityassessment, consistingofserum samples containingantibodies fromotherfour flaviviruses,thatprovednegativeforneutralization titersinourWNVassay,whileduplicatetestingof allWNVantibody-positiveserumsamplesproved positive (1:20) for neutralizationtiters (Niedrig etal.,2007). Second,wealsocompared the neutralizingantibodiestitersof18samplestested inparallelforWNV andUsutuvirus(aclosely relatedJEVgroupavian virus).Innoneofthecases showedhigherantibodytitersmorespecificto Usutu thantoWNV(seeFiguerola etal.,2007a,formore details). We cannot discard that the serology to WNVobservedinsomeofthesamples,particularly
thosefrombirdsflying fromCentralEurope(e.g. Turdusphilomelos,Sylvia atricapilla) couldbe attributedtocross-reactingantibodiestootherflavi- viruses(particularly TBEV)thatcanbeprevalentin CentralEurope.However, thisseemsunlikelysince the technique has shown no cross reactivity to TBEV-positivesera.
Inafirst model,weinvestigatedtheeffectsof taxonomicrelationships onWestNileViruspre- valencebyusingGeneralized LinearMixedModels (GLMM).GLMMallowsamoreversatileanalysis ofcorrelationthanstandardregression methods, becausetheerrordistribution ofthedependent variable and the function linking predictorsto it canbeadjustedtothecharacteristics ofthedata (Littelletal.,1996).Ourresponse variablewasthe antibody status (1present,0absent), andweuseda binomialdistributederrorandalogisticlinkfunction, toensurelinearity,andstatisticsadjustedtomodel dispersion. Binomial errors areadequate toanalyze binaryresponsevariables.Goodness-of-fit ofthe modelwasassessed bycheckingtheoverdispersion parameterandtheGeneralizedChi-Squarestatistic (Littelletal., 1996). Period (athree levelsfactor, summer:birdscapturedinJune–August, autumn: September–November andwinter:December– March)andage(first-yearoradultbird,notincluding unknownagebirds in theanalyses)were includedas fixedfactorsintheanalyses.Specieswasincludedasa repeatedsubjecteffect(i.e.observationsofasame speciesarecorrelated)andtheinteractionbetween speciesandperiodwasincludedasarandom factor. Thestatisticalsignificanceofeachnestedtaxonomic level(Genera,FamilyandOrder)wastestedusing Z-statistics forrandomeffectsusingthemacro GLIMMIXforSAS8.2(Littellet al.,1996).As agehadnosignificanteffect(F1,27 = 1.88,P= 0.18, N=957),wereporttheresultsofanalysesexcluding thisvariable toinclude thefulldataset andrange of species.
Inasecondmodel,weanalyzedtherelevance of differentecological factors.Species bodymass(log transformedmeanvaluestofit anormaldistribution asjudgedbychecking thenormalquantileplot), migratorybehaviour (residentormigratory species), breedingsociality(solitaryorcolonialbreeders), and wintersociality(solitaryorgregarious species)were includedasfixedfactorsintheanalyses.Valuesfor
Table1
Modelanalyzingthe relationshipbetweenhost ecology,periodofcaptureandpresenceofWestNile Virus(WNV)neutralizingantibodiesinthe bloodof1213individualsbirdscapturedinsouth-westSpain
Estimate±S.E. / F / d.f. / PBodymass / 0.746±0.288 / 6.72 / 1,69 / 0.01
Migratorybehaviour / 2.032±1.084 / 0.71 / 1,69 / 0.40
Period / 3.55 / 2,29 / 0.04
Summer / 0
Autumn2.194±1.286
Winter2.287±1.133
Coloniality / 0.67 / 1,68 / 0.42Wintergregarism / 2.82 / 1,68 / 0.10
BodymassxMigratorybehaviour / 0.71 / 1,68 / 0.40
BodymassxPeriod / 1.21 / 2,1135 / 0.30
BodymassxColoniality / 0.65 / 1,68 / 0.42
BodymassxWintergregarism / 3.23 / 1,68 / 0.08
MigratorybehaviourxPeriod
Migratoryspeciesinautumn / —1.021±1.366 / 3.71 / 2,29 / 0.04
Migratoryspeciesinwinter
Others / —3.653±1.431
0
MigratorybehaviourxColoniality / 1.63 / 2,67 / 0.20
MigratorybehaviourxWintergregarism / 1.41 / 2,67 / 0.25
PeriodxColoniality / 1.28 / 3,26 / 0.30
PeriodxWintergregarism / 0.99 / 3,26 / 0.41
ColonialityxWintergregarism / 1.55 / 2,67 / 0.22
Finalmodelwasobtainedafterbackwardsvariableselection.OnlyvariableswithP0.05areinterpreted asstatistically significantand parameterestimatesaregiven.ForvariablesnotincludedinthemodelnoparameterestimateispresentedandtheFandPvaluescorrespondto thevalueswhenaddedtothefinalmodel.
thesevariablesweretakenfromliterature(Cramp,
1982–1994) andwerevalidatedbyfourindependent ornithologistsaccordingtotheecologyofthespecies inSpain.Foreachindividual wealsoincluded the periodofcollectiontocontrolforseasonaldifferences intheprevalenceofantibodies.Totesttherelationship betweenecological factorsandantibodyprevalence wefollowedastepwise-backwardselectionprocedure startingfromaninitialmodelincluding allthetwo- wayinteractionsbetweenfactors.
Weestimatedthesamplesizenecessary todetect increasesof10,20,30and40%inWNV seropre- valencewiththeprogramG-Power(Buchneretal.,
1997).Effectsizeswerecalculated forprevalences between1and55%,andsamplesizenecessary to obtainapowerof0.80whenusingaChi-Square test wasestimated.Apowerof0.80indicates thata significantresult(P0.05)willbeobtainedin80% oftheanalysesofdatasets withstatisticaldifferences ofthatmagnitude, andisthethresholdvalueusually usedinecology(BausellandLi,2002).
3.Results
Ofthe1213individuals tested,126(10.4%of individuals from24outof72species)hadWNV neutralizing antibodies,withtitersrangingfrom1:20 toover1:640(seeElectronicAppendixA).Important interspecific differencesinthepresenceofWNV neutralizing antibodieswerefound,withprevalences rangingfrom0to42.9%.However,taxonomiclevels wereunrelatedtothesedifferences inprevalence (Genera, Z=0.80, P=0.21; Family, Z=1.11, P=
0.14;Order,Z=0.58,P=0.28).
Multivariateanalyses indicatethatantibodypre- valence wasunrelatedtohostsociality(Table1). Prevalenceof antibodieschangedseasonally(Table1) withsignificantlyhigherprevalencesinautumn(mean
±S.E.:10.29%±12.71)thaninsummer(atestof
Least-Squaremeansdifference,2.09±7.72,t29 =2.46, P=0.02), and intermediate prevalences in winter (3.27%±9.12, contrast with autumn, t29 =1.85, P=0.07;contrastwithsummer,t29 =0.61,P=0.55).
Althoughmigratorybehaviourwasnotdirectlyrelated to antibody prevalence (migrants, 5.23%±7.41; residents,3.32%±8.13),asignificantinteractionwith seasonwasfound(Table1).Prevalencedidnotchange withseasoninresidentspecies(F2,29 =2.11,P=0.14) butonlyinmigratoryspecies(F2,29 =6.15,P=0.006). Whencomparing migrantandresidentspecieswithin eachperiod,insummermigrants(i.e.specieswintering inAfrica)tendedtohavehigherprevalences of antibodiesthatresidents(5.56%±8.58vs.0.77%±
8.85; t29 =1.88, P=0.07). Winter migrants (i.e. comingfromcentralandnorthern Europe)tendedto havelowerprevalencesthanresidentspecies(1.48%±
10.49vs.7.06±12.16;t29 =1.70,P=0.09).Large species(asestimatedfromtheirbody size)had higher prevalencesofantibodies(Table1,Fig.1).
Asagemayaffecttherelationshipbetweenantibody prevalenceandbodymass,theanalyseswererepeated usingonlylessthan1yearoldbirds(417individuals), confirmingthattherelationshipbetweenprevalence andbodymasswassignificantalso whenconsidering onlybirdsofthesameage(F1,43 =5.61,P=0.02).
Power analyses indicate that the sample size
necessarytodetectsignificantchangesinseropreva-
Fig.1.Prevalenceof WestNileVirus(WNV)neutralizinganti- bodiesinrelationto bodysize(grams)inresident(418individuals) andmigratory(795individuals)birdssampledin south-westSpain. For illustrationpurposesa regressionlinehasbeenplottedfor migratoryand residentspecies.Opensymbolsanddottedline correspondtomigratoryspeciesandfilledsymbolsandcontinuous linetoresidentspecies.Onlyspecieswithat leasttenindividuals sampledhavebeenincludedintheplot.
Fig.2. Samplesizenecessarytodetectwitha Chi-squaretest anda powerof 0.80increasesby10,20,30and40%intheprevalenceof WestNileVirus(WNV)antibodies.
lencedependsdramatically oninitialseroprevalence (Fig.2).Forexample,4857individualsarenecessary todetecta40%increaseinseroprevalencewheninitial seroprevalence is1%(i.e.inourstudyPasser domesticus hadaprevalenceof0%),butonly74 individualsarenecessary whenfocusinginspecies with40%prevalence (i.e.Fulicaatra,with42.6% prevalenceorLarusridibundus,with42.9%).
4.Discussion
InSpain,clinicalsignsofWNVdiseaseinbirdshas onlybeenreportedrecently(Ho¨fleetal.,2008).WNV neutralizing antibodieshadbeenreportedinhorses (Jime´nez-Clavero etal.,2007),chicksofdifferent colonialbreedingwaterbirds(Figuerolaetal.,2007a), andtherapidseroconversionofcommoncootsduring acapture–recapturestudyhasalsoconfirmedthelocal circulationofWNVinthestudyarea(Figuerolaetal.,
2007b).Infections withclinicalsymptoms inhumans werereportedin2004inBadajoz(Spain)andAlgarve (Portugal)(Estevesetal.,2005;Kaptouletal.,2007), overlapping withthecollection ofsamplesforthis study.Previousrecordsgivea seroprevalenceof upto
30%inhumansinsometownsintheEbroDelta
(LozanoandFilipe, 1998)andof16.5%innorthwest Spain (Gonza´lez and Filipe, 1977), presumably withmaximumepidemicactivityduringthe1970s.
However, theseresultswereobtainedbyhaemagglu- tination-inhibition,atechniqueburdenedbyitscross- reactivitywitharangeofflaviviruses.Recentstudies withhighlyspecificneutralizationassaysshowthat theprevalenceofWNVantibodiesinhumansliving aroundwetlandsinSpainiscurrentlyverylow(Bofill etal.,2006).
Thepresenceofserumantibodies neutralizing WNV inadultbirdsindicatespreviouscontact (infection)withWNV oracloselyantigenically relatedflavivirus,andsurvivaltotheinitialinfection. Consequently, forawildbirdpopulationwithlow pathogenicityWNV infection(suchasthoseusually foundintheOldWorld,ZellerandSchuffenecker,
2004),thehighertheprevalenceofWNVneutralizing antibodies, thehighertheexposuretothevirus.This scenarioisnotapplicablewhenWNVinfectionresults inhighmortality,asobservedinNorthAmerica.
Theresultssuggestthatevolutionaryrelationships areoflittleimportance inexplainingvariationsin exposure toWNV. Thiscontrastswiththeinitial studiesthatidentifiedCorvidae(McLeanetal.,2001), Mimidae,andCardinalidae (Ringiaetal.,2004)as birdfamiliesthatareparticularlyexposedtoWNV infection.InourstudybothRallidae(6.7–42.6%)and Laridae(25.0–42.9%)presentedveryhighantibody prevalence,althoughourresultssuggest thatthese highprevalenceswererelatedtotheecologyofthe species sampled(migratory speciesofmediumand largesize),ratherthantothebirds’taxonomy.
InNorthAmerica, theAmerican Crowappearsto beparticularly susceptibletomortalitybyWNV (Komaretal.,2003).Thishasledsomeresearchersto suggestthatCorvidaeingeneralmightbeveryatarisk forexposuretothevirus.Interestingly,noneofthe35 individualsofCorvusmonedula(theonlyCorvidae includedinourstudy)hadWNVantibodies,even whencapturedtogetherwithindividuals ofother specieswithhighprevalences. Itisimportanttonote thatthislow(zero)prevalenceofantibodiesinCorvus monedulaisnotlikelytoresultfromtherapiddeathof infectedindividuals, giventhatallattemptswehave donetothemomenttodetectthe virus inseveral hundredsdeadwater-birds hadfailed(datanot shown). WesuggestthatthehighincidenceofWest NileinAmerican Crowcanresultnotonlyfromthe transmissionbymosquitoes butalsofromthecon- sumptionofcorpsesofbirdsdyingduringtheviraemic
phaseoftheinfection. Inthiscasetheutilityof Corvidsformonitoring WNVcirculation inthewild couldbereducedinEurope.
Noeffectofwinter orbreeding sociality on antibodyprevalence wasfound.Althoughahigh prevalence ofbloodparasiteshadbeenreported amongsociallivingspecies (Tella,2002), thelow host-specifityof WNVmaymakethedensityofbirds therelevantparameter affecting riskofexposition, regardless whetherornotitconsists ofconspecifics. Interestingly, migratoryspeciesshowedhigheranti- bodyprevalencethanresidentspecies, butonlywhen comparingsummermigrantswithresidents.Although localcirculation ofthevirusistakingplace(since residentspeciesalsohaveantibodies), thishigher prevalenceobservedinmigratorybirdssuggestthat thesebirds spendpartoftheirlives inareasinAfrica wherethecirculationofthevirusmaybehigherthan inthesurveyed areainSpain.Forexample,arecent serosurveyinhorsesdetectedextremely highpre- valences ofantibodies(upto97%)insomesub- Saharancountries(Cabreetal.,2006),areasvisitedby manyEuropean longdistancemigratory species. Our analysessupport theviewthatspeciesoflargerbody massmayhaveincreased opportunityforexposureto WNV.Giventhatweanalyzedantibodyprevalencein free-livingandapparently healthyindividuals, this conclusion isnotmerelyabiascausedbythe difficulties infindingcarcassesofsmallerspecies (Marraetal.,2004),aproblemassociatedwithstudies basedonlyondeadbirds.Thedirect relationship betweenprevalenceofWNV antibodiesandbody masscanbeexplained byseveral non-exclusive factors. Largerspecieslivelonger(Calder,1984), however wehavedemonstrated thattherelationship betweenseroprevalence andbodymassisalso significant whenanalyzingonlyfirst-yearbirds.We suggest thatthelargerprevalenceofantibodiesin largerspeciesistheresultoftheirlargersurface area andhigherCO2production(Nagy,1987),andcanhost and attractahighernumberof ectoparasites(Soliman etal.,2001),mosquitoes,andotherbitingarthropods thattransmitthevirus.
Inconclusion,wesuggest thatmigratorybirdsof largebodymass may provideameansformonitoring WNV prevalenceonalargegeographical scale(e.g. migratoryflyways).Additionally,residentspeciesof largebodymassmayprovideabetterdescriptionof
localWNVprevalence.Further, thepastallegations regardingagreaterprobability ofinfectionbyWNV byparticulartaxonomicgroups shouldbemore carefullyexploredgivenourfindingsandthefact thatmany ofthosestudies werebasedonthe examination of only a few species, and on dead birds,makingdifficult toseparatetheeffectsof exposure,susceptibilityandcarcassdetectionprob- ability.Fromaconservation standpoint,itperhaps wouldbemorebeneficialtofocusourattentiononthe effectsofWNVonspeciesofgreaterbodymassandon migratoryspecies.
Acknowledgements
Spanish MinistryofHealthnetworkEVITAR (G03/059),JuntadeAndaluc´ıa (JA)projects RNM118,RNM157,C03-059,andEuropeanCom- missionsupported thisstudy.Thisresearchwas partially)fundedbyEUgrantGOCE-2003-010284
EDENandthepaperiscatalogued bytheEDEN SteeringCommittee asEDEN95( fp6project.net/). Thecontentsofthispublicationare theresponsibilityoftheauthorsanddonotnecessarily reflect theviewsoftheEuropeanCommission. PermitsweregivenbyJA,ParqueNacionalyNatural deDon˜ana,andParajeNaturalMarismasdeOdiel. SpecialthankstoO.Gonza´lez,M.Va´zquez,andE. Garc´ıaforalltheirhelp.EquipodeSeguimientode ProcesosNaturales, GrupoZamaya,andGosur assistedinthecaptures. P.RodriguezandM.Adrian allowedustoworkontheirproperties.J.L.Arroyo,F. Carro,J.J.Chans,L.Garc´ıa,F.Iba´n˜ez,B.Ja´n˜ez,M. Man˜ez,R.Rodr´ıguez,J.L.delValle,B.Ramos,J.C. Rubio,C.Sa´nchez, J.M.Sayago,M.Vega,other anonymousvolunteersandJAhelpedinthefield. C. DomingoandN.Reyesgaveusastandardizedstockof WNvirusandconfirmed independentlythespecificity oftheneutralization assaywithrespecttootherWN relatedflaviviruses.
AppendixA.Supplementarydata
Supplementarydataassociatedwiththisarticlecan befound,intheonline version,atdoi:10.1016/ j.vetmic.2008.04.023.
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