Screening ofActiveLyssavirusInfection inWildBatPopulations byViralRNA DetectiononOropharyngealSwabs
JUAN E.ECHEVARR´IA,1*ANAAVELLO´N,1 JAVIERJUSTE,2 MANUELVERA,1 AND CARLOS IBA´N˜EZ2
CentroNacionaldeMicrobiolog´ıa,InstitutodeSaludCarlosIII,28220Majadahonda, Madrid,1 andEstacio´n
Biolo´gicadeDon˜ana,ConsejoSuperiordeInvestigacionesCient´ıficas,41013Seville,2 Spain
Brainanalysiscannotbeusedfor theinvestigationof activelyssavirusinfectioninhealthybatsbecause mostbat speciesare protectedbyconservation directives. Consequently, serologyremainstheonlytoolfor performingvirologicalstudies onnaturalbatpopulations;however,thepresenceofantibodiesmerely reflectspastexposuretothevirusandisnotavalidmarkerofactive infection.Thisworkdescribesanew nestedreversetranscription(RT)-PCRtechniquespecificallydesignedforthedetectionoftheEuropean batvirus1onoropharyngealswabsobtainedfrombatsbutalsoabletoamplifyRNAfromtheremaining rabies-relatedlyssavirusesinbrainsamples.Thetechniquewassuccessfullyusedfor surveillanceofa serotine bat(Eptesicusserotinus)colonyinvolvedinacase ofhumanexposure,inwhich15outof71 oropharyngealswabswerepositive.Lyssavirusinfectionwasdetectedon13oropharyngealswabsbutin only5brainsoutofthe34animalsfromwhichsimultaneousbrainandoropharyngealsampleshadbeen taken. The lyssavirus involvedcould be rapidly identified byautomatic sequencing of the RT-PCR productsobtainedfrom14brainsandthreebatoropharyngealswabs.Inconclusion,RT-PCR using oropharyngeal swabswillpermitscreeningofwildbatpopulationsfor activelyssavirusinfection,for researchorepidemiological purposes, in line not onlywithconservationpolicies butalso in a more efficientmannerthanclassical detectiontechniquesusedonthebrain.
Rabies iscausedbydifferent rhabdovirusesincluded within thegenusLyssavirus.Landmammalsareinfectedworldwideby theclassicalrabiesvirus(RABV) orserotype1,aswellasbats inAmerica(11).Thisviruscausesnearlyall thehumancasesof rabies inthe world.AfewAfrican land mammals havebeen foundtobeinfectedbyadifferent lyssavirus,theMokolavirus (MOKV) (serotype 3)(25).Thehostsfortheremaining lyssa- virusesare non-Americanbats:Lagosbatvirus(LBV) (sero- type2)(25)andDuvenhagevirus(DUVV)(serotype4)(29)in Africa,theAustralian batvirus(ABV)(proposedasgenotype
7)inAustralia(12),theEuropeanbatvirustype1(EBV1),and Europeanbatvirustype2(EBV2) (16)inEurope.Both Eu- ropean batviruseswereformerlyclassifiedtogether asDUVV; however,theyhaverecentlybeendividedintotwo different species (3, 10, 16, 19). Phylogenetic reconstructions show closerrelationshipsbetweenEBV1andDuvenhagevirusthan between EBV1andEBV2.Inaddition, EBV2showsacloser relationshipwithserotype1 thanwithEBV1(4).Twodifferent subgenotypeshaverecentlybeendescribedfor each(1).The reservoirofEBV1is theserotine bat(Eptesicusserotinus[Ves- pertilionidae]),whileEBV2isfoundsofarinthespeciesofthe genusMyotis(Vespertilionidae),MyotisdasycnemeandMyotis daubentonii)(1).Other Europeanbatspecieshaverarelybeen found infected bythese viruses(6). More than 500infected batshavebeenfoundinEuropesincethefirstcasewasfound in1957(20),andmore than 95%ofthebatscorrectlyidenti- fiedwereserotines, presumably infected byEBV1(20).How- ever,thefirsthumancasewascausedbyEBV2in1985(17,19).
* Correspondingauthor. Mailingaddress: Centro Nacional deMi- crobiolog´ıa,Instituto deSaludCarlosIII,Ctra.Majadahonda-Pozuelo s/n,28220Majadahonda,Madrid, Spain.Phone: 34-91-5097901.Fax:
34-91-5097966.E-mail:.
The remaining two known cases were caused byEBV1, in Ukraine in1977andRussiain1985(19,24).Allthree human cases were fatal. Hundredsof Europeanswere exposed to rabid batsafter 1985(20),butallreceived postexposurepro- phylaxisandnone wereinfected.
Arapiddiagnosisoflyssavirusinfection shouldbemadeon the animal brain after anyhuman exposure. Direct antigen detection byimmunofluorescence(IF) isthemostwidespread screening method (18).Results are usuallyconfirmed bythe mouse inoculation testorbyviralisolation onmurine neuro- blastoma cells(18).More recently,RNA detection byreverse transcription(RT)-PCRhas been proposed as a rapid and sensitive alternative (13,14,15,21,23,26,31).However, as RT-PCRisnotfasterthanIF,andrabidanimalbrainsusually contain high amounts of virus, very few laboratories have adoptedthistechnique. Ontheother hand,directsequencing ofRT-PCRproducts isthemostcommon techniqueforvirus identification andinmolecular epidemiologystudies(1,5,27). MostEuropeanbatspeciesareprotected(8)andthebrain cannot beusedforthescreening ofactivelyssavirusinfection innatural bat populations.For thisreason, most studies are basedexclusivelyonserology(22,28);however,totalantibody presence merelyreflects past exposure tothe virusand does not demonstrateactiveinfection. Moreover, conclusiveiden- tificationofthelyssavirusinvolved,basedonserologicaltech-
niques, isnotpossibleduetocross-reactivity.
Inthepresent work,anewPCRmethod is describedfor detection of RNA from all known rabies lyssaviruses, with further virusidentification bygenomicsequencing. The pres- enceofviralRNAonbatoropharyngealswabsasamarker of activelyssavirusinfection isevaluated inabatcolonyinvolved inacaseofhuman exposure.
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FIG. 1. Primer sequences and mismatches withthe different rabies-relatedlyssaviruses:RABV (genotype 1),LBV (genotype 2),MOKV (genotype 3),DUVV (DVHV)(genotype 4),EBV1a (subtype a,genotype 5),EBV1b(subtype b,genotype 5),EBV2a (subtype a,genotype 6), EBV2b(subtype b,genotype 6).Sequences ofprimers LISEBL1F, LISEBL1R, LISEBL2F, andLISEBL2R areshown.
MATERIALSANDMETHODS
Samples.Group 1consistedof12RNAextractsfromallsevenrabies-related lyssavirusesdistributed asfollows:OneRABV(CVSstrain),twoLBV,three MOKV, one DUVV, twoEBV1, twoEBV2, and one ABV. Allwere kindly donatedbyJ. Smith from the Centers for Disease Control and Prevention (Atlanta,Ga.).Group 2included47brainsfromdifferent mammals(25bats,15 dogs,sixcats,andonehorse)fromtherecordscenteroftheCentro Nacionalde Microbiolog´ıa (Majadahonda,Madrid, Spain). Nineteenbrains had previously hadpositiveresultsforrabiesantigendetection byIF,andpositiveconfirmation, either usingthe mouse inoculation test orbyvirusisolation inmurine neuro- blastoma cells(30).Fourteenbrains(from ninedogs,fourcats,andonehorse) camefromtheNorthAfricanSpanishcitiesofCeutaandMelilla,whiletheother fivewere from bats from the southernIberian Peninsula. The remaining 28 brainshadpreviouslyhadIF-negative results.Finally,group3 consistedof71 oropharyngealswabsand39brainsfrom69different serotine bats(E.serotinus) capturedorfound dead between June 1999andAugust 2000andbelonging to thesamecolonyinapublicbuildinginSeville(Andalusia, Spain).Sixbatswere capturedtwiceduring follow-up.Thiscolonywasstudied after ahuman being wasbitten bya bat which tested positive for rabies antigen byIF and viral isolation fromthebrain.Thepatient receivedadequatepostexposureimmuno- prophylaxis and remains symptom free at present. Another37samples (one brainand36 oropharyngealswabs)from36 batscapturedorfounddeadinother areas ofSevillewerealsoincluded forcomparison.
Collectionoforopharyngealsamples.Batsweremist-nettedattheexitsused bythe animals forleavingthe building fornight feeding. After capture, each animalwasaged,sexed,measured,weighed,andringidentified. Oropharyngeal samples weretaken withdrycotton swabsstored intubes containing 1.5mlof lysisbuffer (see below) for transportand daytime storage. On arrival at the laboratory,swabswere applied tightlyto the tube wallsand the liquids were divided into twodifferent aliquots whichwerefrozen to—80°Cuntil analysis. Eachswabwasstored withoneofthealiquots. Batswereusuallyreleased after sampling.
Immunofluorescence.Thiswasperformedfollowingstandardprocedures(9). Ammon’shorn andcerebellum wereexamined forlandmammals, whilelongi- tudinal sections of the entire encephalonwere used for bats. At least two
impressionsfromdifferent areaswereobservedbeforegivinganegativeresult.A commercial RABV-derived fluorescent conjugate(Sanofi-Pasteur, Marnes la Coquette,France)wasusedforimmunologic staining.After 1999,apan-lyssa- virus-specificmonoclonal antibody,kindlydonatedby J.CoxfromtheWHO CollaboratingCenter forRabies SurveillanceandResearch(Tu¨bingen,Germa- ny),wasusedinaddition forbatspecimens.
RNAextraction.Brainsampleswerehomogenizedwithsterilizedglassgrind- ersandresuspendedinminimalessentialmedium.RNAwasextracted fromthe samplesasdescribedpreviously(7).Briefly,50llofeachsuspensionwastreated with200llofaguanidiniumthiocyanate extraction buffer,followedbyisopro- panol and 70%ethanol precipitations.AnRNA plasmid, supplied aspositive controlintheAccessRT-PCRkit(Promega, Madison,Wis.),wasincludedinthe extraction bufferaspart ofaninternal control system(seebelow)ataconcen- tration of20 molecules/ll. Pelletsresultingfromthefinalcentrifugationwere resuspendedin10 ll ofdistilledwaterandusedimmediately.Fororopharyngeal swabs,500ll ofsample wasdirectly treatedwith500ll ofisopropanol,con- tinuing theprocedureasbefore.
Primerdesignandpreparation.Sequences ofthenucleoproteingeneofeach rabies-relatedlyssaviruswereobtainedfromgenomicdatabasesandalignedby usingtheMacawprogram (National Center forBiotechnology Information, Bethesda, Md.).Externalandnestedprimersequenceswerechosenfromregions conserved among allrabies-relatedlyssaviruses;however,nucleotides matching withEBV1wereselected invariablepositions (Fig.1).Fortheinternal control system,the 1UPS and 1DSprimers supplied inthe AccessRT-PCRkit(Pro- mega) asapart ofthe positive control system were used asnested primers. External primers were chosen from the plasmid sequence supplied inthe kit insert(CONINT1F,5’CTGGCCTGTTGAACAAGTCT3’;CONINT1R,5’ GATCTGATCCTTCAACTCAGC3’).Primersynthesiswasundertakenby a commercial customer service(PharmaciaBiotech, Freiburg, Germany).
Reversetranscription,amplification,andproductdetection.Single-step ret- rotranscriptionandprimaryamplification wereperformedusingtheAccessRT- PCR kit(Promega).Fivemicroliters ofextracted sample wasadded toanRT- PCR mixturecontaining 10llof5Xreaction buffer;3mMmagnesium sulfate; dATP,dCTP,dGTP,anddTTP,eachataconcentrationof500 lM;LISEBV1F, LISEBV1R, CONINT1F,andCONINT1Rprimers, eachataconcentrationof
FIG. 2. RT-PCRresultsforbatsamples.Theupper band(323bp) istheinternal controlband.Thelowerband(117bp)isthelyssavirus- specificband.Lanes1,2,4to9,and11arelyssavirusnegative,lane10 islyssaviruspositive, lane 3has presence ofenzyme inhibitors, and lanes12 and13 arenegativeandpositivecontrols(withnointernal control).
FIG. 3. Results ofthe PCR test onallrabies-related lyssaviruses. Lanes1and2,LBV;lanes3to5,MOKV;lane6,DUVV; lanes7and
11,EBV1;lanes8and9,EBV2;lane10,ABV;lane12,RABV(CVS strain); lane13,negativecontrol; lane14,molecular sizemarker (123 bp;LifeTechnologies, Gaithersburg, Md.).Nointernal controlwas included inthisassay.
0.2lM;5Uofavianmyeloblastosisvirusreversetranscriptase;5UofThermus flavusDNApolymerase; andRNase-freedistilledwatertoafinalvolumeof50 ll. All reagents except primers were supplied in the kit. Amplification was performedinanAutocyclerplus(Linus,Cultek,Madrid, Spain)thermal cycler, programmedforafirstretrotranscriptionstepof45minat48°C,followedbytwo minat94°Cforreversetranscriptaseinhibition andcDNAdenaturation,and30 repetitivecyclesof1minofdenaturationat93°C,1minofannealingat60°C,and
1minofelongation at72°C.Elongationwasextended for5additional mininthe lastcycle.FornestedPCR,1lloftheprimaryamplificationproductswasadded toanewPCR mixture containing 5llofmagnesium-free10Xreaction buffer (Roche Diagnostics GmbH); 3mMmagnesium chloride; dATP, dCTP, dGTP, anddTTP(PharmaciaBiotech), eachataconcentrationof500lM;LISEBV2F and LISEBV2R primers, each at aconcentrationof0.5lM; 1UPS and 1DS primers,eachataconcentration of0.2lM;1.25UofAmpli-TaqThermus aquaticusDNApolymerase(Roche DiagnosticsGmbH); anddistilledwatertoa finalvolumeof50ll.Thermal cycleswereperformed asbeforebutskippingthe retrotranscriptionstepandusing94°Cfordenaturationand50°Cforannealing. ThePCR products weresizedbygelelectrophoresisin2%agarose containing
0.5gofethidium bromide permlofTBE(Tris-borate-EDTA)bufferandseen under UV light.Standardprecautionsweretaken toavoidcarryover contami- nation. Pipetting wasperformedwithaerosol-resistanttips,and different bio- safetycabinets wereusedformaster mixpreparation,sample andextract han- dling,andnestedreaction. Product detection was undertakeninadifferent area. Samplesshowingboththe323-bpinternal control band andthe117-bplyssavi- rus-specificbandwereconsideredpositive;thoseshowingonlytheinternal con- trolbandwereconsiderednegative;thoseshowingnobandweretestedagainand consideredtocontain enzymeinhibitors, ifnobandwasobserved onrepetition (Fig.2).All samplesshowingpositiveresultsweretestedagain.Inthecaseof oropharyngealexudates, these repetitionsweremade from adifferent aliquot. Onlysampleswithrepetitive resultswerefinallyconsideredpositive.Concentra- tionsofmagnesium, deoxyribonucleotides,andprimerswereoptimized forboth reactions, aswellasdenaturationandannealing temperatures.
Sequencing.First-amplification 262-bpbandsweresequenced forlyssavirus species(genotype)identification. First-amplificationproducts weremixedwith anequal volumeofammonium acetate andprecipitated,firstwithisopropanol andthen with70%ethanol. Finalpelletswereresuspendedin10llofdistilled water.Thesequencingreaction wasperformed withtheABIPRISM bigdye sequencing kit(Applied Biosystems,Foster City,Calif.),followingthemanufac- turer’s indications.Bothforwardandreversestringsweresequenced using LISEBV1FandLISEBV1Ras sequencingprimers,respectively.Sequencing reactions wereperformedinaPTC200(MJResearch,Watertown,Mass.)ther- malcyclerand consisted ofafirst-denaturationcycleof3minfollowedby25 cyclesof10sofdenaturationat96°C,10sofannealing at50°C,and4minof elongation at60°C.Products werepurified bysubsequent80and70%ethanol precipitations.Finalproducts wererunonanABIPRISM 377DNAsequencer (Applied Biosystems).Forward andreversestrainswerefittedusingtheSeqman program ofthe DNASTARpackage (DNASTARINC,Madison, Wis.).Some
landmammal brainsdidnotshowvisiblefirst-amplification bands,andnoneof the bat oropharyngealswabsshowed these, despite their being positive after nested reaction. Reverse transcription-firstamplification reaction wasrepeated asbefore onlandmammal samples,butusingnewprimers withRABV instead ofEBV1 specificnucleotides onvariable positions. Thisreaction wasalsore- peated onbatoropharyngealswabs,butusingother primers (SEQ1F, 5’AAG ATTGTRGAACACCACAC;SEQ1R 5’GCATTGGATGAATAAGGAGA) externaltoLISEBV1F andLISEBV1R. Thenestedreactionwasthenperformed asbeforebutusingLISEBL1FandLISEBL1Rinsteadof LISEBV2Fand LISEBV2R. Sequencing wasperformedasabove, when visible262bp bands wereobtained.
Thereadable220-bpfragments obtainedfromtheautomaticsequencerwere alignedasabove,together withrepresentativestrainsofallrabies-relatedlyssa- virusesobtainedfromgenomic databases. One sequence fromeach ofFrench RABV(genotype1),Moroccan RABV(genotype1),LBV(genotype2),MOKV (genotype 3),DUVV (genotype 4),EBV1a(genotype 5),EBV1b(genotype 5), EBV2a (genotype 6),andEBV2b(genotype 6)wasincluded forcomparison.
RESULTS
RNAextractsfromthedifferentlyssaviruses(group1).PCR products ofthe expected sizewereobtainedforallsevenra- bies-related lyssaviruses (Fig. 3). First-amplificationbands wereapparentforallsamplesexceptfortheLBVinlane1,the DUVV inlane6,andtheABVinlane10.
Brainsamplesfromanimalrabiesdiagnosis(group2).RT- PCR results weretotallycoincident withprevious results. All IF-negative sampleswereRT-PCRnegativeandallIF-positive brainswereRT-PCRpositive.
Batsamplesfrom abatcolonyinvolvedinhumanexposure toEBV1(group3)(Table1).Afterthehumanexposurecasein June1999,27additional animalswerecapturedbetweenJune andAugust,andall testednegative.However,onebatfound moribund inSeptembertested positiveinboth thebrain and ontheoropharyngeal swab,asdidthebrainfromanother moribund batfoundinanotherpartofthecitysomedayslater. Thislastanimalwastheonlyonethatshowedpositiveresults forboth antigen detection and RT-PCRusingthe brain, de- spitebeingnegativeontheoropharyngealswab.Asbatsmoved to hibernationshelters, no more captures could bemade in
1999.Oropharyngealswabsfrom4of12additional batscap- turedon21May2000testedpositivebyRT-PCR. Twoofthese RT-PCR-positiveanimals died during capture. Both brains
TABLE 1. DistributionovertimeofRT-PCRresultsobtainedafter follow-upofaserotine batcolonyfromapublicbuildingafter acaseof human exposure toarabidbatfromthiscolonye
SpecimenResult
No.ofsampleswithresult attimepoint
June 1999 July1999 Aug1999 Sept1999 Dec1999 May2000 June 2000 July2000 Aug2000
Total
BrainPos.1a001b(1)02c 100 5 (1) Neg. 4b 2b 0 1 1b 0 19 7 0 34
NA04170010 (13)0(22)536 (13)
Oropharyngealexudates / Pos. / 0 / 0 / 0 / 1 / 0 / 4 / 10 / 0 / 0 / 15Neg. / 1 / 6 / 17 / 1 (1) / 1 / 8 (13) / 10 / 7 (22) / 5 / 56 (36)
NA / 4 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 4
Total captures56172 (1)112 (13)20d7d(22)575 (36)
aBatcausinghuman exposure.
bBatfounddead onthefloorofthebuilding.
cDeath duringcapture andsampling.
dSacrificedafter sampling.
eResults inparenthesesrefer tobats capturedinlocations other than the publicbuilding. NA,not available; Pos.,positive;Neg.,negative; Aug,August; Sept, September;Dec,December.
wereRT-PCRpositive,andoneofthem wasalsoIFpositive. Thirteenoropharyngealswabsfrom other bats capturedthe sameweekinanotherpartofthecityshowednovirus.Asthe riskforthehumanpopulationwasconsideredhigh,healthand conservationauthoritiesagreedtoremovethebatcolonyfrom the public building. Twenty bats were capturedand slaugh- tered after being sampled inJune 2000.Ten ofthese tested positive for RT-PCRon oropharyngealswabs,but onlyone testedpositiveinthebrain.Thisbrainwashighlypositiveusing IF.Theonlybatknowntobepositivebutstillaliveafter the May2000campaign wascapturedagaininJune. Itremained positiveontheoropharyngealswab,althoughittestednegative inthebrain.Thelast12batscapturedinJulyandAugust2000 tested negative, aswellas22additional batscapturedatan- other locationveryclosetothepublicbuilding.PCRproducts fromsixsamples(three brainsandthreeoropharyngeal swabs) taken atdifferent timesweresequenced forlyssavirusidenti- fication(seebelow).AllwereclassifiedasEBV1(genotype 5). Tosummarize, only5(15%)ofthe33brainsfrombatswith simultaneousoropharyngealsamplewereRT-PCRpositive.In contrast, viruswasdetectedin13(39%) oropharyngeal exu-
dates (Table 2).
Lyssavirus identification(genotyping).Alllyssavirus strains from land mammals were classified as RABV (genotype 1) (Table 3).Incontrast, allbat-derived strainswereclassifiedas EBV1(genotype5). Homologies betweenthesamegenotype ranged from93.6to100%forRABV andfrom89.5to99.5% for EBV1. These values ranged from 68.6 to 77.7% for
TABLE 2. Results obtainedwithbatsforwhichsimultaneousbrain andoropharyngealsampleswereobtaineda
RABV1,andfrom61.8to80.5%forEBV1,comparedwiththe other lyssaviruses.Thus,everystrain couldbeeasilyassigned toonelyssavirusspeciesaccording tonucleotide homology.
DISCUSSION
The RT-PCRmethod described here isable to detect all rabies-relatedlyssaviruses.OnlyfiveofallpreviousPCR-based methodsforrabiesdiagnosishaveasimilarly widerangeof specificity(2,13,14,26,31),and onlytwoofthese provide virusidentification byproduct sequencing(13,14).Astheonly batlyssavirusknownsofarinSpainis EBV1,theprimers described herewereoptimized forthedetection ofthispartic- ularvirus, andsensitivitytotheotherlyssavirusesmaybe suboptimal. Infact,noband wasobtainedafter firstamplifi- cationforsomeRABV-infected brains,despitetheirhighly positiveIFimages.Variable positionsshouldbedegenerated, ormixturesofindividualvirus-specificprimersshouldbeused, toachieveabetter pan-lyssavirusamplification method. Prim- erswerechosenfromconservedpositionstoensure thedetec- tionofallindividualEBV1variants.
Aninternal control systemwasincluded toavoidfalse-neg- ativeresultsineachindividualtube,duetohandlingerrors, or thepresence ofenzymeinhibitors. Inoursamplingdesign,the RNA plasmid wasincluded inthe extraction buffer, and this wasusedasatransportmedium fortheoropharyngealswabs. Therefore,theinternal control systemusedhere madeitpos- sibletomonitorthewholeprocessfromas earlyas sample collection. Onlyone ofthe previous methods includes anin- ternal control system(26).However, inthisstudyrRNA was usedasatarget instead ofthecontrolled lownumber ofplas-
midmoleculesusedhere.Astheamount ofrRNA isexpected
Result forbrain
No.withresult for
oropharyngealexudateTotal no.
PosNeg
tobehighinclinicalsamples,low-gradeRNAlossesorenzyme inactivationcouldbemissedandfalsenegativeresultscouldbe shown.
Pos415
Neg91928
Total132033
aAbbreviations: Pos,positive;Neg,negative.
Both RT-PCRand antigen detection usingIF showed the samerateofefficiency fordetection oflyssavirusesinwell- preservedanimalbrains.However,as themost-widespread commercial antisera are derived fromRABV, reactivitywith other rabies-relatedlyssavirusesisusuallypoorer. Infact,one
TABLE 3. Homology between samplessequenced inthisworkandsequences ofdifferent rabies-related lyssaviruses obtainedfromgenomicdatabasesa
Virusb
Dog
%Homology withisolatefrom:
Horse
Bat
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8RABV (France) / 93.6 / 94.1 / 94.1 / 95 / 95 / 95 / 94.5 / 93.6 / 94.5 / 77.7 / 77.3 / 77.7 / 77.3 / 77.7 / 69.1 / 77.3 / 77.3
RABV (Morocco) / 100 / 98.2 / 98.2 / 99.1 / 99.1 / 99.1 / 99.5 / 100 / 98.6 / 75.5 / 74.5 / 75.5 / 75 / 75.5 / 66.8 / 75 / 75
LBV / 75 / 75.5 / 75 / 75 / 75 / 75 / 75.5 / 75 / 74.5 / 75 / 74.1 / 75.5 / 75 / 75 / 66.4 / 75.5 / 75.5
MOKV / 68.6 / 70 / 70 / 69.5 / 69.5 / 69.5 / 69.1 / 68.6 / 69.5 / 70.5 / 70 / 70 / 70 / 70.5 / 61.8 / 70.5 / 70.9
DUVV / 75.5 / 75 / 76.8 / 75.9 / 75.9 / 75.9 / 75.9 / 75.5 / 75.9 / 78.6 / 77.7 / 79.1 / 78.6 / 78.6 / 69.5 / 78.6 / 78.2
EBV1a / 75 / 76.4 / 76.4 / 76.4 / 76.4 / 76.4 / 77.3 / 75 / 76.4 / 98.2 / 97.3 / 97.7 / 98.6 / 98.2 / 89.5 / 97.7 / 97.7
EBV1b / 76.4 / 77.7 / 77.7 / 77.7 / 77.7 / 77.7 / 74.5 / 76.4 / 77.7 / 99.5 / 99.1 / 99.1 / 99.1 / 99.5 / 90.9 / 99.1 / 99.1
EBV2a / 73.6 / 74.1 / 74.5 / 75 / 75 / 75 / 75.5 / 73.6 / 75 / 80.5 / 80.9 / 80.9 / 80.5 / 80.5 / 70.5 / 80.5 / 80
EBV2b / 74 / 75 / 75.5 / 75.9 / 75.9 / 75.9 / 75.5 / 74.5 / 75.9 / 80 / 80.5 / 80.5 / 80 / 80 / 70 / 80 / 79.5
aAllstrainswereobtainedfromthebrain exceptforthose frombats6,7and8,whichwereamplifiedfromoropharyngealswabs.
bForthefollowingviruses,EMBL accessionnumbers aregiveninparentheses:RABV (France)(U22474), RABV (Morocco) (U22852), LBV(U22842), MOKV (U22843), DUVV (U22848) EBV1a (U22844), EBV1b(U22845), EBV2a (U22847), EBV2b(U22846).
ofthelyssavirus-positivebatbrainswas missedafterafirst examination withone ofthese reagents. Thissample showed fewbutclearfluorescence imageswhentested withanoncom- mercialmonoclonal antibody, andlyssavirusRNA wasclearly amplified bynested RT-PCR. Nevertheless,noband wasob- servedafter primary amplification, whichsuggestsalowviral load. Other worksshowthat RT-PCRismore efficient than antigen detection fordegraded samples(13).
Mostofthepreviousinformationaboutthelyssavirusinfec- tion inwildbat populationswasobtainedfromserology(22,
28)anddirectvirusdetection inthebrain(28).However,brain analysiscannotbeusedforhealthyindividualsbecauseallbats throughouttheEuropean Union areprotected (8),asinmany othercountries. Ascollectionoforopharyngeal exudatesis harmless forbats, RNA detection forthisspecimen seems a validalternative forthedetection ofactivelyssavirusinfection inwildpopulations.Infact,allbatswithneurological infection showed virusinoropharyngealexudates asexpected, incon- cordance withclassicpatternsofrabiespathogenesis,inwhich braincolonization fromperipheralnervesprecedes centrifuge disseminationofthevirustothesalivaryglands(11).Theonly bat showinginfection inthe brain but not oropharyngealex- cretion wastheonewithanapparently lowamount ofvirusin thebrain(seeabove).Itmayhavebeencapturedbefore cen- trifugedissemination fromthebraintothesalivaryglands. However, mostofthe batswithvirusonoropharyngeal swab andavailablebrainsampleshowednovirusinthebrain.Con- sequently, theviruswasunable toreachthesalivaryglandsby axonalspread fromthe brain, andthe infection inthese ani- malsdidnotfollowtheclassicpatternofrabiespathogenesis. Someprevious workshaveshownalowactiveinfection rate, despite highRABV (28)orhighEBV1 (22)antibody preva- lenceinhealthybatpopulations.Somering-identified individ- ualswereevencapturedaliveyearsaftertheyhadtested pos- itiveforlyssavirusantibodies (22).Allthese data suggestthat the clinicalexpression ofthe EBV1 and RABV infection in batsisusuallyamild,nonfatal extraneurologicaldisease.This couldexplainthegreater efficiencyoftheoropharyngealswab comparedtothebrainfordetection ofactiveEBV1infections inbats,asshowninthisstudy.Onlyanimalswithneurological disease canbedetectedbytheusingthebrain. However, the
useoftheoropharyngeal swabalsoallowsdetection ofhealthy carriers ofEBV1,whichisofmajorepidemiologicalinterest. Tosumup,theRT-PCRdescribed hereisaneffectivecom- plement toIFforprimarydiagnosisofrabiesinanimals,also providing rapid identification ofthe lyssavirusbyautomatic sequencing oftheproducts. Theuseofthistechniqueonbat oropharyngealswabs,incombination withantibody detection, willpermit highlyefficientmassscreening ofwildbatpopula- tionsforlyssavirusinfection, aswellasconforming tocurrent conservationpolicies.Thisnewapproachwillpermit notonly newbasicresearch studiesbutalsosurveillanceofbatcolonies
forepidemiologicalpurposes.
ACKNOWLEDGMENTS
We thankJeanSmithfromtheCentersfor DiseaseControland Preventionfor supplying RNA extracts from rabies-relatedlyssavi- ruses.Wealsothank Carlos Ru´iz, Juan LuisGarc´ıa, Juan Quetglas, andElena Migensfortheir helpwithbatcapture andsampling.
Thisworkwassupportedby“Fondo deInvestigaciones Sanitarias” project98/0945and“InstitutodeSaludCarlosIII”grant1364/99,both fromtheSpanishMinistryofHealth, aswellasby the“Delegacio´n ProvincialdeMedio Ambiente” ofSevilleandthe“Cabildo Catedra- licio”ofSeville.
REFERENCES
1. Amengual,B.,J.E.Whitby,J.King,A.Serra-Cobo,andH.Bouhry.1997.
Evolution ofEuropeanbatlyssaviruses.J.Gen. Virol.78:2319–2328.
2. Black,E.M.,L.M.McElhinney,J.P.Lowings,J.Smith,P.Johnstone,and P. R.Heaton. 2000. Molecular methods to distinguish between classical rabies and the rabies-relatedEuropeanbat lyssaviruses.J.Virol.Methods
87:123–131.
3. Bourhy,H.,B.Kissi,M.Lafon,D.Sacramento,andN.Tordo.1992.Anti- genic characterizationof bat rabies virus in Europe.J. Clin. Microbiol.
30:2419–2426.
4. Bourhy,H.,B.Kissi,andN.Tordo.1993.Molecular diversityofthelyssa- virusgenus.Virology194:70–81.
5. Bourhy,H.,B.Kissi, L.Audry,M.Smreczak,M.Sadkowska-Todys,K.
Kulonen,N.Tordo,J.F.Zmundzinski,andC.Holmes.1999.Ecologyand evolution ofrabies virusinEurope.J.Gen. Virol.80:2545–2557.
6. Brass,D.1994.Rabies inbats. Natural history and publichealth implica- tions.LiviaPress,Ridgefield, Conn.
7. Casas,I.,L.Powell,P.E.Klapper,and G.M.Cleator.1995.Newmethod for theextractionofviralRNAandDNAfromcerebrospinalfluidforuseinthe polymerase chainreaction assay.J.Virol.Methods 53:25–36.
8. CounciloftheEuropeanCommunities.1992.Directive 92/43/EEC of21
May1992ontheconservationofnatural habitats andwildfaunaandflora. Off.J.Counc. Eur. Communities1992:7–50.
9. Dean,D. J.,M.K.Abelseth,andP. Atanasiu.1996.Thefluorescent antibody test, p. 88–95.InF. X.Meslin, M. M. Kaplan, and H. Koprowski (ed.),
Laboratorytechniques inrabies, 4thed. World Health Organization,Ge- neva,Switzerland.
10. Diezschold,B.,C.E.Ruppretch,M.Tollis,M.Lafon,J.Mattei,T.Wiktor, andH.Koprowski.1988.Antigenicdiversityoftheglycoproteinandnucleo- capsidproteinsof rabiesandrabiesrelatedviruses:implicationsforepide- miologyandcontrol ofrabies.Rev.Infect. Dis.10(Suppl. 4):S785–S787.
11. Dietzschold,B.,C.E.Ruppretch,Z.F.Fu,andH.Koprowski.1996.Rhab- dovirusesinvirology,p.1137.InB.N.Fields,D.M.Knipe,andP.M.Howley (ed.), Fieldsvirology.Lippincot-RavenPublishers, Philadelphia,Pa.
12. Fraser,G.C.,P.Hooper,R.A.Lunt,A.R.Gould,L.J.Gleeson,A.D.Hyatt, G.M.Russell,andJ.A.Kattenbelt.1997.Encephalitiscausedbyalyssavirus infruitbatsinAustralia. Emerg. Infect. Dis.2:327–331.
13. Heaton,P.R.,P.Johnstone,L.M.McElhinney,R.Cowley,E.O’Sullivan, andJ.E.Whitby.1997.HeminestedPCRassay fordetection ofsixgenotypes ofrabies andrabies related viruses.J.Clin.Microbiol. 35:2762–2766.
14. Heaton,P.R.,L.M.McElhinney,andJ.P.Lownings.1999.Detectionand identification ofrabies andrabies related virusesusingrapid cyclePCR. J. Virol.Methods 81:63–69.
15. Kalmovarin,N.,T.Tirawatnpong,R.Rattansiwamoke,S.Tirawatpong, T.
Panpanich,andT.Hemachudha.1993.Diagnosis ofrabies bypolymerase chainreaction withnested primers. J.Infect. Dis.167:207–210.
16. King,A.,P.Davies,andA.Lawrie.1990.The rabies virusesofbats. Vet.
Microbiol. 23:165–174.
17. Lumio,J., M. Hillbom, R. Roine, L.Ketonen, M. Haltia, M. Velle,E.
Neuvonen,andJ.Lahdevirta.1986.Human rabiesofbatorigininEurope. Lancet i:378.
18. Meslin,F.K.,andM.M.Kaplan.1996.Anoverviewoflaboratory tech- niques inthediagnosis andprevention ofrabies andinrabies research, p.
9–27.InF.X.Meslin,M.M.Kaplan, andH.Koprowski(ed.), Laboratory techniques inrabies, 4thed.WorldHealth Organization,Geneva, Switzer- land.
19. Montan˜o-Hirose,J.A.,H.Bourhy,andM.Lafon.1990.Areduced panelof anti-nucleocapsidmonoclonal antibodies forbatrabiesvirusidentification in Europe.Res.Virol.141:571–581.
20. Mu¨ller, W.W.,J.H.Cox,andK.P.Hohnsbeen (ed.). 1998.Review of reportedrabies casedata inEuropetothe W.H.O.collaboratingcenter
Tu¨bingenfrom1977to1998.Rabies BulletinEurope4/1998.WHO Collab- orating Center, Tubingen, Federal Republic ofGermany.
21. Nadinavis,S.A.1998.Polymerase chainreaction protocols forrabies virus discrimination.J.Virol.Methods 75:1–8.
22. P´erez-Jorda´,J.L.,C.Iba´n˜ez,M.Mun˜oz-Cervera,andA.T´ellez.1995.Lys- savirusinEptesicusserotinus(Chiroptera,Vespertilionidae).J.Wildl.Dis.
31:372–377.
23.Sacramento,D.,H.Bouhry, andN.Tordo.1991.PCR techniqueasan alternative method fordiagnosisandmolecularepidemiologyofrabiesvirus. Mol.Cell.Probe 5:229–240.
24. Selimov, M. A.,A.G.Tatarov,A.D.Botvinkin,E.V.Klueva,L.G.Kulikova, andN.A.Khismatullina.1989.Rabiesrelated Yulivirus:identification with apanel ofmonoclonal antibodies. ActaVirol.33:542–545.
25. Shope,R.E.,F.A.Murphy,A.K.Harrison,O. R.Causey,R.E.Kemp,D.L.
Simpson,andD.L. Moore.1970.TwoAfricanvirusesserologicallyand morphologically related torabies virus.J.Virol.6:690–692.
26. Smith,J.,L.M.McElhinney,P.R.Heaton,E.R.Black,andJ.P.Lowings.
2000.Assessment oftemplatequalityby theincorporationofaninternal controlintoaRT-PCRforthedetection ofrabiesandrabies-relatedviruses. J.Virol.Methods 4:107–115.
27. Smith,J. S.,andD.Seidel.1993.Rabies:anewlookatanolddisease.Prog.
Med.Virol.40:82–106.
28. Steece,R.,andJ.S.Altenbach.1989.Prevalence ofrabiesspecificantibodies intheMexicanfreetailed-bat(Tadaridabrasiliensismexicana)atLavacave, NewMexico.J.Wildl.Dis.25:490–496.
29. Tignor,G.H.,F.A.Murphy,H.F.Clark,R.E.Shope,P.Madore,S.P.
Bauer,S.M.Bucley, andC.D.Meredith.1977.Duvenhagevirus:morpho- logical, biochemical, histopathological,and antigenic relationshipsto the rabies serogroup. J.Gen. Virol.37:595–611.
30. Webster,W.A.,andG.A.Casey.1996.Virusisolationinneuroblastomacell culture, p.96–104.InF.X.Meslin,M.M.Kaplan,andH.Koprowski(ed.), Laboratorytechniques inrabies, 4thed. World Health Organization,Ge- neva,Switzerland.
31. Whitby,J.E.,P.R.Heaton,H.E.Whitby,E.O’Sullivan,andP.Johnstone.
1997.Rapid detection ofrabies andrabies related virusesbyRT-PCRand enzyme-linked immunosorbentassay.J.Virol.Methods 69:63–72.