Interferon批处理for

Viruses2014,6,4880-4901;doi:10.
3390/v6124880virusesISSN1999-4915www.
mdpi.
com/journal/virusesCommentaryImmunologyofBatsandTheirViruses:ChallengesandOpportunitiesTonySchountzArthropod-borneandInfectiousDiseasesLaboratory,DepartmentofMicrobiology,ImmunologyandPathology,CollegeofVeterinaryMedicine,ColoradoStateUniversity,FortCollins,CO80524,USA;E-Mail:tony.
schountz@colostate.
edu;Tel.
:+1-970-491-7350;Fax:+1-970-491-8326ExternalEditor:PatrickC.
Y.
WooReceived:6October2014;inrevisedform:21November2014/Accepted:28November2014/Published:8December2014Abstract:Batsarereservoirhostsofseveralhigh-impactvirusesthatcausesignificanthumandiseases,includingNipahvirus,Marburgvirusandrabiesvirus.
Theyalsoharbormanyothervirusesthatarethoughttohavecauseddiseaseinhumansafterspilloverintointermediatehosts,includingSARSandMERScoronaviruses.
Asisusualwithreservoirhosts,thesevirusesapparentlycauselittleornopathologyinbats.
Despitetheimportanceofbatsasreservoirhostsofzoonoticandpotentiallyzoonoticagents,virtuallynothingisknownaboutthehost/virusrelationships;principallybecausefewcoloniesofbatsareavailableforexperimentalinfections,alackofreagents,methodsandexpertiseforstudyingbatantiviralresponsesandimmunology,andthedifficultyofconductingmeaningfulfieldwork.
Thesechallengescanbeaddressed,inpart,withnewtechnologiesthatarespecies-independentthatcanprovideinsightintotheinteractionsofbatsandviruses,whichshouldclarifyhowthevirusespersistinnature,andwhatriskfactorsmightfacilitatetransmissiontohumansandlivestock.
Keywords:chiroptera;reservoirhost;virusecology;zoonosis;emerginginfectiousdisease;RNA-seq;metabolomicsOPENACCESSViruses2014,648811.
IntroductionBatsbelongtoChiroptera,thesecond-mostspecies-richorderofmammalswithmorethan1100species[1].
Genomicevidencesuggeststhatbatssharemostfeaturesofothermammals[2–4];however,theyhaveotheranatomicandbehavioralfeaturesthatappeartobeuniqueandsomeofthosemaybeessentialforhostinginfectiousagents.
Inparticular,thesizesofcolonies(sometimesinthemillions),torporofsomespecies,theirmutualgroominghabits,gregarioussocialbehavior,andpopulationdensitieslikelyfacilitatetherapidspreadofinfectiousagentsamongbats[1,5–8].
Infectionofanyorganismhasaninherentcosttothehostandinfectiousagent,andabalanceofhostresponseandvirusreplicationisessentialforestablishmentofareservoirhost/virusrelationship.
Thus,itislikelythatbatsandtheirviruseshaveco-adaptedinarelationshipthatlimitsdisease,butalsoimpairsantiviralresponses.
Batsalsoaresusceptibletoinfectiousdiseasesthatcanhavedramaticimpactsontheirhealthandecology.
Whitenosesyndrome,causedbythefungusPseudogymnoascusdestructans,haskilledmillionsofbatsineasternNorthAmerica,threateningsomespecieswithextinction[9–11].
Bartonellaspecieshavebeenisolatedfrommanybatspecies[12–16],althoughtheimpactofthesebacteriaonhumanorveterinaryhealthisunclear.
Severalotherviral,bacterial,protozoal,nematodeandtrematodespecieshavebeenassociatedwithdiseaseanddeathsofbats.
Herpesviruses,Pasteurellaspp.
,Salmonellaspp.
,Yersiniapseudotuberculosis,Burkholderiaspp.
,CedeceadavisaeandClostridiumsordelliihavebeenisolatedfromordetectedindeadbats[17–19].
ArtibeusbatsarenaturallysusceptibletoHistoplasmacapsulatum.
ExperimentalinfectionofA.
literatuswithH.
capsulatumresultedinfatalhistoplasmosisinsomebatsbutchronicsystemicinfectioninothers,suggestingthisspeciescanactasareservoirbutalsodevelopdisease[20],similartowhatisobservedinrabiesvirusinfectionsofbats.
Twoimportantfeaturesofbatsthatareuniqueamongmammalsarepoweredflight,whichpermitslargehomeranges,includingoverlargebodiesofwaterthatcanbetransitedinrelativelyshortamountsoftime,andnocturnalactivity[21].
Thesefeaturespresentdifficultiesforstudyingbatsandtheirinfectiousagentsunderfieldconditions.
Ideally,closedlaboratorycoloniesofbatsshouldbedevelopedtofacilitatemodeldevelopment,butsuchcoloniesaredifficulttoestablishbecauseofthelogisticsofcapturing,quarantiningandtransportingbats(oftentootherhemispheres),theexpenseofadequatefacilitiesforbreeding,theknowledgeandexpertiseofveterinarycarestaff,andthelowfecundityofbats.
Together,thesefeaturesmakequalitybatinfectiousdiseaseresearchdifficultandexpensive.
Becauseoftheselimitations,moststudiesontherelationshipsofbatsandtheirvirusesrelyuponvirologicalandserologicaltestingofnaturalpopulationsofbats.
Thispresentsseveraldifficultiesininterpretingdatabecauseitisfrequentlyimpossibletoknowwhenagivenbatwasinfected,ifitwasinfectedwithotheragentsthatmightconfoundinterpretationofdata,orifhormonesorenvironmentalstressorswereinfluencingimmuneresponses.
Althoughmorethanahundredviruseshavebeendetectedin,orisolatedfrom,bats[1,22–29],onlyafewbatspecieshavebeenidentifiedthatarereservoirsorsuspectedreservoirsofinfectiousagentsthathavehighimpactonhumanhealth(Table1).
Nonetheless,thelargepopulationsizesofbats,huntingofbatsasfoodsources,agriculturalandlivestockpractices,andencroachmentofhumansuponbathabitatwilllikelyleadtocontinuedspillovereventsandoutbreaksofdisease[30,31].
Viruses2014,64882Table1.
Highimpactvirusesandtheir(suspected)batreservoirhosts.
VirusDiseaseReservoirHostRabiesvirusandotherlyssaviruses[1,32]RabiesManybatspecies,world-widedistributionMarburgvirus[33]MarburgvirusdiseaseEgyptianfruitbat(Rousettusaegyptiacus)Ebolaviruses[34]EbolavirusdiseaseHammer-headedbat(Hypsignathusmonstrosus),Franquet'sepaulettedfruitbat(Epomopsfranqueti),littlecollaredfruitbat(Myonycteristorquata)1SARS-CoV[26]SevereacuterespiratorysyndromeChinesehorseshoebat(Rhinolophusspp.
)2MERS-CoV[35]MiddleEastrespiratorysyndromeEgyptiantombbat(Taphozousperforatus)2NipahandHendraviruses[36,37]EncephalitisCertainflyingfoxes(Pteropusspp.
)Sosugavirus[38]SevereAcuteFebrileDiseaseRousettusspp.
11Suspectedreservoirs;2NucleotidesequencesfoundwithhighsimilaritytoSARSorMERScoronaviruses.
Asisusualwithreservoir/virusrelationships,virusesmust:(a)infectandpersistintheirbathostswithoutcausingsubstantialdisease;(b)betransmittedtoothersusceptiblehostsbeforetheimmuneresponseeliminatestheinfection;or(c)thehostdiesfromtheinfection[1].
Forthesereasons,manymicrobialagents,particularlyviruses,haveevolvedimmune-evasionstrategiesthatmanipulatethehostresponseinamannerfavorabletothevirus[39–42].
Theseimmuneevasionmolecules,mostofwhicharevirally-encodedaccessoryproteins,alsoaffectotherspecies,suchashumans,afterspilloverevents.
However,becausethemoleculesareevolutionarily-optimizedforthereservoirhosts,theylikelybehavedifferentlyinthenonreservoirhostsandmaycontributetopathogenesis.
Nearlyalllaboratoryworkexaminingimmune-modulatingproteinsfrombatvirusesisperformedwithimmortalizedrodent,humanornon-humanprimatecelllines.
Thedifficultyininterpretingthesedataisthatnocomparisonshavebeenmadetothereservoirhostcells.
NipahvirusP,VandWproteinstargettheSTAT1pathwaysinhumancells[43],buttheseviralproteinshavenotbeenexaminedincellsfromPteropusgiganteus,anaturalreservoirofNipahvirus.
Iftheybehaveidenticallyinhumansandbats,theirrolesinpathogenesisofhumandiseaseissuspect.
Experimentalexpressionofviralproteinsinmammaliancellsoftenusesviralgenesclonedintomammalianexpressionvectorsthatproduceabundantviralproteinthatisprobablynotreflectiveofthelevelsfoundincellsinfectedwithvirus[42–45].
Thispresentsanotherinterpretationdifficultybecausehostresponsesofreservoirscanbefine-tunedbyevolutionandsubtlequantitativechangesingeneexpressioncandramaticallyimpactinfectiousoutcomes[46].
Itmaybethatactivitiesoftheseviralproteinsarequantitativelydifferentincellsfromreservoirhoststhanwhatoccursinhumancells,andthesedifferencesmayexplainwhyhumansdevelopdiseasewhereasthereservoirdoesnot.
Experimentsexaminingthefunctionsoftheseproteinsshouldusevectorsthathavecontrollablepromotors,suchasthosemediatedbytetracycline,sothatquantitativeeffectscanbemeasuredandcomparedbetweenprimarybatcellsandcellsfromhumanstoidentifydifferencesincellularresponsesthatmightaccountforpathology.
Viruses2014,648832.
ViralDiseasesofBatsPathogenisarelativeterm.
Allzoonoticvirusesare,bydefinition,pathogenictohumans;however,theyareusuallynon-orminimally-pathogenicinthereservoirhosts.
HendraandNipahvirusesdonotcauseclinicaldiseaseinflyingfoxes[47],andMarburgvirussimilarlycausesonlysubclinicaldiseaseinEgyptianfruitbats[48,49].
Severalrecentpublicationshavesuggestedthatbatsarereservoirhostsofzoonoticvirusesbecausetheyaresomehowspecialrelativetoothermammalianspeciesinthattheyaresusceptibletoinfectionbutwithoutdevelopingdisease[50–52].
Butthereareseveralinfectiousdiseasesofbats,someofwhicharediscussedbelow,anditislikelythatmanymoreas-yetundiscoveredinfectiousdiseasesaffectbats.
Moreover,rodentreservoirsofzoonoticvirusesremainpersistentlyinfected,oftenforlife,withoutsignsofdisease,includingthosethathosthantavirusesandarenaviruses[53–56].
Ofthosethathavebeencloselyexamined,aroleforregulatoryTcellsmaycontributetopersistentinfectionwithoutdiseasetotheseotherwiseinnocuousviruses[57,58].
Whethersimilarimmunemechanismsoccurinreservoirbatsthatmayaccountforpersistencewithoutdiseaseisunknown.
2.
1.
RabiesItisbeyondthescopeofthisworktoadequatelydiscussrabiesvirusinfectionsofbats;however,manyexcellentreviewsofrabiesvirusandotherlyssavirusinfectionsofbatshavebeenpublished[59–61].
Fewstudieshavethoroughlyexaminedthehostresponseofbatsinfectedrabiesvirus.
Turmelleetal.
[62]experimentallyinfectedbigbrownbats(Eptesicusfuscus)withrabiesvirusandfoundthatmostsurvivedfortheseveralmonthsofthestudy.
Moreover,thedatasuggestedthatabortiveinfectiondidnotprovidedurableimmunityinbatsandthatrepeatedinfectionscouldcontributetoresistancetodisease.
Consideringthehighdensitiesofwildbatcolonies,repeatedinfectionsprobablyoccurfrequentlyandmayprovideamechanismforresistancetorabiesinbats.
Persistenceofvirusinthisworkmayhavebeenlinkedtolowvirusreplicationthatfailedtostimulatedangersignalstotheimmuneresponse,permittinglong-terminfectionwithoutsignsofdisease.
Davisetal.
[63]experimentallyinfectedlittlebrownbats(Myotislucifugus)withrabiesvirusesandfoundthatintramuscularinfectionledtorapidclinicalprogressionofrabies,whereassubcutaneousinfectionledtodelayedclinicalprogression.
Notably,batsthatdevelopedrabiesaftersubcutaneousinfectionweremorelikelytoshedvirusinsalivaryglands.
Together,thedatasuggestedthatrouteofinfectionofrabiesvirusesamongbatscaninfluenceclinicaloutcomeandpotentialtransmissionamongbats.
2.
2.
TacaribeVirusTacaribearenavirus(TCRV)wasfirstidentifiedintwospeciesofartibeusbatsinTrinidadandTobagointhemid-1950s[64].
WilburDowns,whowasdirectoroftheTrinidadRegionalVirusLaboratory(TRVL),foundanear-deadgreatfruit-eatingbat(Artibeusliteratus)onthefrontporchofhishomethathadbeencapturedbyhispetcat[65].
ThebatexhibitedsignsofrabiesbutsubsequenttestingexcludedrabiesbecauseofthelackofNegribodies.
Tissuehomogenatesoftheunidentifiedagentwereinoculatedintosucklingmiceandcauseddisease.
Duringthenext3years,19isolatesofTCRVweremadefrom6A.
literatusand5A.
jamaicensisdiseasedbatscollectedontheislandofViruses2014,64884Trinidad;however,onlyonestrain,theoriginalTRVL-11573,remains.
DownsproposedthatartibeusbatswerereservoirhostsofTCRV.
However,experimentalinfectionofA.
jamaicensiswithTRVL-11573resultedintwodistinctoutcomes;fataldiseasewithsimilaritiestotheSouthAmericanhemorrhagicfeverswithinthreeweeks,ornosignsofdiseaseandwithoutsubstantialviralburdenorantibodyresponses[66].
ThisworkarguesagainstartibeusbatsasreservoirhostsofTCRV.
Abrahametal.
showedthatasingleaminoacidchangeinhumantransferrinreceptorconferredsusceptibilityofhumancellstoTacaribevirus[67].
TacaribevirusiscloselyrelatedtoJunínvirusandMachupovirus[68],theetiologicagentsofArgentineandBolivianhemorrhagicfevers,respectively.
ArtibeusbatsarefoundthroughoutthetropicalAmericas[69]anditisthoughtthatclimatechangeisexpandingtheirdistribution.
ThebatsarecurrentlyendemictotheFloridaKeysandareexpectedtoexpandtheirrangetomainlandFlorida,wherehighhumanpopulationdensityandagriculturalpractices(fruitorchards)mayprovideanopportunityforspilloverandemergenceintohumans.
AtleastonelaboratoryinfectionofTCRVhasoccurred,resultinginflu-likesymptomsandfeverfollowedbyseroconversionwithintwoweeks[70].
Thus,TCRVhasatleastsomeemergingzoonoticdiseasepotential.
2.
3.
LloviuVirusIntheearly2000s,alargenumberofSchreiber'sbent-wingedbats(Miniopterusschreibersii)werefounddeadincavesintheIberianPeninsula,theborderregionofSpain,PortugalandFrance.
Genesequencesofanovelfilovirus,Lloviuvirus,weredetectedinall5Schreiber'sbatscollected(throatandrectalswabs,brain,liver,lung)inacavenearCantabria,Spain,butinnoneof9deadMyotismyotisbatscollectedatthesite[71].
Theauthorsspeculatedthedie-offwasaresultofLloviuvirusinfection;however,withoutacolonyofSchreiber'sbatsithasnotbeenpossibletofulfillKoch'spostulates.
3.
BatsasReservoirHostsofImportantHumanPathogensThemostnotableexamplesofhumanpathogenicviruseshostedbybatreservoirsarerabiesvirusandotherlyssaviruses,filoviruses,henipavirusesandcoronaviruses.
Virusesfromthesegroupshavehaddramaticimpactsforthediseasestheycause;however,manymorevirusesalsooccurinbatsandtheseposethreatsasfutureemerginginfectiousdiseases[22,27,28,72,73].
Ineachofthesebat/virusrelationships,thevirusescauselittleornoharmtothehost,orcantakemonthstoyearstomanifestdisease[1].
Thesegroupsofhigh-impactagentsareRNAvirusesand,presumably,thehighererrorrateofRNApolymerasesfacilitatesvirusadaptationtonewhosts.
Forexample,rabiesvirusgenotypesarefrequentlyconstrainedwithintaxonomicgroupsofvertebratehosts(e.
g.
,batgenotypes,foxgenotypes),suggestinghostbiologyand/orimmunologyinfluencesviralevolution[63,74,75].
Hypothesesastowhybatsharborsomanyinfectiousagentsbutwithoutdiseasehavebeenproposed.
Onesuchhypothesisisthatelevatedmetabolicbodytemperaturesfromflight,mimicsthefeverresponse[51].
IncreasedbodytemperaturefromexertionisaresultofconsumptionofATPthatincreasesmitochondrialactivitiesthatmayfacilitatehostdefenses[76]andcontributetoreservoirstatusofbats[52].
However,thefeverresponsethatoccursduringinfectionispartoftheimmunologicalcascadeinitiatedbyinfectionorinflammation,inwhichinflammatorycytokines,suchasinterleukin-1,stimulatetheproductionofprostaglandinsthatactuponthehypothalamustoincreasebodytemperature[77,78].
Bythetimefeverfromaninfectionoccurs,theimmuneresponsehasalreadybeguninearnest.
Viruses2014,648854.
BatImmuneResponsestoVirusesVirtuallynothingisknownaboutbatimmuneresponsestoviruses.
Muchoftheexperimentalefforthasbeenconductedwiththeblackflyingfox(Pteropusalecto),areservoirofHendravirus,intheBaker,WynneandWanglaboratoriesatCSIRO[2,4,79–86]andrecentlyreviewedingreatdetailalongwithotherworkonbatimmunology[87].
Asnon-modelorganisms,manyobstaclesinhibitanunderstandingofbatimmuneresponsestoviruses.
Althoughlittleexperimentalworkhasbeenperformed,substantialgeneticandbiochemicalevidencesuggestsbatsaresimilar,butnotidentical,toothermammalsintermsoftheimmunologicalgenesandnoncodingRNAstheypossess[2–4,88],inferringimmuneresponsesthataresimilartoothermammals.
4.
1.
PatternRecognitionReceptorsNucleatedcellshavemanydefensivesystemsforcontainmentofinfectiousagents,andmanyinfectiousagentsencodeproteinstocircumventthesesystems[89,90].
Thiscoevolutionaryarmsraceislargelyresponsibleforshapinghostresponsesandlikelyhasbeenineffectsincetheemergenceofthefirstpathogens.
Infectiousagentsoftensharecommonbiochemicalmotifs,termedpathogen-associatedmolecularpatterns(PAMP),suchaslipopolysaccharides(LPS),lipoteichoicacid,flagellin,ordouble-strandedRNAthatarenotpresentinvertebrates[91].
Consequently,proteinsthatcounteractthesePAMPsarehighlyconservedinvertebrates.
ThepresenceofviralPAMPsincellsandtissuesisaninnatecuethattriggersanumberofcellulardefensemechanisms[92].
Allorganismshaveproteins,termedpatternrecognitionreceptors(PRR),thatsensethepresenceofcertainPAMPsandinduceamplifyingcascadesofhostcelldefensesystems,leadingtophysiologicalterationofthecellandexpressionofavarietyofantiviralgenes.
FourbroadgroupsofPRRshavebeenidentifiedinmammals;Toll-likereceptors(TLRs),retinoicacidinduciblegene-I-likereceptors(RLHs),nucleotide-bindingoligomerizationdomain-likereceptors(NLRs),andtheinterferon-inducibleabsentinmelanoma2(Aim2)[91,93,94].
ManyPRRshavebeenidentified,mostthroughscansofgenomeortranscriptomedatathatarecommonlyfoundinother,well-studiedmammals(e.
g.
,humans,laboratoryhousemice)andsuggestbatsusethesesamesystemsforsurveillanceofinfectiousthreats.
TLRgeneshavebeenclonedfromtwobatspecies,theblackflyingfox(Pteropusalecto)[95]andLeschenault'srousettebat(Rousettusleschenaultii)[96],andarefoundingenomedatafromthelittlebrownbat(Myotislucifugus)andtranscriptomedatafromtheJamaicanfruitbat(Artibeusjamaicensis)[3].
However,therearenoreportsofactivityoftheseproteinsinbatsorbatcellsinfectedwithviruses.
ThreeRLHgenes,Ddx58(RIG-I),Ifih1(MDA5),Dhx58(LGP2),havealsobeenclonedfromtheblackflyingfox[80],butnofunctionalstudieshavebeenperformedwithvirally-infectedbatcells.
ThreeNLRmembers(Ciita,Nod1,Nod2)arefoundinsomeofthebatgeneticdatabases.
However,inthetwobatspeciescloselyexamined,P.
alectoandMyotisdavidii,thePHYINlocus,whichcontainstheAim2andIfit16genesinothermammals,isabsent[4],andorthologousAim2sequencesarenotfoundinatranscriptomeofA.
jamaicensis[3].
Viruses2014,648864.
2.
Interferons4.
2.
1.
TypeIIFNMultipletypeIinterferon-α(IFNα)genesexistinmammaliangenomesthatarosethroughtandemduplications[97].
Ifnbhasonlyasinglecopyinspeciesthathavebeenexamined.
TheseIFNproteinsareexpressedasdownstreameventsofviralinfectionandthePRRcascade.
TypeIinterferonshavebeenfoundinseveralbatspeciesandinfectionofbatcellswithvirusesinducesthetypeIIFNpathway.
Virtueetal.
[82]infectedacelllinefromP.
alectowithhenipavirusesandfoundtheyantagonizedthetypeIIFNresponse.
Biesoldetal.
[98]generatedcelllinesfromtheembryonicandadulttissuesofseveralbatspeciesandimmortalizedthemwithSV40largeTantigengene.
CellswereinfectedwithRiftValleyfevervirusstrain13,whichisdefectiveinitstypeIIFNrepression,andfoundthatbatcellsexpressedIfnb30-foldhigher.
SupernatantsfromthesecellsexhibitedantiviralactivitiesinaVSVreportersystem,demonstratingbiologicalactivityofbatIFNβ.
Ifnwlociarealsopresentinbatgenomesandappeartohavemanytandemcopiesinspeciesexamined,unlikemostothermammals[87].
FunctionalactivitiesofbatIFNωhavenotbeendescribed.
4.
2.
2.
TypeIIIFNInterferon-γ(IFNγ)sequenceshavebeenidentifiedintwopteropid(Pteropusalecto,P.
vampyrus)andoneNewWorld(Myotislucifugus)species[81,87].
Ifngr1andIfngr2arefoundinA.
jamaicensissuggestingthey,too,possessanIfnggene[3].
Aswithotherphylogeneticstudiesofbatgenes,theyhavesomesimilaritytoIFNγofungulates.
IFNγfromP.
alectoimpairsviralreplicationinbatcells,reducingHendravirusreplicationinP.
alectocellsandalsoinhibitsSendaivirusreplicationinaBrazilianfree-tailbat(Tadaridabrasiliensis)lungcellline,indicatingbroadspeciescrossreactivity[81].
4.
2.
3.
TypeIIIIFNThetypeIIIinterferons(IFNλ)arealsoinvolvedinantiviralresponses[99].
Zhouetal.
[100]producedrecombinantP.
alectoIFNλandexamineditsabilitytoinduceIfit1(Isg56)andDdx58(RIG-I),andtointerferewithTiomanvirus-mediatedcelllysis.
Ifit1recognizes5'triphosphate-RNAproducedbysingle-strandedRNAviruses.
TheyfoundelevatedIFNλexpressioninP.
alectosplenocytesinfectedwithTiomanvirus.
IFNλalsoinhibitedPulauvirusreplicationanddramaticallyincreasedexpressionofIfit1and,toalesserextent,Ddx58inaP.
alectocellline.
IFNβandIFNλinducedexpressionofP.
alectoMx1,aGTPasethatmaytargetviralnucleoproteins,andOas1,whichactivatesRNaseLanddegradationofviralRNA,butnotPkr[86],andthatP.
alectosplenocytesarehighlysensitivetoIFNλstimulation[100].
ItislikelythatbattypeIandtypeIIIIFNsinduceexpressionofmanyotherantiviralgenesand,consideringtheirproperties,thatvirusesnaturallyhostedbybatshavecountermeasuresthattargetproteinsoftheIFNpathways.
Viruses2014,648874.
3.
ResponsesofInnateImmuneCellsandLymphocytesSpecializedimmunecellsplayessentialrolesinthehostresponsetoinfection.
Severaldendriticcellsubsets,macrophages,neutrophilsandNKcellsareresponsibleforearlycontainmentofinfectiousthreats.
Whilemorphologicaldescriptionsofsomeofthesecellsandgenomic/transcriptomicevidenceoftheirexistenceinbatshavebeenmade,noworkhasbeenperformedtoexamineresponsesofthesebatcellstoviruses.
BatsalsohaveTandBcellsasevidencedbycellularpurification,theproductionofhightiteredIgG[101–104](whichrequiresTcellhelp)andsequencesinthevariousbatgenomeandtranscriptomedatasets.
However,onlyantibodyresponseshavebeenexaminedbyserologicalassays.
Noinvitroorexvivoworkonantigen-specificTcellsorBcellshavebeenreported.
Asignificantlimitationofconductingresearchintobatlymphocyteresponsestovirusesisthelackofclosedcoloniesforexperimentalinfectionwork,suitablegrowthfactorsforexpansionofantigen-specificlymphocytes(e.
g.
,IL-2,IL-4,GM-CSF,Flt3L,etc.
),andthemethodologiesforbatlymphocyteculture.
IgGisotypesandcopynumbervarybetweenbatspeciesbutexhibitevidenceofsubstantialdiversity[79,105].
OnlyasingleIgGisotypehasbeendetectedinCarolliaperspicillata,whereasEptesicusfuscusappearstohavetwoIgGisotypes,andMyotislucifugushasfiveIgGisotypes[106].
AnotherunusualfeatureofM.
lucifugusisthatithasaveryhighdiversityofVDJHlocusbutlittleevidenceofsomatichypermutation.
Thismayrepresentanevolutionaryalternativeforgenerationofantibodydiversity.
Itisunknownifthesedifferenceshaveimmunologicalsignificance.
5.
ApproachesforStudyingBatImmuneResponsesManysignificantchallengesforunderstandingimmuneresponsesofbatsexist.
Alackofclosedcolonies,thegeneticdiversityofbats(betweenandwithinspecies),fewimmunological,molecularorbiochemicalreagentsandcelllines,thelargenumberofbatspeciesandtheirviruses,andthedifficultyofworkingwithbatsallcontributetothesechallenges.
However,noveltechnologiesandinstrumentationthataresubstantiallyspecies-independentarenowavailable,particularlyRNA-Seqandmetabolomics,thatcanfacilitatetherapiddiscoveryofbat-virusinteractions.
Proteomicstoolsaresubstantiallylimitedbecausemanyoftheserequireligand-specificantibodiestotargetproteinsthatarelargelyunavailableforbats;however,combineduseoftranscritomeandliquidchromatography/massspectrometryanalysismayhelpcircumventthislimitation[84,107].
Someantibodiesarecrossreactivewithbatproteins,butmostappeartobetowardshighlyconservedintracellularproteinssuchasthoseinvolvedintheantiviralresponse.
Fujiietal.
showedthatantibodiestoSTAT1andpSTAT1Y701arecrossreactivewithRousettusaegyptiacusSTAT1[108],andtheseantibodiesmaybeusefulforexamininghowMarburgvirusVP40interfereswithEgyptianfruitbatSTAT1activities[109].
ConsideringSTAT1'sconservation,itislikelytheseantibodiesarealsocross-reactivewithSTAT1fromotherbatspecies.
5.
1.
HusbandryFewbreedingcoloniesareavailableforuseinbatresearch,principallybecauseofalackofsourcesforobtainingbats,expertiseinveterinarycare,adequatefacilitiesandthecostofmaintainingcolonies.
Mostzoosandzoologicalorganizationsareunwillingtoprovidebatsforbiomedicalresearchpurposes,Viruses2014,64888andthispolicyhassignificantlystymiedresearchintobatimmunologyandimpairsunderstandingofinfectiousdiseasesthatkillbats,includingwhitenosesyndrome.
Theestablishmentofclosedbreedingcoloniesforresearchpurposesisanessentialcomponentfordevelopingcontrolled,hypothesis-drivenresearch(Figure1).
Figure1.
Bathusbandryandmanipulations.
Establishmentofbatcoloniesforexperimentalresearchrequiresenrichmentforsuccessfulmaintenanceandreproduction.
(A)Landscapefabricattachedtowallsprovidesroostingsubstrate;(B)Hangingoffruitonskewerscanprovideenrichmentforfrugivorousbatsbystimulatingforagingbehavior.
Awingband(green)ispresentwithauniqueidentificationnumber;(C,D)Hangingofinvertedwickerormetalbasketsprovideadditionalroostingsubstrates;(E)AfemaleSeba'sfruit-eatingbat(Carolliaperspicillata)withoffspring;(F)Bloodcollectionfromabatwing;(G)Oralswabforviruscollection.
PhotosarebytheauthorandareofthecolonyattheUniversityofNorthernColorado[3,66,106].
Viruses2014,64889Free-flightroomsarehighlydesirableforwelfareand,perhapsrequired,forsuccessfulbreedingcoloniesofmostbatspecies.
Theabilitytoflyinanenclosureisprobablynecessaryforthesocialhealthofthebatsandwilllikelyimprovereproductivesuccess.
Thedecisionofwhichbatspeciestouseisdrivenbythevirus-of-interest,andhusbandryrequirementsarefrequentlyspecialized.
Ingeneral,fruitbatsareeasiertocolonizethanareinsectivorousbats,whichmustlearntoeatnon-flyinginsects.
Enrichmentisalsonecessaryforthewelfareofcolonizedbats,includingartificialroosts,hangingoffruitfromwallsorceilingstostimulateforagingbehavior,andplacestohide,suchaslandscapefabrichangingfromawall.
Forexperimentalinfections,batsmayrequirehousingincagesthatmaypreventflight.
ManyofthevirusesofinteresthostedbybatsrequireBSL-3orBSL-4containment,whichlimitsthenumberofinstitutionswheresuchworkcanbeperformed.
Despitethesedifficulties,managingcoloniesofcertainspeciesofbatsforresearchpurposesisreadilyattainable.
5.
2.
RNA-SeqDeepsequencingapproaches[110]forexaminationoftranscriptionalprofilesofbatsandbatcellsinfectedwithvirusescanshedlightonbiochemicalpathwaysthatareactivatedorrepressed,thusinformaboutthehostresponseandpotentialcellulartargetsofviralevasion.
ThecostsofRNAsequencing(RNA-Seq)havebecomeconsiderablylessinthelastseveralyears.
Improvementsininstrumentationhaveincreaseddepthofcoverage,makingthepricepergigabaseevenlessexpensive.
ThecostofcDNAlibraryconstructionhasnotsubstantiallyimproved,butcanbedoneforaboutUS$100persample.
Moreover,preparationofRNAandlibrariesfornucleargenetranscripts,noncodingRNAs(e.
g.
,miRNA)andvirusesrequireddifferentmethodologies.
Transcriptsofsomegenesthatencodepotentproteins,suchascytokines,areoftenexpressedatverylowlevelsandhaveshorthalf-lives[111]thatcanmaketheirdetectiondifficultorimpossible,evenwithdeepercoveragethatismoreexpensive.
Forthesegenes,real-timePCRisnecessarytoidentifydifferencesingeneexpression.
Nonetheless,RNA-Seqoffersinvestigatorsapowerfultooltogeneratelargeamountsofdata,regardlessofthebatspeciesofinterest.
AsignificantcomplicatingfactorofRNA-Seqistherequirementforsubstantialcomputationalresourcesandexpertise.
AssemblyofreadsfromvertebrateRNA-Seqdatarequiresmultiprocessor/multicorecomputers,hundredsofgigabytesofRAM,terabytesofharddrivecapacityandfamiliaritywithLinuxoperatingsystems.
Whilemanyinstitutionshavesuchcomputationalresources,othersdonot.
Fortunately,twosuchresourcesthatuseTrinity(describedbelow)arepubliclyavailable;thePittsburgSupercomputerCenter(https://www.
psc.
edu)andtheDataIntensiveAcademicGrid(http://www.
diagcomputing.
org/).
Thus,thelackofcomputationalresourcesbyresearchgroupsisnotasignificantobstaclesolongasinternetaccessisavailablefordatatransferandawillingnesstowaitforsampleprocessingbecausequeuetimescanbelong.
Ingeneral,whenworkingwithaspeciesthatdoesnothaveanannotatedgenomeavailableitisnecessarytogeneratedenovoassembliesofalltheRNA-Seqsampledatafromanexperiment.
Bydoingso,oneensuresthateverytranscriptandisoformisrepresentedintheassembleddataset.
SeveralRNA-Seqassemblyprogramsareavailableinthepublicdomain,includingTrinity[112]andOases[113].
TheseprogramsaredesignedforRNAassembly;otherapplicationsareavailableforDNAassembly.
Eachprogramhasadvantagesanddisadvantages,soitisessentialtocarefullyexaminepackagestodetermineViruses2014,64890whichismostsuitedforagivendataset.
Oncethedenovoassemblyiscompleted,othersoftwaretoolsareemployedtocountreadsfromeachsampleforuseinstatisticalanalysisoftranscriptandisoformabundances.
Severalabundanceestimationpackagesarefreelyavailable,includingtheBioconductorpackage(composedofedgeRandDESeq)[114,115],andRSEM[116]andeXpressannotation[117]thatprovidequantitativeandqualitativestatisticalanalysisofdifferentially-expressedgenes.
Figure2.
Reactomemappingofdifferentially-expressedgenes.
Abundanceofdifferentially-expressedIFNγpathwaytranscriptsinPiritalarenavirus-infectedhamsterliversrelativetouninfectedgroupwereestimatedbyRSEMandstatisticallyanalyzedwithDESeq.
ThesymbolsfortheelevatedgeneswereimportedintotheReactomepluginofCytoscapeforpathwayanalysis.
Genesinpurpleboxeswereelevatedininfectedanimals(author'sunpublisheddata)andsuggeststhispathwayisimportantinthehostresponsetothevirus.
Itisnotuncommonformorethanathousandgenestobedifferentiallyexpressedinanexperimentandidentificationofthosetranscriptsisrequiredforsubsequentanalysis.
BatchBLASTisusedtoidentifytranscriptsusingtheRefSeq_RNA,mir-BaseandIgProtdatabases,andmanyBLASTtoolsareavailable,includingweb-based,commandlineandGUIprogramsthatarefreeorcommercial.
MosttranscriptswillbeidentifiedbyBLAST,includingsomenoncodingRNAs;however,manyotherswillViruses2014,64891havenohitsinthedatabasesandthesetranscriptscanbedifficulttointerpret.
Determiningtherolesofdifferentially-expressedgenescanbereadilyidentifiedusingDAVID[118]andReactome[119](remotelyorfromwithinCytoscapesoftware[120]).
Theseapplicationsprovidepathwayinformationaboutproteins,includingotherproteinsinthosepathways(Figure2).
Thesepackagesareessentialforrapidlyidentifyingpotentialviraltargetsduringinfectionandcansuggestwhichpathwaysshouldbefurtherscrutinized.
5.
3.
Real-TimePCRAlthoughRNA-Seqcanprovidebroadinformationaboutexpressionofgenesandpathwaysthatmaybeimportantinhostresponsestoviruses,itislimitedinitsabilitytoidentifygenesexpressedinverylowabundances.
Real-timePCRisamaturetechnologythatcanbemoresensitivethanRNA-Seqandisrapidlydeployableformultiplexexaminationofgeneexpression[46,121].
However,becausefewbatgenomeandtranscriptomedatasetsareavailable,obtainingsequencesofgenesofinterestcanbechallenging.
Someimmunegenesarehighlyconserved(e.
g.
,Tnf)whereasothersarelessso(e.
g.
,Ifng).
BecauseRNA-Seqoftenmissesthesetranscripts(becausetheirabundancesaretooloworwerenotexpressedinthetissuefromwhichtheRNAwasextracted)itmaynotbesuitableforobtainingsequencedatafordevelopingprimersetsforgenesofinterest.
Thereareotherapproachesforobtainingsequencesofunknowngenes,principallydegeneratePCRusingprimersdesignedfromorthologoussequences;however,thisapproachoftenfailstoamplifyspecificproducts.
Sequencingofgenomesisoneapproachtomitigatingthisproblemanditscosthasalsodecreasedsubstantially.
Theprincipaladvantageofgenomesequencingisthatallgeneticinformationisessentiallypresentindatasets,unlikeRNA-Seqwheremanygenesarenotdetected.
Newtoolsarenowavailablethatcanrapidlymineunannotatedgenome(andtranscriptome)datathatperformsinsilicoassemblyofcDNAsequencesforautomatedprimerdesign[121].
Withsufficientgenomesequencingcoverage,thisapproachidentifiesnearlyallgenes,manyofwhichmaybeabsentfromRNA-Seqdata.
5.
4.
MetabolomicsMetabolomicsisarecently-emergingfieldthatcanprovideinsightintobiochemicalactivitiesduringinfectionsandimmuneresponses,andisbaseduponthedetectionofspecificmetabolites[122–124].
Forbatresearch,thiscanprovidevaluableinformationbecausemanymetabolitesareidenticalorsimilar,regardlessofspecies.
Becausethebiochemicalpathwaysforproducingthesemetabolitesareoftenconservedamongspecies,profilingprovidesinformationonhostmetabolicresponsestoviralinfections.
Somevirusesremodelthemetabolismofinfectedcellsfortheproductionofprogenythatmaycontributetopathogenesis.
Forexample,hepatitisCvirusaltersphospholipidandsphingomyelinabundanceandleadstoelevatedceramides,whicharetoxictohepatocytes[125].
Theselipidsarereadilydetectedbymetabolicprofilingandsuchapproachesmaybeappliedtostudiesofbatsandtheirviruses.
5.
5.
ImmuneCellCultureSubstantialinformationaboutimmuneresponsescanbegarneredfromstudiesofantigen-specificTandBcells.
However,severalobstaclesimpedesuchresearch,includingthelackofrecombinantbatViruses2014,64892cytokinesforthegrowthandpropagationofimmunecellsandthelackofMHC-matched,syngeneicanimalsandcelllinesforTcellwork.
Itmaybethatcommercially-availablereagentsforspeciesthatarephylogeneticallyclosertobats,suchasthoseforhorseandcattle,maybeusedforestablishinglong-termantigen-specificculturesofbatTandBcells.
Inparticular,helperTcelllinesrequireperiodicrestimulationwithantigenspresentedonsyngeneicMHCclassIIproteinsfoundonantigenpresentingcells(APC),andarequirementofIL-2.
Withoutinbredbats,itisdifficulttoconductsuchwork.
OnesolutiontothisproblemmaybetheuseofautologousAPC,andbonemarrowisarichsourceofprogenitorcellsfortheirgeneration.
Granulocyte-macrophagecolonystimulatingfactor(GM-CSF)isroutinelyusedforgeneratingcommondendriticcells(cDC)thatareefficientAPCforhelperTcells[126,127].
HorseGM-CSFiscommerciallyavailableandsharessubstantialsimilaritytoorthologousGM-CSFinseveralbatspecies,includingthereceptor-bindingdomain(Figure3),andmaybeusefulforpropagationofcDCfrombatbonemarrow.
Figure3.
AminoacidalignmentofmatureGM-CSFfromhorse,P.
alectoandM.
lucifugus.
Identicalaminoacidsindarkgray,similaraminoacidsinlightgray,anddissimilaraminoacidsinwhite,consensusbelow.
TheredboxistheputativehelixAinvolvedinbindingtotheGM-CSFreceptor.
AlignmentperformedwithMacVectorusingthedefaultCLUSTAWsettings[128].
Thehumerusandradiusbonesofbatsarerichsourcesofbonemarrowandareeasilypreparedforbonemarrowretrieval(author'sunpublishedobservation).
Thesmallsizesofmanybatspeciespresentsachallengeforobtaininglargeamountsofbonemarrow;however,manybatspeciesofinterestforzoonoticdiseases(e.
g.
,pteropid,rousette)weighmorethanahundredgramsanditshouldbepossibleViruses2014,64893toobtainlargenumbersofbonemarrowcellsthatcanbealiquotedandstoredfrozenforlong-termuseforTcellantigenrecallexperiments.
6.
ConclusionsAlthoughmanydifficultiesandobstaclesexistforconductinghypothesis-driven,experimentalinfectionresearchofbatsandtheirviruses,newtechnologicaladvancementscanpermitrapiddiscoveryoftheirrelationships.
However,beforesubstantialprogresscanbemade,closedcoloniesofspecificpathogen-freebatsneedtobeestablished.
Fieldstudiesareoflimitedvalueforexaminationofthefinegranularityofreservoirhostresponsestoviruses,andthecaptureofwildbatsforexperimentalinfectionexperimentshasariskofinfectiousandenvironmentalfactorsthatcancomplicateinterpretationofdata.
Susceptiblebatcelllinesandantibodiesforusewithbatproteinsmustbeidentifiedorgeneratedforcloserscrutinyofhowthesevirusesinteractwiththeirhostcellmachinery,andhowtheymightmanipulatethehostresponseinamannerthatisfavorableforboththevirusandthehost.
Untilthen,itwillbenearlyimpossibletoanswerthequestion:ArebatsspecialAcknowledgmentsTheauthorthanksCharlesH.
Calisher,AnnCogswell-Hawkinson,SethFrietze,TimothyI.
ShawandRushikaPereraforhelpfuldiscussionsonvarioustopicsofbatsandtheirviruses.
ConflictsofInterestTheauthordeclaresnoconflictofinterest.
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