ww w . e l s e v i e r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Original
article
Evaluation
of
short-interfering
RNAs
treatment
in
experimental
rabies
due
to
wild-type
virus
Camila
Michele
Appolinario,
Susan
Dora
Allendorf,
Marina
Gea
Peres,
Clovis
Reynaldo
Fonseca,
Acacia
Ferreira
Vicente,
João
Marcelo
Azevedo
de
Paula
Antunes,
José
Carlos
Figueiredo
Pantoja,
Jane
Megid
∗DepartamentodeHigieneVeterináriaeSaúdePública,FaculdadedeMedicinaVeterináriaeZootecnia(DHVSP-FMVZ),
UniversidadeEstadualPaulista“JúliodeMesquitaFilho”,Botucatu,SP,Brazil
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t
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c
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o
Articlehistory:
Received3February2015 Accepted13May2015 Availableonline5August2015
Keywords: Rabies Dogvirus Batvirus siRNA
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Wehaveevaluatedtheefficacyofshort-interferingRNAstargetingthenucleoproteingene andalsothebrainimmuneresponseintreatedandnon-treatedinfectedmice.Micewere inoculatedwithwild-typevirus,classifiedasdog(hv2)orvampirebat(hv3)variantsand bothgroupsweretreatedorleavedascontrols.Nodifferencewasobservedinthelethality ratebetweentreatedandnon-treatedgroups,althoughclinicalevaluationofhv2infected miceshoweddifferencesintheseverityofclinicaldisease(p=0.0006).Evaluationofbrain immuneresponse5dayspost-inoculationintreatedhv2groupshowednodifferenceamong theanalyzedgenes,whereasafter10dayspost-inoculationtherewasincreasedexpression of2,5-oligoadenylatesynthetase1,tumornecrosisfactoralpha,interleukin12,interferon gamma,andC-X-Cmotifchemokine10associatedwithhigherexpressionofNgeneinthe sameperiod(p<0.0001).Inhv2non-treatedgrouponlyhigherinterferonbetaexpressionwas foundatday5.Theobserveddifferencesinresultsoftheimmuneresponsegenesbetween treatedandnon-treatedgroupsisnotpromisingastheyhadneitherimpactonmortality norevenareductionintheexpressionofNgeneinsiRNAtreatedanimals.Thisfinding suggeststhattheuseofpre-designedsiRNAalonemaynotbeusefulinrabiestreatment.
©2015ElsevierEditoraLtda.Allrightsreserved.
Introduction
Rabiesvirus(RABV)causesacuteencephalitisandhasa case-fatalityrateapproaching100% beingconsideredoneofthe mostdeadlyexistentinfectiousdiseases.1Thesurvivalofa
15-year-oldgirlfromWisconsin,bittenbyabatthatreceived
∗ Correspondingauthorat:UNESP-FMVZ-DHVSP,DistritodeRubiãoJr,semnumero,Botucatu,SãoPaulo,CEP18.618-970,Brazil.
E-mailaddress:[email protected](J.Megid).
novaccination,ledphysiciansworldwidetoapplythe proto-col known asthe “MilwaukeeProtocol”2 but after10 years it has been shown to be ineffective.There are at least 26 reported cases in which this protocol was tested without success.3Therefore,continuouseffortsshouldbemadetofind
someeffectivetreatmentforrabies,includingnew technolo-giessuchasRNAinterference.
http://dx.doi.org/10.1016/j.bjid.2015.05.008
RNAinterference(RNAi)isanendogenousmechanism,first described in the late90s that leads topost-transcriptional gene silencing. It is well conserved in a broad variety of species,includingplantsandanimals.4,5 Ashortnucleotide
sequence (approx. 21–23 nucleotides length), also known asshort-interferingRNA (siRNA),associatedwiththe RNA-inducedsilencingcomplex(RISC),recognizesandbindstoa complementarymRNA,causingitscleavageintosmaller frag-ments andinactivating its expressionand,thus,inhibiting proteinsynthesis.6TheRNAimechanismplaysanimportant
roleincellulardefenseagainstviralinfectionsinadditionto otherimportantcellularfunctions,includingthemobilityof geneticelementsand regulationofgene expressionduring animaldevelopment.7 Thegeneralpotentialofthis
mecha-nismhasstimulatedstudiesoftheuseofsiRNAandmicroRNA asatherapeuticoptionfornon-infectious8–10 andinfectious
diseases,includingdengue,11,12 respiratorysyncytialvirus,13
influenza,14 tuberculosis,15 SARS,16AIDS,10andherpes
sim-plextype2.17
Despite the antiviral effect of siRNAs, they are potent activatorsofthe mammalianinnateimmune system. Syn-theticsiRNAduplexescaninducehighlevelsofinflammatory cytokines and type I interferons, after systemic admin-istration in mammals and in primary human blood cell cultures.18,19 The production of antiviral agents such as
type I interferons, including interferon alpha (IFN␣) and interferonbeta(IFN),isanimportantimmunemechanism againstrabiesvirusinfectionthatoccurssoonafterthecell infection.20,21
In2007,Brandãoandcolleaguespublishedastudyin BHK-21cellsshowingtheefficacyofanoveltherapyagainstrabies virusbasedontheuseofsiRNAsdesignedagainsttheNgene sequenceofPasteurvirus(PV).Theresultsdemonstratedthat cells treated with three different sequences of siRNA had a five-fold drop in the amount of infected cells evaluated bydirectimmunofluorescence testwhencomparedto con-trols,withnocytopathogenicityduetothetreatment.22Those
samesequencesweretestedbythesamegroupinvivoand demonstratedreductioninthelethalityratewhencompared tountreatedanimals.23
StudiestestingsiRNAsinvitroandinvivousuallyhaveas targetsrabiesnucleoprotein(N),glycoprotein(G),and/or poly-merase(L)genes;thesequencesaredeliveredbyavectorsuch asadenovirus,24lentivirus,25orassociatedwithaliposome.26
siRNAsalways inhibitviral replication atsomelevel, how-everitisdifficulttopreciselydeterminetheirrealefficacyand possibleapplicationinmedicalpractice.Thisisbecause in almostallstudies,thesiRNAstestedarethosedesignedand checkedinexperimentalinfectionduetoexactly thesame RABVstrains(usuallyalaboratorystrain)usedastemplates todesignthesiRNAsequences.7,22,24,26
Thisstudyaimedtotesttheclinicalefficacyofthree differ-entsequencesofsiRNAdesignedagainsttheRABVNgenein thetreatmentofmiceinfectedwithtwodifferentwildstrains ofRABV,isolatedfromrabidhumanpatientsinfectedbyadog orbyavampirebatvariant.Inaddition,consideringthe differ-enceofpathogenicitybetweendogandbatvariants27andthe
immunestimulationthat siRNAadministrationcaninduce, thebrainimmuneresponseofinfectedandnon-infected ani-malswasevaluated.
Table1–NucleotidesequencesofsiRNAsdesigned againstPasteurvirusNgene.22
siRNA Duplexsequence
RNA124 Sense5GCCUGAGAUUAUCGUGGAG3 Antisense5AUCCACGAUAAUCUCAGGC3 RNA750 Sense5GCACAGUUGUCACUGCUUC3
Antisense5UAAGCAGUGACAACUGUGC 3
RNAB Sense5GACAGCUGUUCCUCACUCG3
Antisense5AGAGUGAGGAACAGCUGUC 3
Materials
and
methods
Experimentaldesign
Twogroupsof60C57/BL6miceeach,4–6week-oldfemales, S.P.F,wereinoculatedinthegastrocnemiusmusclewith100L ofviralinoculumwithsameviraltitration(LD5010−6.66/30L)
forvariant2[dog(hv2)]orvariant3[vampirebat(hv3)].Thirty animalsweretreatedintraperitoneally24hp.i.,withaunique doseofamixtureconsistingofthreesiRNAsequences(3.3M concentration each)designedagainstthe Ngeneofthe PV strain,usinglipofectamineasthedeliverymethod22(Table1);
theother half(n=30)were leftuntreated,andjustreceived salineintraperitoneallyatthesametimeofthesiRNAtreated group.Anon-inoculatedgroupofanimals(n=30)wereusedas controlsforbasalimmuneresponseandreceived intramus-cularofsterilesalineaswellasintraperitonealinoculation. Anon-inoculatedsiRNAtreatedgroup(n=30)wasincluded to evaluatepossibleside effects ofthe treatmentand also theimmunestimulationofsiRNA.Forallgroups,10animals wereobservedfor30daysand10wereeuthanizedafter5and 10daysp.i.,whenwholebrainswereremovedandstoredat −80◦Cuntilfurtherreal-timePCRanalyses.
Animalsofallgroupswereweightedandevaluateddailyfor theonsetofrabiesclinicalsigns,suchasruffledfur, hunch-ingback,hypo/hyperexcitability,paralysisofoneorbothhind limbortetraplegia,28andforanyotherabnormalityinthecase
ofthenon-inoculatedgroups.
The animalstudy wasapproved bythe SãoPaulo State UniversityEthicalCommittee(registrationnumber238/2008), which follows the guidelines established by the COBEA – BrazilianCollegeofAnimalExperimentation).
RNAextractionandRealTime-RT-PCR(RT-qPCR)
BraintissueRNAswereextracted withthe Invitek® kit and
storedat−80◦C.ThereactionforcDNAsynthesisconsisted
of1gofextractedRNA,1LofOligo-DTprimer(Invitrogen®)
and1LofSuperScriptII(Invitrogen®)accordingtothe
man-ufacturer’sinstructions.TheRT-qPCRreactionwasperformed with2Lof1/50dilutedcDNA,1Lof0.1gofeachprimer andMasterMixSyberGreen(Promega®)inafinalvolumeof
25Laccordingtothemanufacturer’s instructions. Primers forthe 18S murinegeneswere supplied byIDT® and used
as housekeepinggenes, and primers forthe RABV N gene were manufactured as described previously.29 The mouse
0 20 40 60 80 100 Lethality rate (%) 0 50 100 (%) Animals
presenting clinical signs
• • • Lethality rate hv2 x hv3 hv2 Control hv3 Control hv2 Control hv3 Control hv3 siRNA hv2 siRNA hv2 siRNA hv3 siRNA
A
Clinical signs hv2 x hv3B
Fig.1–(A)LethalityrateofcontrolsandsiRNA-treatedgroupsinoculatedwithvariant2(hv2)andvariant3(hv3);Cox
proportionalhazardswereusedtoestimatelethalityratesandhazardratiosbetweengroups.Nostatisticaldifferencewas
found.(B)Percentageofanimalsineachgroupshowingclinicalsigns,whichincludedweightloss,ruffledfur,hunched
back,hypoexcitability,hyperexcitability,paralysis,andtetraplegia;Kruskal–Wallistestshowedastatisticaldifference
(p=0.0006)betweenthehv2controlandsiRNA-treatedgroup.
Quantitect® PrimerAssayfromQiagen® wasusedto
evalu-atetheexpressionofchemokineC-Cmotifligand2(CCL2), 2-5-oligoadenylatesynthetase1(OAS1),interleukin2(IL2), interleukin6(IL6),interleukin12(IL12),tumornecrosis fac-toralpha (TNF␣), interferon gamma (IFN␥),interferon beta (IFN),C-X-Cmotifchemokine10(CXCL10),cellsurface gly-coproteinCD200receptor1(CD200R)andinsulin-likegrowth factor1(IGF-1).
All thermal cycling and detection was performed using anAppliedBiosystemsStepOneFast(ABI7500Fast)thermal cycleremployingathermalprofileof40cyclesof50◦Cfor20s, 95◦Cfor10min,95◦Cfor15sand60◦Cfor1min.
Dataanalysis
Coxproportionalhazardswereusedtoestimatelethalityrate andhazardratios(HR)betweengroups.Kruskal–Walliswith
p<0.05 asthe significancelevel was chosenfor evaluation ofgeneexpressionofcytokines/chemokinesand the RABV
Ngene.Graph-Prism® 5.0andInstat® softwareswereused asanalysistools.Geneexpressionofcytokines/chemokines werefirstcomparedbetweentreatedandnon-treatedgroups inoculatedwiththesamevariant,atthesameperiod,5or10 daysp.i(ex:hv3non-treated5daysvshv3siRNAtreatedat5 days);asecondcomparisonwasmadebetweengroupsatthe samecondition,inoculated(withsameviralvariant)treated andnon-treatedbutatdifferentperiods,5and10days(ex:hv2 siRNAtreatedat5daysvshv2siRNAtreatedat10days).
Results
and
discussion
Therewasanonsignificantstatisticaldifferenceinmortality rateinthegroupstreatedwithsiRNAsirrespectiveofthe vari-antsused.Forhv2,thelethalityratewas100%innon-treated and 70% intreated groups (p=0.27; HR=0.57); for hv3,the
lethalitywas60%innon-treatedand80%intreatedgroups (p=0.21;HR=1.97)(Fig.1A).
Clinicalevaluationofanimalsinfectedwithvariant2and treatedwithsiRNAshowedlessseverity ofclinicaldisease, whichincludedweightloss,paralysis,anddeath(p=0.0006) comparedtothecontrols.However,noclinicaldifferencewas observedamonghv3-infectedanimalseithertreatedor non-treatedwithsiRNA(Fig.1B).
TheNgeneexpressionofallgroupscomparedatthesame period,eitherat5or10daysp.i.,showednosignificant dif-ference.However,Ngeneexpressioninthehv2treatedgroup wassignificantlyincreasedatday10vsday5p.i.(p<0.0001).
RABV-N at 5 days and 10 days
5d hv2 Control 5d hv2 siRNA 5d hv3 Control 5d hv3 siRNA
10d hv2 Control 10d hv2 siRNA 10d hv3 Control 10d hv3 siRNA 100 105 1010 1015 Fold increase
***
Fig.2–RABVNgeneexpressioninthebrainofmice
infectedwithhv2orhv3,treatedornon-treated(control)
withsiRNA.Kruskal–Wallistestwasappliedtocompare
theresultsbetweendifferentgroupsatday5andday10p.i.
Therewasnodifferencebetweenthehv2andhv3groupsat
day5oratday10.However,hv2siRNA-treatedgroups
hv2 at 5 days Control (NT) x siRNA (T) NT CCL2 NT OAS1 NT IL6 NT TNF α NT IFN β NT IFN γ NT IL12 NT CXCL10 NT IGF1 NT CD200R T CCL2 T OAS1 T IL6 T TNF α T IFN β T IFN γ T IL12 T CXCL10 T IGF1 T CD200R 0 20 40 60 80 100 500 1000 1500 2000 100 000 200 000 300 000 400 000 Fold increase •••
A
NT CCL2 NT OAS1 NT IL6 NT TNF α NT IFN β NT IL12 NT IFN γ NT CXCL10 NT IGF1 NT CD200R T CCL2 T OAS1 T IL6 T TNF α T IFN β T IL12 T IFN γ T CXCL10 T IGF1 T CD200R 0 20 40 60 80 100 500 1000 1500 2000 100 000 200 000 300 000 400 000 Fold increase hv3 at 5 days Control (NT) x siRNA (T) •• •••C
hv2 at 10 days Control (NT) x siRNA (T) NT OAS1 NT IL6 NT TNF α NT IFN β NT IFN γ NT IL12 NT CXCL10 NT IGF1 NT CD200R T OAS1 T IL6 T TNF α T IFN β T IFN γ T IL12 T CXCL10 T IGF1 T CD200R 0 20 40 60 80 100 500 1000 1500 2000 100 000 200 000 300 000 400 000 Fold increase •• • • • • •B
NT OAS1 NT IL6 NT TNF α NT IFN β NT IL12 NT IFN γ NT CXCL10 NT IGF1 NT CD200R T OAS1 T IL6 T TNF α T IFN β T IL12 T IFN γ T CXCL10 T IGF1 T CD200R 0 20 40 60 80 100 500 1000 1500 2000 100 000 200 000 300 000 400 000 Fold increase hv3 at 10 days Control (NT) x siRNA (T)D
Fig.3–Relativegeneexpressionofcytokinesandchemokinesindifferentgroups(hv2andhv3)atday5andday10p.i.The
Kruskal–Wallistestwasappliedtoanalyzetheresults.(A)Expressionatday5p.i.inthehv2control(NT)andsiRNA-treated
(T)groups;IFNwashighlyexpressedintheNTgroup(***p<0.001).(B)Expressionatday10p.i.inthehv2control(NT)and
siRNA-treated(T)groups;IL12washighlyexpressedintheNTgroup(**p<0.01);inthetreatedgroup,OAS1,TNF˛,IL12,IFN
andCXCL10expressionlevelswereincreased(*p<0.05).(C)Expressionatday5p.iinthehv3control(NT)andsiRNA-treated
(T)groups;IFNwashighlyexpressedintheNTgroup(**p<0.01).(D)Expressionatday10p.i.inthehv3control(NT)and
siRNA-treated(T)groups;nosignificantdifferencewasfound.
TheincreaseinNgeneexpressionisexpectedtofollow dis-easeprogression.28However,despitenotreachingstatistical
significance,hv2siRNA-treatedanimalsatday5hadverylow expressioncomparedtoallothergroupsatthesameperiod, reflectingsomeinterferenceofsiRNAinvirusreplicationin thisstudy(Fig.2).
Thebrainimmuneresponseofdifferentgroupsshowed highexpressionofIFN-(p<0.001)atday5p.i.inhv2 non-treated group and IFN- (p<0.01) and CCL2 (p<0.001) in hv3non-treatedgroup;atday10p.i.hv2non-treatedgroup showedincreasedIL12expression(p<0.01).Therewasno sta-tisticaldifference in the analyzedimmune markers inthe treatedhv2 group atday5,whereas atday 10OAS1, TNF-␣,IL12,IFN-␥, andCXCL10were increased(p<0.05) (Fig. 3). InfectedcellsshowedarapidproductionandreleaseofIFN-, whichisimportantforhostsurvival.Thisinducesthe expres-sionofseveralIFN-stimulatedgenes(ISGs),suchasOAS1,that
exertanantiviraleffectsimilartoIFN-,butatdifferentstages ofviralreplication.28DamagedneuronsalsoproduceIFN-␥,
IL1,IL6,IL12,CCL2,CCL4,CCL5,CCL7andtheIFN-inducible protein, CXCL10. All these cytokines and chemokines are responsible for the upregulated expression ofmajor histo-compatibility complex (MHC) molecules on the surface of microgliaandalsofortheincreasedexpressionofadhesion moleculesbyendothelialcells.Alltheassociatedfactorsare importantfor inductionofthe adaptive immuneresponse, whichinvolvestheactivationandmigrationofTcells,aswell astheproductionofspecificantibodies.30–32EnhancedNgene
expressionatday10vsday5inhv2-infectedmicemay indi-catethatatday5siRNAinterferedwithviralreplication,which wasassociatedwithnodifferenceamongcytokine/chemokine geneexpressionatday5inthisgroup(e.g.,IFN-),suggesting thatvirallevelswerenotsufficienttotriggerthehostimmune response.Thispatternofresponsedidnotoccurinthehv2
Non-inoculated
siRNA treated 5 days and 10 days
CCL2 5d OAS1 5d IL6 5d TNF α 5d IFN β 5d IL12 5d IFN γ 5d CXCL10 5d IGF1 5d C200R 5d CCL2 10d OAS1 10d IL6 10d TNF α 10d IFN β 10d IL12 10d IFN γ 10d CXCL10 10d IGF1 10d C200R 10d 0 2 4 6 8 10 200 400 600 800 1000 2000 4000 6000 8000 10 000
*
5 days 10 days Fold increaseFig.4–Relativeexpressionofimmunemarkersatdays5
and10inmicebrainsafteradministrationofsiRNAs.The
treatmentledtoanincreaseingeneexpressionofIFN
(*p<0.05)atday5.TheKruskal–Wallistestwasusedto
comparetheresultsbetweengroups.
non-treatedgroupthatshowedahigherIFN-expressionat day5norinthehv3treatedgroup,whichhasasimilargene expressionprofilecomparedtonon-treatedgroups.
Consideringthedifferencesintheresultsbetweenvariant 2and3,theidentityofsiRNAsequencesandthebatanddog virusesusedinthisstudywereblastedshowingidentity ran-gingfrom95to100%forhv2and86to95%forhv3.ThesiRNAs testedweredesignedbasedonPastervirus(PV),22whichisa
fixedstrain,andtheidentityresultsindeedshoweda differ-encebetweenstreetrabiesvirusandsiRNAsequences,with loweridentityforthebatvariant.
AlthoughtheNgeneisconsideredaconservedsite, stud-ieshaveshownasignificantgeneticvariabilityinstreetrabies virusstrains,from5upto49%.33,34Itisimportantto
remem-berthatalmostaperfectcomplementarysequencebetween siRNA molecule and the viral RNA target is necessary to inducecleavageofmRNA.35 Thisrequirement may explain
thedifferencefoundintheresultsfordogandbatvariants observedinthisstudyandalsothelackofdifferenceinthe
Ngeneexpressionamongthegroupsevaluatedinthesame period.
ToassessiftreatmentwithsiRNAcouldatleastbe par-tiallyeffectiveinthe symptomaticphaseofthedisease,an additionalstudyinmicewasdevelopedadministeringa sin-gledoseofsiRNAsonlyaftertheonsetofclinicalsigns.No differenceinmortality,clinicalevaluation,orprolongationin theevolutionperiodwasobservedinanyofthetreatedgroups (datanotshown).
Theimmuneresponseevaluationinnon-inoculated ani-mals treated with siRNA showed an up-regulation of all immunemarkers,inparticularthatofIFN-␥atday5compared today10.Thisincreasedisappearedat10days,reflectingthe reductionintheactivityofsiRNA,whichcanlastupto6days36
(Fig.4).
Conclusion
Therapy with siRNAhas neitherreduced the lethality rate in two different street rabies virus infections nor N gene expression. However, there were less severe clinical signs using siRNAtherapy in variant2infection. Theefficacy of siRNAtherapyiscloselyassociatedwiththe identityofthe siRNAdesignand itstarget.Inthisstudy,ahigheridentity wasreachedwithvariant2despitevariant3,justifyingthe obtainedresults.Areduced expressionofimmunemarkers withsiRNAtherapyininfected,butnotinnon-infectedmice, may haveoccurred asaresultoftheantiviral effect. How-ever,theresultswerenottranslatedonreducedmortalityor expressionofNgene,whichcanindirectlyreflectvirus repli-cationsuggestingthatpre-designedsiRNAmaynotbeuseful inrabiestreatment.Thepotentialinapplyingthis technol-ogyislimitedbecauseinmedicalpracticethetypeofvirus infectingthepatientisunknowninalmost100%ofthecases. Morestudiesarenecessarytoovercomethislimitationandto showwhetherthistechnologycouldbeappliedeitheralone orassociatedwithothertherapeuticmeasures.
Funding
Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP–No.08/11446-1)andConselhoNacionalde Desen-volvimentoCientíficoeTecnológico(CNPq482726/2012-1).
Conflicts
of
interest
Theauthorshavedeclaredthereisnoconflictofinterest.
Acknowledgments
WethankDr.MariaLuizaCarrieriandDr.IvaneteKotaitfor kindlyprovidingbothrabiesvirusstrains.
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1.FooksAR,BanyardAC,HortonDL,JohnsonN,McElhinneyLM, JacksonAC.Currentstatusofrabiesandprospectsfor elimination.Lancet.2014,
http://dx.doi.org/10.1016/S0140-6736(13)62707-5.
2.WilloughbyRE,TievesKS,HoffmanGM,etal.Survivalafter treatmentofrabieswithinductionofcoma.NEnglJMed. 2005;352:2508–14.
3.JacksonAC.Currentandfutureapproachestothetherapyof humanrabies.AntiviralRes.2013;99:61–7.
4.NapoliC,LemieuxC,JorgensenR.Introductionofachimeric chalconesynthasegeneintopetuniaresultsinreversible co-suppressionofhomologousgenesintrans.PlantCell. 1990;2:279–89.
5.FireA,XuS,MontgomeryMK,KostasSA,DriverSE,MelloCC. Potentandspecificgeneticinterferencebydouble-stranded RNAinCaenorhabditiselegans.Nature.1998;391:744–5.
6.AkhtarS,BenterI.Toxicogenomicsofnon-viraldrugdelivery systemsforRNAi:potentialimpactonsiRNA-mediatedgene silencingactivityandspecificity.AdvDrugDelivRev. 2007;59:164–82.
7. MeshramCD,SinghNK,SonwaneAA,etal.Evaluationof singleanddualsiRNAstargetingrabiesvirusglycoprotein andnucleoproteingenesforinhibitionofvirusmultiplication invitro.ArchVirol.2013;158:2323–32.
8. ZhengX,SuzukiM,IchimTE,etal.Treatmentofautoimmune arthritisusingRNAinterference-modulateddendriticcells.J Immunol.2010;184:6457–64.
9. AraújoR,FernandesM,Cavaco-PauloA,GomesA.Biologyof humanhair:knowyourhairtocontrolit.AdvBiochemEng Biotechnol.2011;125:121–43.
10.ChenSH,ZhaoriG.PotentialclinicalapplicationsofsiRNA technique:benefitsandlimitations.EurJClinInvest. 2011;41:221–32.
11.Sanchez-VargasI,TravantyEA,KeeneKM,etal.RNA interference,arthropod-borneviruses,andmosquitoes.Virus Res.2004;102:65–74.
12.MukherjeeS,HanleyKA.RNAinterferencemodulates replicationofdenguevirusinDrosophilamelanogastercells. BMCMicrobiol.2010;10:127.
13.ChangZ,BabiukLA,HuJ.Therapeuticandprophylactic potentialofsmallinterferingRNAsagainstsevereacute respiratorysyndrome:progresstodate.BioDrugs. 2007;21:9–15.
14.ScullMA,RiceCM.AbigroleforsmallRNAsininfluenza virusreplication.PNAS.2010;107:11153–4.
15.JayaswalS,KamalA,DuaR,etal.Identificationof host-dependentsurvivalfactorsforintracellular MycobacteriumtuberculosisthroughansiRNAscreen.PLoS Pathog.2010;6,e1000839.
16.WangY,CaoYL,YangF,ZhangY,WangSH,LiuL.Small interferingRNAeffectivelyinhibitstheexpressionofSARS coronavirusmembranegeneattwonoveltargetingsites. Molecules.2010;15:7197–207.
17.KatakowskiJA,PalliserD.siRNA-basedtopicalmicrobicides targetingsexuallytransmittedinfections.CurrOpinMolTher. 2010;12:192–202.
18.JudgeA,MaclachlanI.Overcomingtheinnateimmune responsetosmallinterferingRNA.HumGeneTher. 2008;19:111–24.
19.RobbinsM,JudgeA,AmbegiaE,etal.Misinterpretingthe therapeuticeffectsofsmallinterferingRNAcausedby immunestimulation.HumGeneTher.2008;19: 991–9.
20.NakamichiK,SaikiM,SawadaM,YamamuroY,MorimotoK, KuraneI.Double-strandedRNAstimulateschemokine expressioninmicrogliathroughvacuolarpH-dependent activationofintracellularsignalingpathways.JNeurochem. 2005;95:273–83.
21.FaulEJ,WanjallaCN,SutharMS,GaleM,WirblichC,Schnell MJ.RabiesvirusinfectioninducestypeIinterferonproduction inanIPS-1dependentmannerwhiledendriticcellactivation reliesonIFNARsignaling.PLoSPathog.2010;6,e1001016.
22.BrandãoPE,CastilhoJG,FahlW,etal.Short-interferingRNAs asantiviralsagainstrabies.BrazJInfectDis.2007;11:224–5.
23.DurymanovaOnoEA,IamamotoK,CastilhoJG,etal.Invitro andinvivoinhibitionofrabiesvirusreplicationbyRNA interference.BrazJMicrobiol.2013;44:879–82.
24.GuptaPK,SonwaneAA,SinghNK,etal.Intracerebraldelivery ofsmallinterferingRNAs(siRNAs)usingadenoviralvector protectsmiceagainstlethalperipheralrabieschallenge.Virus Res.2012;163:11–8.
25.SinghNK,MeshramCD,SonwaneAA,etal.Protectionofmice againstlethalrabiesviruschallengeusingshortinterfering RNAs(siRNAs)deliveredthroughlentiviralvector.Mol Biotechnol.2014;56:91–101.
26.YangYJ,ZhaoPS,ZhangT,etal.SmallinterferingRNAs targetingtherabiesvirusnucleoproteingene.VirusRes. 2012;169:169–74.
27.LafonM.Batrabies–theAchillesheelofaviralkiller?Lancet. 2005;366:876–7.
28.ChopyD,DetjeCN,LafageM,KalinkeU,LafonM.ThetypeI interferonresponsebridlesrabiesvirusinfectionandreduces pathogenicity.JNeurovirol.2011;17:353–67.
29.SoaresRM,BernardesF,SakamotoSM,etal.Aheminested polymerasechainreactionforthedetectionofBrazilian rabiesisolatesfromvampirebatsandherbivores.MemInst OswaldoCruz.2002;97:109–11.
30.GriffinD.ImmuneresponsetoRNA-virusinfectionsofthe CNS.NatRev.2003;3:493–502.
31.KoyamaS,IshiiKJ,CobanC,AkiraS.Innateimmuneresponse toviralinfection.Cytokine.2008;43:336–41.
32.SteelCD,BrevingK,TavakoliS,KimWK,SanfordLD,Ciavarra RP.Roleofperipheralimmuneresponseinmicroglia activationandregulationofbrainchemokineand proinflammatorycytokineresponsesinducedduringVSV encephalitis.JNeuroimmunol.2014;267:50–60.
33.DenduangboripantJ,WacharapluesadeeS,LumlertdachaB, etal.TransmissiondynamicsofrabiesvirusinThailand: implicationsfordiseasecontrol.BMCInfectDis.2005;5:52.
34.IsrasenaN,SupavonwongP,RatanasetyuthN,KhawplodP, HemachudhaT.Inhibitionofrabiesvirusreplicationby multipleartificialmicroRNAs.AntiviralRes.2009;84:76–83.
35.IsrasenaN,MahavihakanontA,HemachudhaT.Rabiesvirus infectionandmicroRNAs.AdvVirusRes.2011;79:329–44.
36.ChiuYL,RanaTM.siRNAfunctioninRNAi:achemical modificationanalysis.RNA.2009;9:1034–48.
