Limited Effects of Type I Interferons on Kyasanur Forest Disease Virus in Cell Culture

19 Pages·2016·11.06 MB·English

RESEARCHARTICLE
Limited Effects of Type I Interferons on
Kyasanur Forest Disease Virus in Cell Culture
BradleyW.M.Cook1,2,CharleneRanadheera3,AidanM.Nikiforuk1,2,ToddA.Cutts1,
DarwynKobasa3,4,DeborahA.Court2,StevenS.Theriault1,2*
1 AppliedBiosafetyResearchProgram,NationalMicrobiologyLaboratoryattheCanadianScienceCentre
forHumanandAnimalHealthandNationalMicrobiologyLaboratoryattheJ.C.WiltInfectiousDiseases
ResearchCentre,PublicHealthAgencyofCanada,Winnipeg,Manitoba,Canada,2 Departmentof
Microbiology,TheUniversityofManitoba,Winnipeg,Manitoba,Canada,3 HighContainmentRespiratory
VirusesGroup,SpecialPathogensProgram,NationalMicrobiologyLaboratoryattheCanadianScience
CentreforHumanandAnimalHealth,PublicHealthAgencyofCanada,Winnipeg,Manitoba,Canada,
4 DepartmentofMedicalMicrobiology,TheUniversityofManitoba,Winnipeg,Manitoba,Canada
*[email protected]
Abstract
a11111
Background
Thetick-borneflavivirus,KyasanurForestdiseasevirus(KFDV)causesseasonalinfections
andperiodicoutbreaksinsouth-westIndia.Thecurrentvaccineofferspoorprotectionwith
OPENACCESS reportedissuesofcoverageandimmunogenicity.Sincetherearenoapprovedprophylactic
therapeuticsforKFDV,typeIIFN-α/βsubtypeswereassessedforantiviralpotencyagainst
Citation:CookBWM,RanadheeraC,NikiforukAM,
CuttsTA,KobasaD,CourtDA,etal.(2016)Limited KFDVincellculture.
EffectsofTypeIInterferonsonKyasanurForest
DiseaseVirusinCellCulture.PLoSNeglTropDis10
(8):e0004871.doi:10.1371/journal.pntd.0004871 Methodology/PrincipalFindings
Editor:SunitKumarSingh,MolecularBiologyUnit ThecontinuedpassageofKFDV-infectedcellswithre-administeredIFN-α2atreatmentdid
(MBU),INDIA noteliminateKFDVandhadlittleeffectoninfectiousparticleproductionwhereastheIFN-
Received:March4,2016 sensitive,greenfluorescentprotein-expressingvesicularstomatitisvirus(VSV-GFP)infec-
tionwascontrolled.FurtherevaluationoftheotherIFN-α/βsubtypesversusKFDVinfection
Accepted:June30,2016
indicatedthatsingletreatmentsofeitherIFN-αWAandIFN-αΚappearedtobemoreeffec-
Published:August1,2016
tivethanIFN-α2aatreducingKFDVtitres.Concentration-dependentanalysisoftheseIFN-
Copyright:©2016Cooketal.Thisisanopen α/βsubtypesrevealedthatregardlessofsubtype,lowconcentrationsofIFNwereableto
accessarticledistributedunderthetermsofthe
limitcytopathiceffects(CPE),whilesignificantlyhigherconcentrationswereneededfor
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany inhibitionofvirionrelease.Furthermore,expressionoftheKFDVNS5incellculturebefore
medium,providedtheoriginalauthorandsourceare IFNadditionenabledVSV-GFPtoovercometheeffectsofIFN-α/βsignalling,producinga
credited.
robustinfection.
DataAvailabilityStatement:Allrelevantdataare
withinthepaper.
Conclusions/Significance
Funding:Theauthorsreceivednospecificfunding
forthiswork. TreatmentofcellculturewithIFNdoesnotappeartobesuitableforKFDVeradicationand
theassayusedforsuchstudiesshouldbecarefullyconsidered.Further,itappearsthatthe
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. NS5proteinissufficienttopermitKFDVtobypasstheantiviralpropertiesofIFN.We
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004871 August1,2016 1/19
InterferonEffectsonKyasanurForestDiseaseVirus
suggestthatotherprophylactictherapeuticsshouldbeevaluatedinplaceofIFNfortreat-
mentofindividualswithKFDVdisease.
AuthorSummary
Since1957KyasanurForestdiseasevirus(KFDV)hascausedseasonalinfectionsandperi-
odicoutbreaksinsouth-westIndia.Itisestimatedthatnearly500peopleacquireKFDV
annuallyand3–5%ofthoseinfectedsuccumbtothedisease.Thevaccinestrategyiscom-
plicatedbythelackofcoverage,complianceandefficacy,highlightedbythefactthatless
thanhalfofthetargetpopulationreceivedtherecommendedthreedose-regimen.Besides
thepreventionoftickbitesandvaccination,therearenoapprovedantiviralsforKFDV
infection.Basedontheseobservations,thecommonly-used-IFN-α2awasassessedand
wasnotcapableoflimitingKFDVvirustitres.FurthercharacterizationoftheotherIFN-
α/βsubtypesusedatdifferentconcentrationsrevealedthatKFDVreplicationwasinsensi-
tivetoallsubtypes,eventhoughsignsofcellulardamagewerereduced.Thus,infectious
titre,ratherthanmonolayerstainingorcytopathiceffect(CPE)monitoring,ismorereli-
ableforIFNanalyses.ThecapabilityofKFDVtoovercometheantiviralpropertiesofIFN
wasattributedtotheNS5protein.Thus,othertreatmentoptionsneedtobeevaluatedfor
patientssufferingwithKyasanurForestdisease.
Introduction
KyasanurForestdiseasevirus(KFDV)isatick-borneflavivirusthatwasidentifiedin1957fol-
lowingamonkeyepizooticandacoincidinghumanoutbreakinsouth-westIndia[1].KFDV
casespreviouslywerelocalizedwithintheShimogadistrictofKarnataka;howeverKFDVhas
beenrecentlydiscoveredintheneighboringstatesofKerala,TamilNadu,GoaandMaharash-
tra[2–5]and,possiblyChinain1989[6]increasingthepotentialpublichealthriskassociated
withthispathogen.AvaccineforKFDVisavailableforthoselivinginaffectedareasandthose
livingwithina5kilometerradiusofapositivecasefromeitherhumans,monkeysortickpools
[7],buttherehasbeenissueswithimplementationandefficacy.Themosttroublingaspectof
vaccineuseisthatlessthanhalfofthetargetpopulationactuallyreceivethefullthree-dosereg-
imenthatisrequiredforprotection[8,9].Withtheannualnumberofcasesrangingfrom400–
500andanassociatedfatalityrateof3–5%[10],thereisaneedforalternativetherapeutic
options,besidesthecurrentvaccineandtickbitepreventionmeasures.
KFDVisamemberofthetick-borneencephalitisserocomplexwhichincludes:tick-borne
encephalitis,theformerRussianspring-summerencephalitis,Omskhemorrhagicfever,
Powassan,LangatandLouping-Illviruses[11].AvariantofKFDV,Alkhumrahemorrhagic
feverviruslocatedinSaudiArabia[12]andinEgypt[13–15],alsoispartofthiscomplex[16].
Thesingle-strandedpositive-polarityRNAgenomeofKFDVis10,774basesinlengthand
encodesasinglepolyprotein:C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5[17].KFDV,
AlkhumrahemorrhagicfevervirusandOmskhemorrhagicfevervirusareuniquetothiscom-
plexastheyprimarilycausehemorrhagicfevermanifestationswithneurologicalinvolvement
[18].
Interferon(IFN)wasfirstdescribedforitsabilitytointerferewithvirusinfectionin1957by
IsaacsandLindenmann[19,20].Inresponsetoviralinfection,IFNisreleasedfrominfected
cellstosurroundinguninfectedcells.Uponbindingtoitsreceptorandsubsequentactivationof
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004871 August1,2016 2/19
InterferonEffectsonKyasanurForestDiseaseVirus
theJak/STATsignalingcascade,thecellular“antiviralstate”isestablished.Theantiviralstate
allowsnaïvecellstobecomeresistanttoviralinfectionviaexpressionofIFN-stimulatedgenes
(ISG)thathavemanyrolesinprotectingthehostfrominfection[21–23].KFDV,likemany
otherflavivirusesincludingdengue,yellowfever,Langat,WestNile,tick-borneencephalitis
andJapaneseencephalitisviruses[24–27]employstheNS5proteintopreventantiviralstate
establishmentandcompromisedIFNsignalling[28],whereasNS4B-2khasbeenalsoimpli-
catedfordengue,WestNileandyellowfeverviruses[26,29,30].Theramificationsofacompro-
misedJak/STATpathwaywerehighlightedinmicelackingtheIFN-α/βreceptor.Infectionof
thesemicewithWestNile[31],dengue[32]andLangatviruses[33]ledtoincreasedvirusbur-
den,earlieronsetofclinicalsignsandhighermortalitywhencomparedtocontrolmock-
infectedtypemice.
Ofthetype1IFNs(14subtypesofαandoneofβ),onlyIFN-α2aandα2bareFDA-
approvedforclinicaltreatmentinhumans[22].Casereportsindicatearelativelynarrowwin-
dowforpost-exposuretreatmentagainstotherflavivirusessuchasWestNile,SaintLouis
encephalitisandJapaneseencephalitisviruseswitheitherIFN-α2aor2b,especiallyinthe
immune-compromisedorduringtheseverestagesofdisease[34–37].Intissueculture,both
IFN-α2aandα2barepotentatreducingthereplicationofdenguevirus2[38,39],WestNile
[31,40,41],Langat[25]andotherflaviviruses[42].Interestingly,strain-specificresponseswere
observedbetweendenguevirus1,3and4,yellowfevervirus(vaccinestrainsFNVand17D)
[42]andJapaneseencephalitisvirus(strainsVip,KE-093,KE-094andKE-095)[43].Similarly,
theantiviralactivitiesofotherIFN-α/βsubtypesdifferdramaticallyforvesicularstomatitis
virus(VSV),humanimmunodeficiencyvirus[44]andhumanrhinoviruses[45].Toour
knowledge,withtheexceptionofIFN-α2a,theotherIFN-α/βsubtypeshavenotbeenevaluated
againstKFDV.
CurrenttreatmentoptionsforKFDVarelimitedtosupportivecare,becausenothera-
peuticdrugshavebeenclinicallyapproved.UsingVSVasanIFN-sensitiveviruscontrol
forcomparison,theantiviralefficacyoftheIFN-α/βsubtypeswasassessedagainstKFDV
infectionincellculture.WeconcludethatIFN-α2awasunabletoeradicateKFDVfromcell
culture,butappearedtoofferprotectionformcytopathiceffects(CPE).AlthoughsomeIFN
subtypesathigherdosesdidrestrictKFDVreplication,albeitmarginally,furtherscrutiny
couldnotdemonstrateastrongdose-dependentresponseintermsofvirionproduction
Thus,itisnotappropriatetouseprotectionfromCPEasthesoledeterminantofpotency.
Inconclusion,KFDVisnotsensitivetotheantiviraleffectsofIFNanddespitethelackofa
concisemechanismforIFNresistance,itisclearthattheNS5proteinisamajor
contributor.
Methods
Cells,VirusesandInterferon
Cells. Forallexperiments,humanlungcarcinoma(A549CCL-185)cells(ATCC,Burling-
ton,Ontario,Canada)andAfricangreenmonkeykidney(VeroE6CRL-1586)cells(ATCC)
werepropagatedinDulbecco’smodifiedeaglemedium(DMEM)(HyClone,Ottawa,Ontario,
Canada)growthmediumsupplementedwith10%serum(Gibco,Burlington,Ontario,Canada)
and1%antibiotics(Penicillin-Streptomycin)(Gibco).Babyhamsterkidney(BHK-21CCL-10)
cells(ATCC)weregrowningrowthmedium,Eagle’sminimumessentialmedium(EMEM)
(HyClone)with10%serumand1%antibiotics.Alternatively,duringinfectionstheserumcon-
tentineachmediatypewasreducedto2%andtheresultingformulationisreferredtoasvirus
maintenancemedium.Allcelllineswereincubatedduringpropagationorinfectionat37°C/
5%CO ,unlessstatedotherwise.
2
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004871 August1,2016 3/19
InterferonEffectsonKyasanurForestDiseaseVirus
Viruses. KFDV(P9605strain,GenBankaccessionnumber:HM055369)stockswereprop-
agatedinVeroE6cellsinContainmentLevel4(CL-4)laboratoryoftheNationalMicrobiology
Laboratory(NML)attheCanadianScienceCentreforHumanandAnimalHealth(CSCHAH)
inWinnipeg,Canada.Supernatantswereharvestedat96hourspost-infection(hpi).Stocksof
vesicularstomatitisvirusexpressinggreenfluorescentprotein(VSV-GFP)werepropagatedin
VeroE6cellsandharvestedat72hpi.
Interferon. Humantype1IFN(α-1through14,β-1)andUniversalIFN(recombinant
αA/D)wereobtainedfromPBLAssayScience(Burlington,On.,Canada),dilutedto50,000U/
mlandstockswerestoredat-80°C.
VirusClearancebyIFN-α2aTreatment
A549andBHKcellswereseededin6-wellplatesfor80–90%confluencyattimeofinfection.
Thecellswereinfectedwith11TCID units(equivalenttoamultiplicityofinfection(MOI)of
50
0.00001)ofKFDVorVSV-GFPfor1hour,thenvirusmaintenancemediumsupplemented
withafinalconcentrationof2,000U/mlofIFN-α2awasaddedandincubatedfor96hours
(KFDV)and48hours(VSV-GFP);thiswasdesignatedaspassage0.Controlswellswere
treatedsimilarlywithouttheadditionofIFN.Supernatantswereharvestedandstoredat-80°C
fortitration.Theinfected-cellswerewashedwithPBS,passagedbyuseoftrypsin(HyClone)
andthecellsweresplit(1:3)intotwonew6wellplatewells.Thesepassage1sampleswere
treatedagainwith2,000U/mLIFN-α2aorwereleftuntreatedandre-incubatedfor72hours
(KFDV)and48hours(VSV-GFP).Theprocedurewasrepeatedfrompassage1topassage2.
Picturesfromvirus-infectedcellswerecapturedusinganEVOSmicroscopeforKFDVsamples
underCL-4conditionsandwithanAxiovert40CFLmicroscope(CarlZeiss,Toronto,Ontario,
Canada)forVSV-GFPsamplesunderCL-2conditions.
ScreeningofIFNα/βSubtypesagainstKFDV
A549cellswerepreparedin24-wellplateswithDMEMgrowthmediumfor80–90%con-
fluencyattimeofinfection.Inthepre-treatmentscenario,thecellsweretreatedwithorwith-
out1,000U/mloftheselectedIFN-α/βsubtypeswasaddedandincubated24hoursbefore
virusinfection.ThenthecellswereinfectedwithKFDVataMOIof1for1hour,inoculum
wasremoved,cellmonolayerswerewashedandvirusmaintenancemediumwasadded.Inthe
post-treatmentexperiments,KFDVataMOIof1wasadsorbedfor1hour,inoculumwas
removed,monolayerswerewashed,andthenfreshvirusmaintenancemediumsupplemented
withorwithout1,000U/mloftheselectedIFNα/βsubtypes.Supernatantsfromthree-inde-
pendentbiologicalreplicateswereharvestedandstoredat-80°Cforvirusquantificationafter
anincubationperiodof72hours.StatisticalsignificancewasdeterminedusingOne-way
ANOVAanalysisfollowedbyTukey’spost-test.
Dose-DependentAntiviralActivityofIFN-αWA,andIFN-α2aagainst
KFDVandVSV-GFP
Inhibitoryconcentrationsandtitrereduction. A549andBHK-21cellcultureswerepre-
paredin96-wellplatesfor80–90%confluence.Cellswereinfectedwith50μl/wellof11
TCID unitsofKFDVorVSV-GFP(equivalenttoaMOIof0.0003)inthreeindependent,
50
triplicatereplicates.Afteranadsorptionperiodof1hour,150μloffreshvirusmaintenance
mediumwasaddedwithIFN-α2aorαWApreparedfollowingatwo-folddilutionscheme(4,
000.0U/ml—3.9U/ml).Controlsamplesweremock-treatedandinfectedand,mock-treated
anduninfected.Whenmock-treatedinfectedcontrolreached90–100%CPEat96hpi(KFDV)
or48hpi(VSV-GFP),supernatantsfrom2,000.0,500.0,62.5,7.8U/mLandmock-treated
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004871 August1,2016 4/19
InterferonEffectsonKyasanurForestDiseaseVirus
controlswereharvestedandstoredat-80°Cfortitrationandthemonolayerswerefixedfor
IC andIC determination.Cellmonolayersweretreatedwith10%phosphate-bufferedfor-
50 90
malin,washedwithPBSandstainedwith0.5%(wt/vol)crystalviolet(FisherScientific,Ottawa,
Ontario,Canada)dissolvedinasolutionof70%methanol/30%PBS.Afterincubationfor30
minutesatroomtemperature,theexcessdyewasremovedwithtapwater.Onceair-dried,crys-
talvioletdyewaselutedin100μlof95%ethanolandscannedwithaMultiSkanAccentmicro-
platereaderat570nm(ThermoScientific,Mississauga,Ontario,Canada).Theinhibitory
concentrationistheamountofIFN(αWAorα2a)neededtoprotect50%(IC )and90%
50
(IC )ofcellsfromvirus-inducedCPE(KFDVandVSV-GFP).Thesevalueswerecomparedto
90
theun-infectedcontrolswhichrepresentedcellswithoutCPE(100%)andtotheinfectedcon-
trolsdefinedasfullCPE(0%protection).Valueswerecalculatedwithcurvefittingusing
GraphPadsoftware(Prism5).Forthereductionofvirustitres,un-infectedcontrolsrepre-
sentedalackofvirusproductionandfullvirusinfectiontitresweredefinedbytheinfected/
mock-treatedcontrols.
Virusquantification. BHK-21cellswereseededinto96-wellplatesfor80–90%con-
fluency.Virus-containingsupernatants(KFDVorVSV-GFP)wereseriallydiluted10-fold.
Fiftyμlfromeachdilutionwasaddedtotriplicatewellsandincubatedfor1hour.Then,150μl
ofvirusmaintenancemediumwasaddedandre-incubatedforCPEdevelopmentthatwaseval-
uated5dayspost-infectionforKFDVor72hpiforVSV-GFP.The50%tissuecultureinfec-
tious(TCID /mL)dosewascalculatedfollowingtheReedandMuenchformula[46].
50
Cellularcytotoxicity. A549andBHK-21cellscellcultureswerepreparedin96-wellplates
for80–90%confluencyandIFN-αWAandα2awereaddedfollowingatwo-folddilution
schemefrom16,000.0–3.9U/mL.Forcontrolpurposes,lowtoxicity(mock-treated)andhigh
toxicity(10%TritonX-100)(Sigma,Oakville,Ontario,Canada)wereincludedfortheanalysis.
Afterincubationfor96hours,monolayerswerepreparedandsubjectedtocrystalvioletstain-
ing,asdescribedabove.TheconcentrationofIFNthatcaused50%celldeathwasdefinedas
thecytotoxicityconcentration(CC ).ValueswerecalculatedwithcurvefittingusingGraph-
50
Padsoftware(Prism5).
IFNα/βSubtypesAntagonismAssays
Cloning. KFDVNS5,NS4B-2kandEbolavirusVP24geneswereclonedintothepCAGGS
mammalianexpression-vectorunderchickenbeta-actinpromotercontrol,asdescribedprevi-
ously[28].
VSV-GFPinfectionassays. VeroE6cellsweretransfectedusingFugene6(Promega,
Madison,Wi.,USA)withpCAGGSexpressingKFDVNS5,NS4B-2k,VP24proteinsormock
transfectedwithemptypCAGGSvectorfollowingthemanufacturer’srecommendations.Cells
werethentreated,post-transfectionwithvirusmaintenancemediumcontaining1,000U/mLof
eachIFN-α/βsubtypesorUniversalIFNfor24hours.Thesupernatantswereremovedandcells
wereinfectedwithVSV-GFP(MOIof2)in500μlofvirusmaintenancemediumfor1hourat
37°C/5%CO .Virusinoculumwasremovedandmonolayerswerewashedoncewithvirusmain-
2
tenancemediumandfreshmaintenancemediumwasadded.Afteranincubationperiodof24
hours,picturesweretakenwithlightandfluorescentmicroscopywithanAxiovert40CFLmicro-
scope(CarlZeiss)andsupernatantswereharvestedandstoredat-80°Cuntilquantification.
Results
IFN-α2aTreatmentDoesNotClearKFDVInfectionInVitro
Usingvirusclearanceassaysthatinvolvedsuccessivepassagingofinfectedcellculturesalong
withthere-applicationofantivirals[47,48],weinvestigatedtheefficacyofIFN-α2aagainst
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004871 August1,2016 5/19
InterferonEffectsonKyasanurForestDiseaseVirus
KFDVandforcontrolpurposes,againstVSV-GFP.Toaddressthis,bothA549andBHK-21
cellswereinfectedwitheitherKFDVorVSV-GFPat11TCID units(MOIof0.0001).After
50
infectionthecellsweretreatedwith2,000U/mLofIFN-α2aat1hpi(passage0).Virussuper-
natantswereharvestedfortitration,andpassage0infected-cellsweresubculturedintotwo
wellsandIFNwaseitheraddedorwithheld(passage1)andthisprocedurewasrepeatedfor
passage2.
Initiallythepassage0,IFN-treatedcellsshowedareductioninKFDVtitrefromthemock-
treatedcontrolsof100.8TCID /mL(p<0.1)and100.4TCID /mL(p<0.1)inbothA549and
50 50
BHK-21cellsrespectively.However,decreasesweremorestrikingfortheIFN-sensitive
VSV-GFPasreductionsof105.2TCID /mL(p<0.01)and106.5TCID /mL(p<0.01)were
50 50
observedinA549andBHK-21cellsrespectively(Fig1AandFig2A).
WhencomparingtheIFN-treatedcellsfrompassage0topassage1andpassage1topassage
2,onlyVSV-GFPshowedmarkeddifferencesintitres.Therewerenoapparentdecreasesin
KFDVtitresproducedfromIFN-treatedA549cells(passage1:decreaseof100.1TCID /mL
50
(notsignificant)frompassage0,andforpassage2:increaseof100.7TCID /mL(p<0.1)from
50
passage1)(Fig1A)andfromIFN-treatedBHK-21cells(passage1:increaseof100.6TCID /
50
mL(notsignificant)frompassage0,andpassage2:decreaseof100.3TCID /mL(notsignifi-
50
cant)frompassage2)(Fig2A).TheresultsfromKFDVcontrastthatofVSV-GFPinwhich
sharpdeclinesintitreswereobservedinA549cells(passage1:decreaseof101.6TCID /mL
50
(p<0.1)frompassage0,andareductionof101.7TCID /mL(p<0.01)frompassage1)(Fig
50
1A)andBHK-21cells(passage1:adecreaseof102.9TCID /mL(p<0.01),andpassage2:no
50
virusdetected)cells(Fig2A).
Asanticipated,whenIFN-treatedcellsfromeitherpassage0orpassage1weresubcultured
andnotfurthertreatedwithIFN,virustitresandCPEforbothKFDVandVSV-GFPwere
characteristicofthemock-treatedpassage0controls.DespitetheapparentinabilityofIFNto
significantlyimpactKFDVpropagation,theappearanceofCPEwasnotablyreducedcompared
tomock-treatedinbothcelltypestested(Fig1BandFig2B).EvenwiththelackofvisibleCPE
intheIFN-treatedcells,KFDVcouldnotbeeliminatedfrominfectedcellculturebytreatment
withIFN-α2a.
PreliminaryScreeningofIFN-α/βSubtypeswithTimeofAdditionagainst
KFDV
PreviousstudiesevaluatingIFNtreatmentagainstflavivirusesincellculturehaverevealedthat
IFNismoreeffectivewhenaddedtocellsbeforevirusinfectionandbecomeslesseffectiveover
timeonceinfectionhasbeenestablished[25,31,39,41].Tofurtherexpandourunderstanding
oftheefficacyofIFNtreatmentagainstKFDVinfection,weevaluatedtheantiviralactivityof
theIFN-α/βsubtypesinbothpre-infectionandpost-infectiontimeframes.
A549cellswereinfectedwithKFDV(MOIof1)andtreatedwith1,000U/mLofeachIFN-
α/β,whichwasaddedeither24hoursbeforeor1hourafterinfection.WhenCPEreached90–
100%inmock-treatedcontrols,supernatantswereharvestedandtitrated.Regardlessoftimeof
addition,bothIFN-αKandIFN-αWAweremoreeffectivethanIFN-α2aatreducingKFDV
titres(Fig3).Whencomparedtothemock-treatedcontrols,foldreductionsinpre-infection
andpost-infectiontimeframeswere16-fold(IFN-αK),14-fold(IFN-αWA)and3-fold(IFN-
α2a)(Fig3,greybars)and,132-fold(IFN-αKp<0.01),37-fold(IFN-αWAp<0.01)and6-fold
(IFN-α2ap<0.1)(Fig3,blackbars),respectively.
ThesedatasuggestthatIFN-αKandIFN-αWAmayinduceresponsesthataremoreeffec-
tivethanthoseproducedbyIFN-α2aforrestrictingKFDVinfectionregardlessofthetimeof
treatmentrelativetoinfection.
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004871 August1,2016 6/19
InterferonEffectsonKyasanurForestDiseaseVirus
Fig1.IFN-α2adoesnotclearKFDVinfectioninA549cells.A549cellswereinfectedwitha11TCID units(MOIof0.00001)oftheindicatedvirus,and
50
eithertreatedormock-treatedwith2,000U/mLofIFN-α2a(designatedasP0).MonolayerswerepassagedwhenuntreatedcontrolsreachedCPEof90%,
96and48hpiforKFDVandVSV-GFP,respectivelyand2,000U/mLofIFN-α2awaseitheraddedoromitted(P1)andafter72hpiforKFDVand48hpifor
VSV-GFP.Thisprocedurewasrepeatedagainforpassage2(P2).(A)Beforeeachpassage,supernatantswereharvestedfortitrationbyTCID assay
50
determinationonBHK-21cells.Theaveragesandstandarddeviationfromthreebiologicalreplicatesareshowngraphicallyandexpressedinlog scale
10
TCID /mL.Statisticalsignificanceisdenotedas*P<0.1,**P<0.05,***P<0.01.(B)Cellmonolayerswerevisualizedwithlightmicroscopypriorto
50
passaging.Mock,non-IFNtreated/infectedcontrols.UI,Un-infectedcontrols.
doi:10.1371/journal.pntd.0004871.g001
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004871 August1,2016 7/19
InterferonEffectsonKyasanurForestDiseaseVirus
Fig2.IFN-α2adoesnotclearKFDVinfectioninBHK-21cells.BHK-21cellswereinfectedwitha11TCID units(MOIof0.00001)oftheindicatedvirus,
50
andeithertreatedormock-treatedwith2,000U/mLofIFN-α2a(designatedasP0).MonolayerswerepassagedwhenuntreatedcontrolsreachedCPEof
90%,96and48hpiforKFDVandVSV-GFP,respectivelyand2,000U/mLofIFN-α2awaseitheraddedoromitted(P1)andafter72hpiforKFDVand48
hpiforVSV-GFP.Thisprocedurewasrepeatedagainforpassage2(P2).(A)Beforeeachpassage,supernatantswereharvestedfortitrationbyTCID
50
assaydeterminationonBHK-21cells.Theaveragesandstandarddeviationfromthreebiologicalreplicatesareshowngraphicallyandexpressedinlog
10
scaleTCID /mL.Statisticalsignificanceisdenotedas*P<0.1,**P<0.05,***P<0.01.(B)Cellmonolayerswerevisualizedwithlightmicroscopyprior
50
topassagingCellmonolayerswerephotographedpriortopassaging.Mock,non-IFNtreated/infectedcontrols.UI,Un-infectedcontrols.
doi:10.1371/journal.pntd.0004871.g002
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004871 August1,2016 8/19
InterferonEffectsonKyasanurForestDiseaseVirus
Fig3.Screeninginterferon-α/βsubtypesagainstKFDV.CulturesofA549cellswerepre-treated(greybars)24hourspriortoinfectionorpost-treated
(blackbars)1hourafterinfectionwith1,000U/mLofIFN-α(B2,C,D,F,G,H2,I,J1,K,WA,2a,2bor4b),IFN-β(beta-1)andarecombinantIFN-α
(Universal)subtypesandinfectedwithKFDVataMOIof1.Supernatantswereharvestedforeachtreatmentafter72hoursofincubationandquantified
(expressedinlog scaleTCID /mL)onBHK-21(ATCC)whenthemock-treatedcontrolcellsdisplayedCPEnear100%.Pre-infectiontreatment
10 50
experimentswereassayedintwobiologicalreplicatesandpost-infectiontreatmentexperimentswereassayedinthreebiologicalreplicates;theresulting
averagesandstandarddeviationsarepresented.Mock,Mock-treatedwithIFN.UI,Un-infectedcontrol.IFN-α2bwasexcludedfrom24-hourpre-
infectiontreatment.*Significantcomparedtomock-treatedsamples(P<0.1).***Significantcomparedtomock-treatedsamples(P<0.01).
doi:10.1371/journal.pntd.0004871.g003
Dose-DependentAntiviralActivityofIFN-αK,IFN-αWAandIFN-α2a
ThetwocandidateIFNsubtypes,IFN-αKandIFN-αWAwereobservedtobemoreeffective
thanIFN-α2aatrepressingKFDVreplicationataconcentrationof1,000U/mL.Tofurther
explorethisfinding,wesoughttodetermineifthepotencydifferencesofIFN-αWAcompared
toIFN-α2acanbemaintainedoverarangeofconcentrations.Thiswastestedbyanalyzingthe
abilityofeachIFNsubtypestoprotectA549andBHK-21cellsfromthecytopathologyof
KFDV(11TCID unitsequaltoMOIof0.0003)andaIFN-sensitivecontrolvirus(VSV-GFP)
50
(11TCID unitsequaltoMOIof0.0003)followingatwo-folddilutionschemeforeachIFN.
50
ThetraditionalapproachtodefiningIFNpotencywasutilized;theseassaysinvolvetheuseof
multiplecelltypesincludingA549cells,treatedwithIFNandchallengedwithastrongcytolytic
viruslikeVSV,thenanalyzedempiricallywithmonolayerstaining/spectroscopy[21,49–51].
Asanadditionalmeasure,IFNstrengthwasassessedbyinfectiousvirionproductiondeter-
minedbyTCID titrationfromselectIFNconcentrations.
50
Forthecytopathologyassessment,upperandlowerparametersweredefinedaseitherfull
CPE(0%cellprotection)frommock-treatedsamplesandabsenceofCPE(100%cell
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004871 August1,2016 9/19
InterferonEffectsonKyasanurForestDiseaseVirus
Table1. Antiviralactivityofinterferon-α/βagainstthecytopathologyofKFDVandVSV-GFP.
Interferon(IFN)Species KFDVIC (U/mL) VSV-GFPIC U/mL) KFDVIC (U/mL) VSV-GFPIC (U/mL) CC (U/mL)
50 50 90 90 50
A549a
WA 5.2 5.1 406.8 48.5 >16,000.0
2a 7.4 5.6 125.4 50.0
K 18.8 NDc 108.0 NDc
BHK-21b
WA 23.3 NDc 23,711.0 3,808.0 >16,000.0
2a 6.9 988.1 2,048.0 56.2
aIC ,CC andIC valuesfromthreetechnicalreplicatesofeachofthreebiologicalreplicatesinA549cells.
50 50 90
bIC ,CC andIC valuesfromthreetechnicalreplicatesofeachofonebiologicalreplicatesinBHK-21cells.
50 50 90
cND=notdetermined.
doi:10.1371/journal.pntd.0004871.t001
protection)fromun-infectedsamples,therebyallowingIC andIC tobedeterminedfor
50 90
eachIFN.InitiallybothIFNsubtypesappearedequallyaspotent,astheIC valuesforIFN-
50
αWAandIFN-α2awerecomparableagainstKFDV(5.2and7.4U/mL)andVSV-GFP(5.1
and5.6U/mL)inA549cells(Table1).However,thesametrendwasnotobservedinBHK-21
cellswithIC valuesof23.3(IFN-αWA)and6.9U/mL(IFN-α2a)forKFDV.AnIC of988.1
50 50
U/mLwasdeterminedforIFN-α2aagainstVSV-GFPandavaluecouldnotbedetermined
(ND)forIFN-αWA.ThismayhavebeenbecausetheamountofIFNrequiredtoreachtheIC
50
wasgreaterthanthehighestconcentrationtestedandthereforecouldnotbedeterminedusing
thecurvefittingsoftware(Table1).IC valuesobtainedfromKFDV-infectedA549cellsthat
90
weretreatedwithIFN-αWA(406.8U/mL)andIFN-α2a(125.4U/mL)weresignificantly
higherthanVSV-GFP-infectedA549cellsthatweretreatedwithIFN-αWA(48.5U/mL)and
IFN-α2a(50.0U/mL)(Table1).AsimilartrendwasobservedforKFDV-infectedBHK-21
cells.The90%protectionfromIFN-αWA(23,711.0U/mL)wasextrapolatedbycurvefitting
softwareasitwasmuchgreaterthanthehighestamounttested(16,000.0U/mL).Thispre-
dictedvaluewassignificantlyhigherthanthatofIFN-α2a(2,048.0U/mL).Withrespectto
VSV-GFP-infectedBHK-21cellsthatweretreatedwithIFN,theIC wasdeterminedtobe3,
90
808.0U/mLforIFN-αWAand56.2U/mLforIFN-α2a(Table1).IFN-inducedcytotoxicity
(CC )wasnotdeterminedasaconcentrationof16,000.0U/mL,anamountthatwasthree
50
timeslargerthanthehighestconcentrationusedforexperimentsledtocelldeathinA549
(25%cytotoxicity)andBHK-21(11%cytotoxicity)cells.Forcontrolparameters,un-treated
(0%cytotoxicity)and10%TritonX-100-treated(100%cytotoxicity)samplesdefinedthe
upperandlowerlimitsofCC (Table1).Whileitwasapparentthathighconcentrationsof
50
bothIFN-αWAandIFN-α2aarerequiredtopreventKFDV-inducedCPE,theexperiments
utilizingBHK-21cellshighlighttheproblemsofobtainingqualitativedataforIFN-protection
viaCPE,especiallyagainstVSV-GFP.
TofurtherdefineIFNpotencywithrespecttothevariationofICvaluesanddose-depen-
dence,virustitresfromarangeofIFNconcentrations(2,000.0,500.0,62.5and7.8U/mL)
selectedfromthetwo-foldIFNdilutionexperimentsinbothA549andBHK-21cellswerecom-
paredtoun-treatedcontrols.ThiswascomparedastitrereductionsforbothKFDVand
VSV-GFP(controlvirus).Forthehighest(2,000.0U/mL)andlowest(7.8U/mL)concentra-
tionsassessedforKFDV-infectedA549cells,titrereductionsof100.7and100.5TCID /mL
50
(IFN-αWA)and,100.5and100.4TCID /mL(IFN-α2a),respectively,weremeasured.InBHK-
50
21cellsinfectedwithKFDV,titrereductionswerenotobservedfor2,000.0and7.8U/mLof
IFN-αWA;however,reductionsof100.9and100.1TCID /mLwereobservedforIFN-α2a.By
50
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0004871 August1,2016 10/19