IFNc plasmid expression of Protection salmon of Atlantic against virus infection by intramuscularinjection Vaccine

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Vaccine

jo u rn al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e

Protection of Atlantic salmon against virus infection by intramuscular injection of IFNc expression plasmid

Chia-Jung Chang, Camilla Robertsen, Baojian Sun, Børre Robertsen

NorwegianCollegeofFisheryScience,UniversityofTromsø,9037Tromsø,Norway

a r t i c l e i n f o

Articlehistory:

Received13March2014

Receivedinrevisedform8May2014 Accepted20May2014

Availableonline2July2014

Keywords:

Interferon Antiviral Virus Atlanticsalmon Fish

DNAvaccine

a b s t r a c t

InthisworkwehavetestedtheinvivoantiviralactivityoftypeIinterferons(IFNs)inAtlanticsalmon byinjectingpresmoltsintramuscularly(i.m.)withplasmidsencodingIFNa1,IFNborIFNcunderthe controlofaCMVpromoter,andmeasuredexpressionofantiviralgenesinorgansandprotectionagainst infectionwithinfectioussalmonanemiavirus(ISAV)infection.AllthreeIFNplasmidsinducedexpression ofantiviralgenes(Mx,Viperin,ISG15andIFIT5)atthemuscleinjectionsitewhilethecontrolplasmid hadlittleeffect.OnlyIFNbandIFNcplasmidsinducedexpressionofantiviralgenesinheadkidney,liver andheart.ThissuggeststhatIFNbandIFNcaredistributedsystemicallywhileIFNa1isactiveonlyatthe injectionsite.InjectionofIFNcplasmidwasfoundtoinduceexpressionofantiviralgenesandreceptors forvirusRNA(RIG-I,TLR3andTLR7)inheadkidneyfrom1toatleast8weeks.Immunoblottingshowed increasedexpressionofISG15andMxproteininliverwithtimeduringthistimeperiod.Challengeof presmoltswithISAV8weeksafterinjectionofIFNplasmids,showedstrongprotectionoftheIFNcplasmid injectedfish,lowprotectionoftheIFNbplasmidinjectedfishandnoprotectionoftheIFNa1plasmid injectedfish.CluestothedifferenceinprotectionobtainedwithIFNbandIFNcplasmidswerefound byimmunohistochemicalandimmunoblotstudiesofMxprotein,whichindicatedthatIFNcplasmid stimulatedstrongerMxproteinexpressioninhearttissuesandliverendothelialcellsthanIFNbplasmid.

Takentogether,thesedatasuggestthati.m.injectionoftheIFNcexpressionplasmidmaybeanewmethod forprotectingAtlanticsalmonagainstvirusinfection.

©2014TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

FarmedAtlanticsalmonisattackedbyseveralviruses,which representacontinuousthreattotheindustry.Traditionalvaccines based on inactivated virus are available for infectious pancre- aticnecrosisvirus(IPNV),salmonpancreasdiseasevirus(SPDV) andinfectioussalmonanemiavirus(ISAV)andasubunitvaccine basedonrecombinantproteinisavailableforIPNV[1],butthese vaccinesdonotappeartogivesatisfactoryprotectioninthefarm- ingsituation.DNAvaccinationprovidesahighlevelofprotection against infectious hematopoieticnecrosis virus(IHNV), but not

Abbreviations: IFN,interferon;ISG,interferon-stimulatedgene;TLR,Toll-like receptor;RIG-I,retinoicacid-inducibleproteinI;Viperin,virusinhibitoryprotein endoplasmatic reticulum associated interferon-inducible; ISG15, interferon- inducedproteinencodedbytheISG15gene;IFIT,interferon-inducedproteinwith tetratricopeptiderepeats;Mx,myxovirusresistance;ISAV,infectioussalmonane- miavirus;i.m.,intramuscular;i.p.,intraperitoneally;RT-qPCR,reversetranscription quantitativePCR;RPS,relativepercentsurvival.

∗Correspondingauthor.Tel.:+4777644487;fax:+4777644900.

E-mailaddress:borre.robertsen@uit.no(B.Robertsen).

otherviruses[1].Thiscallsforimprovedmethodsforprotection offarmedsalmonagainstvirusdiseases.

ThediscoveryoftypeIIFNsinfishopensapossibilityforusing theminprophylaxisagainstvirusinfectionsinfish.TypeIIFNsare induceduponhostcellrecognitionofviralnucleicacids[2],and protectothercellsagainstinfectionbyinducingnumerousantiviral proteinssuchasMx,ISG15,IFIT5(ISG58)andViperin[3–5].Infish, fourtypeIIFNsubtypes,namedIFNa,IFNb,IFNcandIFNd,haveso farbeencharacterized[6,7].IFNaandIFNdcontain2cysteines(2C- IFNs)whileIFNbandIFNccontain4cysteines(4C-IFNs).Thelargest clusterofIFNgeneshasbeenfoundinAtlanticsalmon,encoding twoIFNa,fourIFNbandfiveIFNcgenes[6].

AtlanticsalmonIFNa,IFNbandIFNcandIFNdhaveonly22–37%

aminoacidsequenceidentityandshowmajordifferencesincellu- larexpressionpropertiesandantiviralactivities[6,8].IFNa1and IFNc induced similar strong antiviral activity against IPNV and inducedsimilartranscriptlevelsofantiviralgenesincelllines,IFNb waslessactiveandIFNdshowednoantiviralactivity[8].IFNa1, IFNbandIFNcprovidedonlytransientinhibitionofISAVreplication inTOcells[9].

Inhumans,pegylatedrecombinantIFN-␣,mostlyincombina- tionwithribavirin,isusedfortreatmentofchronichepatitisCvirus http://dx.doi.org/10.1016/j.vaccine.2014.05.059

0264-410X/©2014TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).

arescarce.TreatmentofrainbowtroutwithrecombinantAtlantic salmonIFNa2injectedintraperitoneally(i.p.)providedprotection againstIHNVinfectionforupto7days,whichisnotenoughforpro- phylaxisoffarmedfish[14].Inthepresentworkwehaveuseda morenovelapproachbystudyingantiviraleffectsofintramuscular (i.m.)injectionofIFNexpressingplasmidsinAtlanticsalmon.The resultsshowedsurprisingdifferencesamongIFNa,IFNbandIFNc plasmidsintheirabilitytoinducesystemicexpressionofantiviral genesandtoprotectsalmonfrominfectionwithahighvirulent strainofISAV.Notably,i.m.injectionofIFNcplasmidprovidedsys- temicup-regulationofantiviralgenesinsalmonforatleast8weeks accompaniedbyahighlevelofprotectionagainstISAVinfection.

2. Materialsandmethods 2.1. Fish

Atlanticsalmon(SalmosalarL.)presmolts(35–45g)ofthestrain Aquagenstandard(Aquagen,Kyrksæterøra,Norway)werekeptat TromsøAquacultureResearchStation,Norwayin300ltankssup- pliedwithfreshwaterat10^◦Candwerefedcommercialdryfood.

Priortotreatments,thefishwereanesthetizedwith0.005%ben- zocaine(ACDPharmaceuticals,Norway).Fishgroupswerelabeled bytattooing(2%alcianblue,Panjetinoculator).Thefishwerekilled byanoverdosebenzocainepriortoharvestoforgans.Allhandling offishwasinaccordancewiththeNorwegian“RegulationonAni- malExperimentation”andallfishexperimentsweresubmittedto andapprovedbytheNorwegianAnimalResearchAuthority(NARA) beforeinitiation.

2.2. Plasmidsusedforintramuscularinjection

Interferonplasmidsencodingtheopenreadingframe(ORF)of AtlanticsalmonIFNa1,IFNbandIFNcwereavailablefromapre- viousstudy[15].AllthethreeIFNORFsweresub-clonedintothe pcDNA3.3-TOPOvector(Invitrogen)downstreamoftheCMVpro- moter.AreligatedpcDNA3.3plasmidwithoutinsertwasusedas negativecontrol.Plasmidsweretransformedandgrown inOne ShotTOP10Escherichiacoli(Invitrogen)andpurifiedbyEndoFree plasmidpurificationkit(Qiagen).

2.3. Antibodies

PolyclonalantibodiesagainstAtlanticsalmonMxandISG15pro- teinswereasdescribed[16,17].

2.4. FishexperimentsforRT-qPCR,immunoblottingand immunohistochemistry

Threeexperimentswereperformedwherefivegroupsofpres- molts kept in one tank were injected intramuscularly (i.m.) approximately1cmbelow thedorsal finwith15␮gplasmidin 50␮lsterilephosphate-bufferedsaline(PBS)atpH7.4orwithPBS only.In Experiments1–3,fishgroupswereinjectedwithIFNa1, IFNbor IFNc plasmidor controlplasmid. In Experiment4, fish groupswereinjectedwithIFNc,controlplasmidorPBS.Muscle tissueattheinjectionsiteand organswereharvested atdiffer- enttimeintervalsafterinjectionandstoredinRNAlater(Ambion) forRNAextractionorstoredinliquidnitrogenforproteinextrac- tion.Experiment1(Fig.1):muscle,headkidneyandliverwere

andheadkidneyweresampled(n=5)atalltimepointsforRT- qPCR(Fig.2A,BandC).Muscle,liver,spleen,gut,heartandgill wereharvested(n=5)forRT-qPCRat7dpi(SupplementaryFig.2).

Liverswereharvested(n=4)forimmunoblottingat7,21and56dpi (Fig.3).

2.5. ChallengeexperimentwithISAV

Groupsofpresmolts(50fishpergroup)keptinonetankwere injectedi.m.withIFNplasmids,controlplasmidorPBSasdescribed in2.3.Eightweeksafterinjectioneachfishwasinjectedi.p.with 100␮l L-15 medium containing 10⁴ TCID50 units of the ISAV Glesvaer/2/90strain[9].Mortalitywasrecordedeverydayand28 dayspost-virusinjectionrelativepercentagesurvival(RPS)inthe groupswascalculatedas[1−(%mortalityintestgroup/%mortality incontrolplasmidgroup)]×100.

2.6. ReversetranscriptionquantitativePCR(RT-qPCR)

OrgansamplesorleukocyteswerecollectedinRLTbufferand RNAwasisolatedwiththeRNeasyMinikit(Qiagen).Onemicro- gram RNA was subjected to cDNA synthesis using QuantiTect ReverseTranscriptionKit(Qiagen).TranscriptsofIFNs,Mx,ISG15, Viperin,IFIT5(alsonamedISG58),RIG-I,TLR7,TLR3incDNAfrom organsorleucocyteswereanalyzedbyqPCRusing7500FastReal- TimePCR System(Applied Biosystems)as described previously [15].Relativequantificationsofgenetranscriptswereperformed bythePfafflmethod[18],usingElongationFactor1␣B(EF1␣B)as referencegene[19].

2.7. DetectionofMxandISG15proteinexpressionby immunoblotting

Frozenorganswereweighedandtransferredto2mlmicrotubes andtissuelysisbuffer(TissueExtractionReagentI,Invitrogen)was added(100mgtissuein100␮llysisbuffer).Homogenizationwas performedwithPrecellysbeadsandhomogenizer(Precellys^®24, BertinTechnologies)at5900rpmfor20s.Aftercentrifugationfor 5min at10,000×g at 4^◦C, proteinconcentration in thesuper- natantswasmeasuredwithBCAproteinassaykit(Pierce,Thermo Science).Supernatants(10␮gproteinperwell)weresubjectedto LDS-electrophoresisona4–12%NuPAGEBis-TrisGel(Invitrogen).

Blotting,antibodyincubationsanddevelopmentofblotsweredone asdescribedpreviously[9].

2.8. DetectionofMxproteinexpressionbyimmunohistochemistry

Organswerefixedin4%paraformaldehydeinPBSfor24hat 4^◦Candembeddedinparaffinwaxbyroutineprocedures.Tissue sections(4␮m)werecutandmountedontopoly-l-lysinecoated slides,driedandclearedwithHistoClearsolution(NationalDiag- nostics).Afterrehydration,slideswereboiledin10mMsodium citratebuffer (pH6.0) for 30minfollowed by incubationin 1%

hydrogenperoxidefor 15min.Theslideswereblockedwith5%

nonfatdriedmilkpowder(AppliChem)for2handsubsequently incubatedwithanti-Mxantibody(1:500)for16hat4^◦Candwith HRP-conjugatedantibody(1:2000,goatanti-rabbitIgG,Invitrogen) for1h.RedcolorshowingMxstainingwasdevelopedbyincuba- tionwith100␮lAECSubstrateChromogen(Dako)for10minand thesectionswerethencounterstainedwithMayer’shematoxylin (Sigma).

Fig.1.Inductionofantiviralgenesinsalmonpresmoltsafteri.m.injectionofIFNexpressionplasmids.Fivegroupsofpresmoltswereinjectedi.m.withPBS,controlplasmid (CP)orplasmidsexpressingIFNa1,IFNborIFNc,respectively(n=5).ExpressionofIFNa1,IFNb,IFNc,Mx,Viperin,ISG15andIFIT5wasmeasuredbyRT-qPCR7daysafter injection.AandB,muscletissueattheplasmidinjectionsite.CandD,headkidney.EandF,liver.TranscriptionofIFNa,IFNbandIFNcinmuscleispresentedasrelativemRNA abundancecomparedtotheElongationFactor1␣Breferencegene,whileothervaluesarepresentedasfoldincreaseintranscripts(mean±SD)comparedtoPBSinjected fish.Statisticalsignificantdifferences(p<0.05)betweenIFNplasmidgroupsandcontrolplasmidgroupareindicatedwithastar(*).

2.9. Statisticalanalysis

Statistical analyses were performed using GraphPad Prism vision6.01forWindows.Genetranscriptsinorgansorleukocytes werecomparedusinganunpairedStudent’st-testandconsidered asstatisticallysignificantatp≤0.05.Thedifferencesinmortality andsurvivalratewerecomparedusingchisquaretestandconsid- eredasstatisticallysignificantatp≤0.01.

3. Results

3.1. Expressionofantiviralgenesinorgansafterintramuscular (i.m.)injectionwithIFNexpressionplasmids

Asexpected i.m. injectionof expressionplasmids for IFNa1, IFNbandIFNcintoAtlanticsalmonpresmoltsresultedinstrong expressionoftherespectiveIFNsinthemuscletissue (Fig.1A).

Consequently,allthreeIFNplasmidscausedstronginductionofthe antiviralgenesMx,Viperin,ISG15andIFIT5atthemuscleinjection

site(Fig.1B).ThisismostlikelyduetoreleaseofIFNfrommus- clecellsthathavetakenupplasmid,sincetransfectionoftheIFN expressionplasmidsintoHEK293cellsresultedinsecretionoffunc- tionalIFNs[8].IFNa1plasmidseemedtohaveasomewhatstronger effectcomparedtotheIFNbandIFNcplasmids,whichhadsimilar effects.

Interestingly,i.m.injectionsofbothIFNbandIFNcplasmidsalso causedup-regulationoftheantiviralgenesinheadkidney(Fig.1D) andliver(Fig.1F)whilstneitherIFNa1norcontrolplasmidhadany effect.Similarresultshavebeenobservedinfourindependentfish experiments.InjectionsofIFNbandIFNcplasmidscausedaminor up-regulationofIFNaandIFNbinheadkidneywhileIFNcexpres- sionwasunchanged(Fig.1C).NoneoftheIFNswereup-regulated inliverbyinjectionsoftheIFN-plasmids(Fig.1E).Takentogether, thissuggeststhati.m.injectionofIFNbandIFNcplasmidscause systemicup-regulationofantiviralgenesduetoreleaseofIFNsat themuscleinjectionsite whileIFNa1plasmidonlyup-regulates ISGsattheinjectionsite.Mxexpressionwascomparedinseveral organsoffish7daysafterinjectionofIFNcplasmid,whichshowed

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Fig.2.TimecoursestudyofIFNcandISGstranscriptlevelsinfishinjectedwithIFNcplasmid,controlplasmidorPBS.(A)ExpressionofIFNc,Mx,ViperinandISG15inmuscle tissueattheplasmidinjectionsite.(B)Expressionofantiviralgenesinheadkidney.(C)ExpressionofviralRNAreceptorsinheadkidney.Geneexpressionwasmeasuredby RT-qPCRattheindicateddayspost-injection.ValuesarefoldincreaseintranscriptscomparedtoPBSinjectedfish(n=5).Blackbars:IFNcplasmidgroup,whitebars:control plasmidgroup.

highestincreaseinliverfollowedbyheart,headkidney,spleen,gut andgills(Suppl.Fig.1).

Supplementary Fig. 1 related to this article can be found, intheonlineversion,athttp://dx.doi.org/10.1016/j.vaccine.2014.

05.059.

3.2. EffectofIFNa1andIFNconexpressionofISGsinheadkidney leucocytes

Since the IFNc plasmid, but not theIFNa1 plasmid induced expression of ISGs in head kidney, we wanted to study if

Fig.3.TimecoursestudyofMxandISG15proteinexpressioninliverofpresmoltsinjectedi.m.withIFNcplasmid,controlplasmidorPBS.Livertissueswereharvested at7,21and56daysafterinjectionandanalyzedforMxandISG15expressionbyimmunoblotting.Gelswereloadedwith10␮gproteinfromeachoffourfishfromPBS group(lanes1–4),IFNcplasmidgroup(lanes5–8)andcontrolplasmidgroup(CP,lanes9–12).Blotswereincubatedwithanti-Mxandanti-actinantibodyoranti-ISG15and anti-actinantibodyandarerepresentativeoftwoindependentexperiments.

recombinant IFNa1 and IFNc might have different effects on inductionofISGsinheadkidneyleucocytes.However,recombi- nantIFNa1andIFNcup-regulatedtheantiviralgenesMx,ISG15, ViperinandIFIT5(ISG58)tosimilarextentsinheadkidneyleuco- cytes(Suppl.Fig.2A).Moreover,IFNa1andIFNcalsoup-regulated similarly the viral RNA receptors RIG-I, TLR3 and TLR7, which activateIFNtranscriptionuponbindingofvirusRNA(Suppl.Fig.

2B).LackofsystemicinductionofISGsbyIFNa1plasmidisthus notlikelytobeduetolackofresponsetoIFNa1inorgans.

SupplementaryFig.2relatedtothisarticlecanbefound,inthe onlineversion,athttp://dx.doi.org/10.1016/j.vaccine.2014.05.059.

3.3. Timecoursestudyofantiviralgeneexpressioninmuscleand headkidneyafterinjectionofIFNcplasmid

Tostudyifi.m.injectionofIFNcplasmidhadaprolongedeffect onexpressionof antiviralgenesinsalmon,groupsofpresmolts werei.m.injectedwithIFNcplasmid,controlplasmidorPBSand measuredforgenetranscriptsinmuscleattheinjectionsiteand headkidneyforaperiodof5–56days.IFNc,Mx,ViperinandISG15 expressionwereincreasedinmuscleofIFNcplasmidinjectedfish throughouttheexperimentalperiod(Fig.2A).IFNcshowedhigh- estexpressioninmuscleatday14afterinjectionandadeclining expressioninthefollowsamplingdays.Mxexpressioninmuscleof IFNcplasmidinjectedfishwashighestatday7andthendeclined whileISG15waselevatedthroughday35anddeclinedatday56.

Mxexpressioninheadkidneywashighestatday7,declinedtoa lowlevelatday14andthengraduallyincreased(Fig.2B).Asimilar trendofexpressioninheadkidneywasfoundforISG15,IFIT5and Viperin,andthevirusRNAreceptorsRIG-I,TLR3andTLR7(Fig.2C).

3.4. TimecoursestudyofMxproteinexpressioninliverafter injectionofIFNcplasmid

SinceweobservedincreasedISGlevelsinheadkidneythrough- outthe56daysafterinjectionofIFNcplasmid,wewantedtostudy ISGproteinlevelsininternalorgans.Forthispurpose,weperformed immunoblottingofMxandISG15proteinsinliverat7,21and56 daysafteri.m.injectionofIFNcplasmid,controlplasmidandPBS.

AsshowninFig.3,Mxproteinwashardlydetectedinliverfrom controlplasmidandPBSinjectedfishatanytimepoint.Incontrast, Mxproteinwasdetectedinliverofall4individuals7daysafter injectionofIFNcplasmidandincreasedatday21and56.Asimilar increaseinexpressionpatternwasobservedforISG15(Fig.3).

3.5. EffectofIFNplasmidsonprotectionofsalmonagainst infectionbyISAV

SinceinjectionofIFNbandIFNcplasmidinducedantiviralgenes systemicallyinAtlanticsalmon,wewantedtofindoutiftheIFN plasmidsmightprovideprotectionofsalmonagainstvirusinfec- tion.Forthispurposewechosetochallengethefishwithahigh virulentstrainoftheorthomyxovirusISAV,whichisknowntocause ahighlevelofmortalityinsalmoninchallengeexperiments[20].

Groupsofpresmoltswereinjectedi.m.withIFNa1plasmid,IFNb plasmid,IFNcplasmid,controlplasmidorPBSandkeptinafresh watertankfor8weeksbeforeinjectionwith10⁴TCID₅₀Unitsof ISAV4.Mortalitystartedtodevelopatday16post-infectionand reached82%and91%inthePBSandcontrolplasmidgroups,respec- tively,atday28whentheexperimentwasterminated(Fig.4).The mortalityintheIFNa1plasmidinjectedfishdevelopedatasimilar rateasinthecontrolgroupsandreached86%whilethemortality intheIFNbplasmidinjectedfishdevelopedsomewhatslowerand reached75%,whichgivesarelativepercentsurvival(RPS)of5.5%

(IFNa)and17.6%(IFNb)(p>0.05).Incontrasttotheothergroups, theIFNcgroupdidnotshowmortalityuntilday26andreached atotalmortalityofonly6%attheendoftheexperiment,which gives aRPS of93.4% (p<0.01).Similarresultswereobtainedin anotherchallengeexperiment.Theseexperimentsthusconfirmed theantiviralgeneresults,whichsuggestedthatinjectionofIFNa1 plasmiddoesnotprovidesystemic protectionofsalmonagainst virusinfectionwhileinjectionofIFNcplasmidgivesahighlevel ofprotectionevenafter8weeks.Surprisingly,however,theIFNb plasmidonlyprovidedalowlevelofprotectiondespitethefactthat italsocausedsystemicinductionofantiviralgenes.

3.6. Comparisonofantiviralproteinexpressioninliverandheart afterinjectionofIFNplasmids

Asthe IFN plasmids showedsucha large differencein pro- tectiveeffect8weeksafterinjection,wewantedtostudyifthey induced differentlevelsof antiviral proteinsin liverand heart, whicharestronglyaffectedbyISAVinfection.Immunoblottingof MxandISG15wereusedforthispurpose.AsshowninFig.5Aand B,fishinjectedwithIFNbandIFNcplasmidsshowedsimilarstrong

Fig.4.EffectofIFNplasmidsonprotectionofsalmonagainstinfectionbyISAV.

Groupsofpresmolts(n=50)wereinjectedi.m.withIFNa1plasmid,IFNbplasmid, IFNcplasmid,controlplasmidorPBS.Eightweeksafterinjectionthefishwere injectedi.p.with10⁴TCID50unitsofISAV.Mortalityinthegroupswasrecordedeach dayfrom1to28daysafterinjectionandRPSwascalculatedfromthecumulative mortalityatday28.

Fig.5. ImmunoblottingofMxandISG15proteininliverandheartofpresmoltsafter i.m.injectionofIFNa1,IFNb,IFNcorcontrolplasmid.Livertissueswereharvested atday56afterinjectionandanalyzedforMx(A)andISG15(B).Lanes1–3:control plasmid(CP).Lanes4–6:IFNa1plasmid.Lanes7–9:IFNbplasmid.Lanes10–12:IFNc plasmid.Non-specificbindingwiththeanti-ISG15antibodywasobservedwitha 90kDaproteinband(indicatedwith*)inallfishsamples.(C)Hearttissueswere harvestedat14daysafterinjectionandanalyzedforMxexpression.Lane1:Control plasmid(CP).Lanes2–5:IFNa1plasmid.Lanes6–9:IFNbplasmid.Lanes10–13:IFNc plasmid.Eachlanewasloadedwith10␮gproteinfromoneindividualexceptlane1 in(C),whichrepresentspooledproteinextractsfromfourfishinjectedwithcontrol plasmid.Actinwasusedasacontrolforequalloadingofprotein.

expressionofMx,freeISG15orISG15conjugatesinliver8weeks afterinjectionwhilefishinjectedwithIFNa1plasmidorcontrol plasmidshowedfaintornoexpressionoftheseproteins.Thesedata didthusnotresolvethedifferenceinprotectionobtainedwiththe IFNbandIFNcplasmids.However,IFNcplasmidinducedahigher levelofMxproteininheartcomparedtoIFNbplasmidalthoughthis experimentwasconducted14daysafterplasmidinjection(Fig.5C).

Mxproteinwasatsimilarlowlevelsinheartoffishinjectedwith IFNa1andcontrolplasmid.

3.7. ImmunohistochemistryofMxproteininheartandliverfrom fishinjectedwithIFNa1,IFNbandIFNcplasmids

ThedifferenceinprotectiveeffectsbetweenIFNbandIFNcplas- midsmightbeduetodifferencesininductionofantiviralproteins incelltypes,whichareimportantforISAVinfectivity.Accordingly, wedecidedtodoimmunohistochemistryofMxproteininliverand heartoffish8weeksafterinjectionwithPBSorIFNa1,IFNband IFNcplasmids(Fig.6).Mx-stainingwasobservedthroughoutthe livertissuefromIFNbandIFNctreatedfish(Fig.6CandD)while littleMx-stainingwasseeninliverofPBSandIFNa1treatedfish (Fig.6Aand B).IntheIFNbandIFNcgroups, Mxwasrelatively stronglystainedinsomecellsresemblingmammalianKuppfercells andmoreweaklystainedinhepatocytes.Interestingly,endothelial cellsofbloodvesselsappearedtobemorestronglystainedforMx inliverfromfishtreatedwithIFNcplasmidthanfromfishtreated withIFNbplasmid.Inheart,stratumcompactumandstratumspon- giosumwasstronglystainedinIFNcplasmidtreatedfish(Fig.6H), butmoreweaklystainedinfishtreatedwithIFNbplasmid(Fig.6G).

HeartfromfishtreatedwithPBSorIFNa1plasmidshowedlittleor nostaining(Fig.6EandF).

theseIFNsdeliveredasgenesinexpressionplasmidsinjectedi.m., whichdemonstratedthatIFNbandIFNcplasmids,butnotIFNa1 plasmidinducedsystemicup-regulationofantiviralgenesinlive Atlanticsalmon.Notably,onlyi.m.injectionofIFNcplasmidpro- videdahighlevelofprotectionofAtlanticsalmonagainstinfection byahighvirulentstrainofISAVforatleast8weeksafterinjection.

ThefactthatallthreeIFNexpressionplasmidsinducedsimi- larlevelsofISGtranscriptsatthemuscleinjectionsite,suggests thatsimilaramountsofIFNa1,IFNcandIFNbwereproducedby themusclecells.Incontrast,onlyIFNbandIFNcplasmidsinduced antiviralgenesinheadkidney,liverandheart.Thelackofinduction ofantiviralgenesbyIFNa1plasmidinjectionisnotduetolackof effectofIFNa1onheadkidneycells,sincerecombinantIFNa1and IFNcinducedsimilarlevelsofISGtranscriptsinheadkidneyleuco- cytes.TheseresultsthussuggestthatIFNcandIFNbaredistributed throughthecirculationandinduceantiviralgenessystemicallyin thefishwhile IFNaisonlyactiveattheproductionsite. During avirusinfection,IFNais thusprobablymainlyimportantatthe virusinfectionsitewhileIFNcandIFNbmaybedistributedsystemi- callyandtriggersynthesisofantiviralproteinsincellsthroughout thefishbody.InthiscontextIFNcappearstobeamainplayerin innateantiviralresponsesofAtlanticsalmonsinceitisproduced byavarietyofcelltypes,isinducedbybothviraldsRNAandssRNA analogsandhasequallystrongantiviralactivityasIFNa1[8].While IFNbisalsodistributedsystemically,ithaslessantiviralactivity thanIFNaandIFNc,isproducedmainlybyspecializedleukocytes andwasmainlyinducedbythessRNAanalog[8].Thedifference indistributionpropertiesofIFNacomparedtoIFNbandIFNcmay haveseveralexplanations.Thenumberofdisulphidebridgesmight possiblyinfluencethedegradationrateoftheIFNs.IFNaisa2C- IFN,whichcontains onedisulphidebridge,whileIFNbandIFNc are4C-IFNs,whichcontaintwodisulphidebridges[21].However, theisoelectricpointsofIFNa1(pI9.2)andIFNb/IFNc(pI6.9/pI5.1) arealsoquitedifferentandmightinfluencetheirdistributionand degradationproperties.

ThetimecoursestudyshowedthatIFNcplasmidinducedup- regulationofnotonlyantiviralgenes(Mx,ISG15,Viperin,IFIT5), butalsogenesforreceptorsofvirusRNA(RIG-I,TLR3andTLR7)in headkidneythroughoutthe8weekexperimentalperiod.Thissug- geststhatfishinjectedwithIFNcplasmidindeedpossessincreased innateimmunitytovirusinfectioncomparedtofishinjectedwith IFNa1orcontrolplasmid.IncreasedexpressionofMxandISG15 protein wasconfirmedboth in liver and heart ofIFNc plasmid injectedfish8weeks afterinjection.It isthushighlylikelythat injectedIFNcplasmidmaycontinuetoprovidesystemicexpres- sionofantiviralgenesbeyondthe8weeksexperimentalperiod.

ThisfindinginspiredustoinvestigateifinjectionofIFNcplasmid mightinfactprovideprotectionofAtlanticsalmonagainstvirus infectionevenat8weeksafterplasmidinjection.Forthispurpose wechoseahighvirulentstrainofISAV,whichisanorthomyxovirus thatcauseshighmortalityinAtlanticsalmonpresmolts.Thechal- lengeexperimentindeedshowedthatIFNcprovidedahighlevel ofprotectionagainstISAVinducedmortalityinsalmon8weeks afterplasmidinjectionwhileIFNa1andcontrolplasmidprovided noprotectioncomparedtoPBSinjectedfish.Surprisingly,injection ofIFNbplasmidgavealowlevelofprotectionagainstISAVinfection despitethefactthatIFNbandIFNcplasmidsinducedcomparable amountsofMxandISG15proteininliver8weeksafterinjection.

ThismaybeduetothatIFNbandIFNcusedifferentreceptorsand consequentlyinduceantiviralproteinsindifferentcelltypes.This

Fig.6. ImmunohistochemistryofMxproteininliverandheart8weeksafteri.m.injectionofIFNplasmids.Liversectionsfromfishinjectedwithcontrolplasmid(A),IFNa1 plasmid(B),IFNbplasmid(C)andIFNcplasmid(D).Heartsectionsfromfishinjectedwithcontrolplasmid(E),IFNa1plasmid(F),IFNbplasmid(G)andIFNcplasmid(H).Red colorshowsstainingforMxprotein.Arrowsindicatemacrophagelikecellsin(C)andendothelialcellsin(D),whichshowstrongstainingforMxprotein.

ideawasexaminedbyimmunohistochemistryof Mxproteinin heartandliver,whicharestronglyaffectedbyISAVinfection.Focal necrosisinliverofISAVinfectedfishiscommonlyfound,butthe maintargetcellsforinfectionbyISAVareendothelialcellslining thecirculatorysystemandnothepatocytes[22].Sectionsofliver fromIFNbandIFNctreatedfishshowedsimilarMx-stainingexcept thatendothelialcellsappearedtobemorestronglystainedinIFNc treatedfishcomparedtoIFNbtreatedfish.Thismaythusinpart explainthedifferencesinprotectionobtainedwithIFNccompared toIFNbplasmid.Moreover,hearttissueshowedstrongerMxstain- ingthroughoutinfishtreatedwithIFNcplasmidcomparedtoIFNb plasmid,whichwasconfirmedbyimmunoblottingofMx.Thissug- geststhatIFNcinducesantiviralproteinsmorestronglythanIFNbin

severaldifferentcelltypesinheart.Otherexplanationsmay,how- ever,alsobepossiblesincemammaliantypeIIFNsareknownto haveawiderangeofbiologicalactivitiessuchassensitizingcells toapoptosisuponsubsequentviralinfection[23],stimulationof cytotoxicactivityofNKcells[24]andstimulationofcellsinvolved inadaptiveimmuneresponses[25].ThedifferenceineffectofIFNb andIFNcmaybeduetodifferencesinuseofreceptors,whichis currentlyunderinvestigationbyourgroup.

Whetheri.m.injectionofIFNcplasmidmightbeausablemethod forcombatingvirusinfectionsinfarmedsalmondependsonsev- eralquestions,whichhavetobeansweredinfuturestudies.Among those are the duration of the antiviral effects of IFNc plasmid injection, whether IFNc plasmid protects against other viruses

thatitinduces antiviralgeneswithabroadspectrum ofantivi- ralpropertieswhileconventional DNAvaccines aredesignedto induceadaptiveimmuneresponsesthataredirectedtowardspe- cificpathogens.

Acknowledgements

ThisworkwasinpartfundedbytheRegionalResearchFundfor NorthernNorway(grant212426)andtheAquacultureprogramme ofTheResearchCouncilofNorway(grant185248).

Conflictsofintereststatement:Therearenoconflictsofinterest.

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