Prostaglandin A1 inhibits the replication of bovine viral diarrhea virus

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

Prostaglandin

A

1

inhibits

the

replication

of

bovine

viral

diarrhea

virus

Lúcio

Ayres

Caldas

_,

_Tânia

_Rosária

_Pereira

_Freitas

_,

_Renata

_Campos

_Azevedo

_,

Wanderley

de

Souza

a_{InstitutoNacionaldeMetrologia,QualidadeeTecnologia(INMETRO),RiodeJaneiro,RJ,Brazil}

b_{MinistériodaAgricultura,PecuáriaeAbastecimento,LaboratórioNacionalAgropecuário–LANAGRO–MG,PedroLeopoldo,MG,Brazil}

c_{UniversidadeFederaldoRiodeJaneiro,InstitutodeMicrobiologiaProfessorPaulodeGóes,RiodeJaneiro,RJ,Brazil}

d_{UniversidadeFederaldoRiodeJaneiro,InstitutodeBiofísicaCarlosChagasFilho,RiodeJaneiro,RJ,Brazil}

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Articlehistory:

Received22September2017 Accepted28December2017 Availableonline1March2018 AssociateEditor:FernandoSpilki

Keywords:

Bovineviraldiarrheavirus BVDV

Prostaglandins ProstaglandinA1

Virus-inducedvacuolization

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Bovineviraldiarrheaviruscancauseacutediseaseinlivestock,leadingtoeconomiclosses. WeshowthatProstaglandinA1 inhibitsbovineviraldiarrheavirusreplicationin Madin-Darbybovinekidneycells(94%inhibitionusing5␮g/mL).Lightandelectronmicroscopyof infectedcellsshowsthatProstaglandinA1alsopreventsvirus-inducedvacuolization,butat higherconcentrations(10␮g/mL).

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Bovineviral diarrheavirus(BVDV)isaninsidious,complex andubiquitouspathogen thatcanaffectcattleofallbreeds andallages,aswellasothersdomesticandwildruminants.1 ThediseasecausedbyBVDVrangesinseverityfrom asymp-tomatictoacuteinfectionandfatalmucosaldisease,witha mortalityindexofapproximately5%,whichleadstoan eco-nomicimpactofBVDVinfectionsofupto£993percowper year.2

∗ _{Correspondingauthorat:UniversidadeFederaldoRiodeJaneiro,InstitutodeBiofísicaCarlosChagasFilho,LaboratóriodeUltraestrutura}

CelularHerthaMeyer,RiodeJaneiro,RJ,Brazil. E-mail:lucio@biof.ufrj.br(L.A.Caldas).

In pregnant cows,BVDV can reach the fetus and cause abortion,stillbirth orteratogenicdefects,depending onthe gestationalperiodwheninfectionwasestablished.1_Whenthe infectionisestablishedinthefinalperiodofgestation,calves can givebirthtoimmunotolerantandpersistently infected calvesthatareconsideredreservoirsofthevirus.3_Also,the serumofinfectedanimalsmaycontainactiveBVDVviral par-ticles, whichmayresultinpermanentinfection inprimary

https://doi.org/10.1016/j.bjm.2017.12.010

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

usingbovineserum,inthelaboratory.

BVDVisthetype-speciesofthePestivirusgenus(fromthe

Flaviviridaefamily)thatalsoincludestheborderdiseasevirus

(BDV) and the classical swine fever virus (CSFV), responsi-bleforsignificanteconomiclossesworldwide.4_{Thepestivirus} capsidhasanicosahedralsymmetryformedbyasingle pro-tein (capsid protein C), and is enveloped by a lipoprotein membrane.Itsgenomeconsistsofapositivesense, single-strandedRNAofabout12,000nucleotides,containingalarge openreadingframecodingforasinglepolyproteinwithabout 4000aminoacids.Thislargepolypeptideiscleaved(byhost andviralproteases)togeneratethecapsidprotein(C),the gly-coproteinsErns,E1andE2,anon-structuralprotease(Npro) andanumberofothernon-structuralproteins(p7,NS2/NS3, NS4A,NS4B,NS5AandNS5B).TheE2proteinappearstobeof majorimportanceforpestivirusneutralization.5

BVDVisolatesaredividedintothegeneticgroupsBVDV1 and2,whicharegeneticallyrelatedtoaseparatespecies,the BVDV-3orHobigroup,6whichwasinitiallyidentifiedinfetal bovineserumfromBrazil.7 _{BVDVisolatesarealsoclassified} intocytopathogenic(cpBVDV)ornon-cytopathogenic (ncpB-VDV)dependingontheirabilitytocausecytopathiceffectin cellculture.3_Birketal.8_{showedthatcpBVDVinduced} exten-sivevacuolizationinMadin-Darbybovinekidney(MDBK)cells; however,thisphenomenonappearedmorphologically differ-entfromthatobservedinapoptosisandnecrosis.

Prostaglandinsarenaturaleicosanoidssecretedbya vari-ety of human tissues and capable of causing profound anddiversephysiologicaleffectsatverylowconcentrations. All eicosanoids function locally at the site of synthesis, throughreceptor-mediatedG-proteinlinkedsignaling path-ways. Prostaglandin A1 (PGA1) is capable of blocking the

replicationofawidevarietyofRNAandDNAviruses,including CSFV.9–11_{DespitetheimportanceofBVDVforanimalhealth,} theeffectofPGA1onBVDVreplicationhasnotbeenexamined.

Therefore,weevaluatetheeffectofPGA1onthereplicationof

cpBVDVinMDBKcells,focusingonchangesinBVDV-induced vacuolization.

MDBKcellswereculturedinvitroinDulbeccoModifiedEagle Medium (DMEM), supplemented with 10% BVDV-free fetal bovineserum(FBS,Gibco),penicillin(500U/mL),streptomycin (100␮g/mL)andamphotericinB(fungizone,at2.5␮g/mL).

Monolayers of MDBK cells were infected with cpBVDV-1 (NADL strain) at a multiplicity of infection of 0.1. Viral particles wereallowedtointeractwithhostcells for1h in theabsenceofFBS,andthenthesupernatantwasreplaced with complete growth mediumcontaining 0.1, 1, 2.5, 5or 10␮g/mLProstaglandinA1(PGA1,SigmaChemicalCo.;stored

asa1mg/mLsolution in100%ethanol).Aftertreatmentof monolayers withPGA1 for 36h, culture supernatants were

testedforthepresenceofvirusby50%tissuecultureinfective dose(TCID50)titration,andcell monolayerswerefixedand stainedwiththePanopticSolutionKit® (LaborclinLtda. Pin-hais/PR,Brazil).Panoptic-stainedmonolayerswereobserved in a Zeiss-Observer D1 light microscope equipped with a Nomarskidifferentialinterferentialcontrastsystem.

Cellviabilitywasdeterminedbyincubatingmonolayersin asolutioncontaining5␮g/mLNeutralred(dilutedinPBS)for 3hat37◦C,inwellsof96-wellplates.Then,thesolutionwas

Table1–EffectofprostaglandinA1(PGA1)onbovine

viraldiarrheavirus(BVDV)replicationinMDBKcells, expressedastheTCID50andthepercentageof

replicationinhibition.

PGA1concentration(␮g/mL) TCID50/mL %Inhibition

0 1×105 ₀

0.5 5.6×104 ₄₄

1.0 5.0×104 ₅₀

2.5 1.9×104 ₈₁

5 6.3×103 ₉₄

MDBKcellswereinfectedwithBVDVandtreatedwithdifferent con-centrationsofPGA1.ThepercentageofBVDVreplicationinhibition wascalculatedbymeasuringthevirusyieldinthesupernatant ofinfectedcells36hafterPGA1treatment.While1.0␮g/mLPGA1 inhibited50%ofBDVDproduction,thehighestconcentrationtested inthisassay(5␮g/mLPGA1)blockedreplicationby>90%.

discarded,andcellswereincubatedwith4%formaldehyde(in PBS)for1min,and100%methanolfor20min.Sampleswere analyzedbyabsorbanceat490nm,inanELISAplatereader (SpectraMaxM2).

For transmission electron microscopy (TEM) analysis, treated anduntreatedmonolayersin25cm2 _{plasticculture} flaskswerefixedin2.5%glutaraldehydein0.1Mcacodylate buffer(pH7.2),post-fixedfor1hin1%OsO4/0.8%potassium ferrocyanideinthesame buffer,dehydratedinethanoland ‘flat-embedded’inPolybedresin(Polysciences1),topreserve thecellarchitecture.Ultrathinsections(bothroutine,anden face)were stained withuranylacetateand leadcitrateand observed ina FEITecnai T20 transmissionelectron micro-scope.

For the visualization of extracellular lamellae by TEM, 0.5mg/mLrutheniumredwasaddedtotheTEMfixationand post-fixationsolutions(aswellastotheintermediatewashes in cacodylate buffer),and cells were processedfor TEMas describedabove.Thisprocedurewasalsoperformedduring enfacesectioning,inordertoavoidremovalofthecellsfrom thesubstratum,whichcoulddisruptanddisorienttheir archi-tecture.

TreatmentofBVDV-infectedMDBKmonolayerswithPGA1 at concentrations of 0.1 and 0.5␮g/mL reduced the virus yieldinculturesupernatantsby50%,andtreatmentwiththe highestconcentrationofPGA1testedinthisassay(5␮g/mL) reducedtheviralyieldby94%(Table1).Thus,PGA1treatment

resultedinstronginhibitionofBVDVreplicationinMDBKcells. Inlightmicroscopyimages,similarcytoplasmicalterations were observed inuntreatedand PGA1-treated cells (Fig.1).

Althoughtreatmentwith5␮g/mLdecreasedbyalmost2logs thevirustitrationinthesupernatantofinfectedcells(Table1), vacuolization wasnotinhibited byPGA1 atthis

concentra-tion(Fig.1E).Areductioninvacuolizationwasonlydetectable whencellsweretreatedwith10␮g/mLPGA1(Fig.1F).Atthis

concentration, treatment ofMDBKcells withPGA1 for36h

didnotresultincytotoxicity,asdeterminedbymicroscopic examinationorvitaldyeexclusion(datanotshown).

The cytosolic vacuoles induced byBVDV infection were further investigated by transmission electron microscopy analysis(Fig.2).BVDVinfectioninducedadramaticincrease invacuolarstructuresinMDBKcells(Fig.2B).Whenviewed

A

C

B

D

E

F

Fig.1–Lightmicroscopyanalysisofbovineviraldiarrheavirus(BVDV)infectedcellstreatedwithprostaglandinA1(PGA1).

MDBKcellsweretreatedwithPGA1for36h,andthensubjectedtopanopticstaining.Lowandhighmagnificationimagesof

untreatedmock-infectedcells(A,B),andofinfectedcellskeptuntreated(C,D)ortreatedwith5(E)and10(F)␮g/mLPGA1.

Although5␮g/mLPGA1stronglyblockedBVDVreplication(E),itdidnotinhibitthevacuolizationprocess(arrows).

Vacuolizationinhibitionwasonlyachievedaftertreatmentwith10␮g/mLPGA1(F).Scalebars:8␮m.

inboth traditional and en face sections, many ofthe vesi-clesininfected cells were positivefor rutheniumred and, thus,contained extracellularmaterial(Fig. 2C and D). One ofthemainfeaturesthatweobservedinBVDV-infectedcells wasthepresenceofmultilamellarstructuresinsidevacuoles (Fig.2C,EandF),whilesmallervacuoleswithinternal vesi-clessurroundedthosewithmultilamellarstructures(Fig.2E, F).Multilamellarstructureswerepreviouslyreportedby Sanz-Sanchez and Risco, where they were linked to a cellular responsetodetachmentfromsubstratumduetobunyavirus infection.12_{DespitethestrongreductioninBVDVreplication} achievedbytreatmentwith5␮g/mLPGA1(Table1),thisPGA1

concentrationdidnotpreventvacuolizationinBVDV-infected cells(Fig.1E).Astrongreductionofvirus-induced vacuoliza-tionwasonlyachievedaftertreatmentwith10␮g/mLPGA1

(Fig.2G).

Although prostaglandins (PGs) are capable of inhibiting the replication ofa varietyofRNA and DNA viruses, their mode of actionis not entirely clear.9–11 _{In some systems,} PGAsexertpotentantiviralactivitybyinducingthe synthe-sisofproteinsfromtheheatshockprotein70(HSP70)family, through cycloheximide-sensitiveactivation ofa heat-shock triggerfactor.13

Thereductionofvirus-inducedvacuolizationafterPGA1

treatment(Fig.2G)isinagreementwiththewidelydescribed antiviraleffectofthisdrug.10_{Thisactivity,initiallyattributed} totheinductionofHSP-70proteins,canalsobetriggeredby otherpathways,suchasnuclearfactor-␬Bregulation13,14_and, morerecently,byamechanismtargetingthesmallribosomal subunit(40S)andtheeukaryoticinitiationfactorseIF3s.11_This virus-inducedvacuolizationseemsnottobecrucialforviral replicationandisprobablycausedbytheinterferenceofBVDV

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A

C

D

F

E

G

B

Fig.2–Transmissionelectronmicroscopy(TEM)analysisofbovineviraldiarrheavirus(BVDV)-infectedcellstreatedwith prostaglandinA1(PGA1).Infectedandnon-infectedMDBKcellsweretreatedwithPGA1for36h,andthenprocessedfor

routineTEM.Vacuoles(v)wererareorabsentinthemajorityofsectionsfromnon-infectedcells(A),butwereabundantin BVDV-infectedcellsnottreatedwithPGA1(B).Manyofthesestructuresexhibitedextracellularcontent,asshownby

rutheniumredlabeling(arrows)intraditional(C)andenface(D)sections.Treatmentwith1␮g/mL(E)or2.5␮g/mL(F)PGA1

couldnotpreventBVDV-inducedvacuolizationininfectedcells,whichhadlargevacuoles(arrows)withmultilamellar structures(arrowheads),andoftensurroundedbysmallervesicles-containingvacuoles(thinarrows).(G)Treatmentwith 10g/mLPGA1,however,dramaticallyreducedvirus-inducedvacuolization.n,nucleus.Scalebars:(A)5␮m;(B,G)2␮m;(C,E)

500nm;(D,F)1␮m.

NS3 protein on the smooth endoplasmic reticulum.15 Fur-thermore,thetargetingofviralproteinsbycyclopentenone prostaglandinswasalreadyreportedbyKalantarietal.16

Thefactthattreatmentwith5␮g/mLPGA1inhibitedBVDV

production94%,but didnotblockvirus-induced vacuoliza-tion in host cells suggests that the vacuoles are, indeed,

not involved in viral replication and that PGA1 probably

reacheditssaturationpoint.Ourdatasuggestthatthe antivi-ral activity of PGA1 is not associated with the inhibition

ofvacuole production,althoughthe viral-induced vacuoles almostdisappearedwhenhigherconcentrationsofPGA1were

OurworkshowsthattheantiviralactivityofPGA1during

BVDVinteractionwithMDBKcellsdoesnotrelyona signif-icantreductioninvirus-inducedcytoplasmicvacuolization, butintheviraltitersreduction.Sincetheantiviraleffectsof PGsonBVDVhadnotbeendescribed,thisisthefirstreportof theparametersinvolvedintheprotectionofMDBKcellsfrom BVDVinfection.

Funding

This work was supported by Programa Nacional de Apoio aoDesenvolvimento da Metrologia,QualidadeeTecnologia (Pronametro)andCoordenac¸ão deAperfeic¸oamentode Pes-soaldeNívelSuperior(CAPES).

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