Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

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

Genetics

and

Molecular

Microbiology

Use

of

homologous

recombination

in

yeast

to

create

chimeric

bovine

viral

diarrhea

virus

cDNA

clones

Sandra

Arenhart

a,b

_,

_José

_Valter

_Joaquim

_Silva

_Junior

a

_,

_Eduardo

_Furtado

_Flores

b

_,

Rudi

Weiblen

b

_,

_Laura

_Helena

_Vega

_Gonzales

_Gil

a,∗

a_{Fundac¸ãoOswaldoCruz(Fiocruz),CentrodePesquisasAggeuMagalhães(CPqAM),DepartamentodeVirologiaeTerapia}

Experimental,Recife,PE,Brazil

b_{UniversidadeFederaldeSantaMaria,CentrodeCiênciasRurais(CCR),DepartamentodeMedicinaVeterináriaPreventiva(DMVP),}

SantaMaria,RS,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3December2015 Accepted25April2016 Availableonline26July2016 AssociateEditor:MaurícioLacerda Nogueira Keywords: BVDV Pestivirus Reversegenetics Infectiousclone

Yeasthomologousrecombination

a

b

s

t

r

a

c

t

TheopenreadingframeofaBrazilianbovineviraldiarrheavirus(BVDV)strain,IBSP4ncp, wasrecombinedwiththeuntranslatedregionsofthereferenceNADLstrainbyhomologous recombinationinSaccharomycescerevisiae,resultinginchimericfull-lengthcDNAclonesof BVDV(chi-NADL/IBSP4ncp#2andchi-NADL/IBSP4ncp#3).Therecombinantcloneswere suc-cessfullyrecovered,resultinginviableviruses,havingthekineticsofreplication,focussize, andmorphologysimilartothoseoftheparentalvirus,IBSP4ncp.Inaddition,thechimeric virusesremainedstableforatleast10passagesincellculture,maintainingtheirreplication efficiencyunaltered.Nucleotidesequencingrevealedafewpointmutations;nevertheless, thephenotypeoftherescuedviruseswasnearlyidenticaltothatoftheparentalvirusin allexperiments.Thus,geneticstabilityofthechimericclonesandtheirphenotypic similar-itytotheparentalvirusconfirmtheabilityoftheyeast-basedhomologousrecombination tomaintaincharacteristicsoftheparentalvirusfromwhichtherecombinantviruseswere derived.Thedataalsosupportpossibleuseoftheyeastsystemforthemanipulationofthe

BVDVgenome.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

Bovineviraldiarrheavirus(BVDV)isanimportantpathogen of cattle, belonging to the genus Pestivirus in the family

∗ _{Correspondingauthorat:LaboratóriodeVirologiaeTerapiaExperimental(LaViTE),DepartamentodeVirologiaeTerapiaExperimental,}

CentrodePesquisasAggeuMagalhães(CPqAM),Fundac¸ãoOswaldoCruz(Fiocruz),Recife,PE50670-420,Brazil. E-mail:laura@cpqam.fiocruz.br(L.H.Gil).

Flaviviridae. Pestiviruses are small (40–60nm) enveloped viruses containing a single-stranded, positive-sense RNA

genome ofapproximately 12.3kb. The BVDV RNA genome

contains a single,long open reading frame (ORF)encoding

a polyprotein of approximately 4000 amino acids.1 _The

http://dx.doi.org/10.1016/j.bjm.2016.07.022

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ORF is flankedby 5 and 3 untranslatedregions (UTRs) of approximately 385 and 229 nucleotides (nt), respectively.2 TheviralRNAgenomeisuncappedatits5 end,and trans-lationbyhost-cellribosomesoccursthroughrecognitionof theinternalribosomalentrysite(IRES),atertiary structure located within the 5 UTR. Direct translation of the viral genomegivesrisetoapolyproteinthatiscleavedbycellular andviral-encodedproteases,resultinginfollowingproteins: NH2–(Npro–C–Erns–E1–E2–p7–NS2/3–NS4A–NS4B–NS5A–NS5B)– COOH.1

Field BVDV isolates are classified into two genotypes, BVDV-1andBVDV-2,basedonthedifferencesinthe5 UTR andon thediversityofglycoproteinE2.3 _{Inadditiontothe} recognizedspecies,anadditionalPestivirusspecieshavebeen proposed:Hobi-like or BVDV-3.4 _{Most field isolatesof both} genotypesare non-cytopathic(ncp) forculturedcells. Non-cytopathicisolatesareresponsibleformostinfectionsinthe fieldandareassociatedwiththemainclinicaland reproduc-tiveconsequencesofBVDVinfection.5

Thedevelopmentofreversegeneticstoolsforpestiviruses inthelate1990sfacilitatedstudiesconcerningmanyaspects ofBVDVbiology6_{anditsinteractionswiththehost,}7_aswell asthedevelopmentofvaccinestrategies.8_{Afterthepioneer} description ofarecombinant cDNA clone ofthe pestivirus classicalswinefevervirusbyMoormannetal.9_andthefirst BVDVclone(CP7)byMeyersetal.10_{in1996,anumberofBVDV} cDNAcloneshavebeenconstructed,includingNADL,Oregon, SD-1,NY’93,Pec515,and 890,11–13 _{amongothers.Molecular} strategiestogenerateinfectiousBVDVfromcDNAweremainly derivedfromtheclassicalmethoddescribedforflaviviruses byRiceetal.,14_{basedontheassemblyoffull-genome-length} cDNAinbacterial plasmidvectors.Instability offull-length pestivirusescDNAinbacterialhostshasbeenpartially over-comebytheuseoflow-copy-numberplasmids,butnotyet solved.13,15,16 _{To improve this system, a bacterial artificial} chromosomestrategy(BAC)wasusedtogeneratefull-length cDNAcopyofpestiviruses,whichseemedtobemorestable duringpassagesinEscherichiacoli.17–19

Thisstudy describesthe constructionofchimeric BVDV cDNAclonesbyhomologousrecombinationinyeast

(Saccha-romycescerevisiae).Thisstrategyhasseveralapplicationsand hasbeenused,inparticular,toovercometheproblemof insta-bilityofsomeflaviviruscDNA clonesinE. coli.20 _Usingthis easyand simplemethod,chimeric full-lengthcDNA clones

containingthe5and3 UTRsoftheBVDV-1referencestrain NADLandtheentireORFofarepresentativeBrazilianBVDV-1 strain,IBSP4ncp,21_{weresuccessfullyconstructedthroughjust} one-stepassembly.

Materials

and

methods

Cellsandviruses

Pestivirus-freeMadin-Darbybovinekidney(MDBK)cells(ATCC CCL-22) were used for all viral manipulation procedures. Cells weremaintainedinminimalessentialmedium(MEM, Sigma–Aldrich,St.Louis,MO,USA)supplementedwith10% equineserum,1%penicillin(10,000UI/mLstock),and strepto-mycin(10,000␮g/mLstock)(Gibco,Langley,OK,USA)at37◦C, 5%CO2.MDBKcellsweremonitoredforpestiviruses contam-inationbyindirectfluorescentantibodyassay(asdescribedin Section‘Fluorescentantibodyandperoxidaseassays’)before andduringalltheexperiments.Thevirususedforthe con-struction was a Brazilian non-cytopathic BVDV-1b strain, IBSP4ncp(GenBankaccessionnumberKJ620017).22

Oligonucleotidesandplasmidvector

Oligonucleotidesforamplificationandhomologous recombi-nation(Table1)wereinitiallydesignedbasedonthesequences ofconservedregionsofreferenceBVDV-1strains,availablein the GenBank.Thesequences werealignedusing ClustalW2

(http://www.ebi.ac.uk/Tools/msa/clustalw2/). Additionally,

some oligonucleotides were designed to add 25nt to the terminiofampliconsforhomologousrecombinationinyeast

(Table1).Theyeast/E.colishuttlevectorpBSCNADLHDR

con-tainingthewholegenomeoftheBVDVreferencestrainNADL was kindlyprovidedbyDr. RubenO. Donis(CDC Influenza Division,Atlanta,GA,USA).

Fluorescentantibodyandperoxidaseassays

Anindirect fluorescentantibody(IFA)assay wasperformed using MDBKcells deposited overglass coverslips. Acetone-fixed cells were incubated for 1h at 37◦C with a pool of

monoclonal antibodies (MAbs) to BVDV (15c5, 12g4, and

20.10.6),23_{followedbywashinginphosphate-bufferedsaline}

Table1–OligonucleotidesusedontheconstructionoftherecombinantcDNAcloneofBVDVstrainIBSP4ncpby homologousrecombination.

Oligonucleotide Sequence

1–BVDVqm5UTRNADLIBSP4-Fa _{CTAAAAATCTCTGCTGTACATGGCACATGGAGTTGATTGCAAATGAAC}

2–BVDV-Osloss-4458Rb _{TGAGGGGCAAGAGTATGCTGAC}

3–BVDV1-4121F ACYATMCCRAACTGGAGRCCAC

4–BVDV1-8893R CATCTCATAGCCACATGGGCAC

5–BVDV-Osloss-8520F TTGAAGCAGTYCAGACAATTGG

6–BVDVqm3UTRNADLIBSP4-R ATTTATTTACAATATATACATTTTGTCTCAACTGCTGGCACCGACAG

Oligonucleotidesareidentifiedaccordingtothesequencestheyamplify/possess.Thehomologoussequencesforrecombinationareinbold.

a _F–sense. b _{R–antisense.}

(PBS) and incubation with an anti-mouse IgG fluorescein isothiocyanate-conjugatedsecondaryantibody(1:100inPBS; Sigma–Aldrich).Slideswereexaminedunderultravioletlight inaDMI4000Bfluorescencemicroscope(Leica,Wetzlar, Ger-many).Immunoperoxidase(IPX)stainingforBVDVantigens wasperformedusingcellmonolayersgrowninsix-wellplates andinoculatedtherespectiveviruses.Cellmonolayerswere fixedin30%coldacetoneby13minandincubatedwiththe sameMAbsforIFA(1hat37◦C),washed,andincubatedwith ananti-mousehorseradishperoxidase(HRP)-conjugated anti-body(1:1000inPBS;Sigma–Aldrich).Afterwashing,anHRP substrate(aminoethylcarbazole–AEC,inacetatebuffer50mM –pH5.0andhydrogenperoxide0.2%)wasaddedand incu-batedforapproximately30minat37◦C.

ConstructionofBVDVcDNAclonesbyhomologous

recombinationinyeast

RecombinantcDNAclones,pBSCIBSP4ncp,wereconstructed byhomologous recombinationin yeastusing threereverse transcription-polymerase chain reaction (RT-PCR) products

encompassing the IBSP4ncp ORF and a BamHI-digested

pBSCNADLHDRvector.Afterthedigestion,theNADL5and 3UTRsinthevectorremainedintact.Thestrategyofcloning isdepictedinFig.1.

AmplificationoftheIBSP4ncpORF

ViralRNAwasextractedfromthesupernatantofMDBKcells infectedwiththe IBSP4ncpBVDVstrain, usingtheQIAamp ViralRNAMiniKit(Qiagen,Valencia,CA,USA).RTwas per-formedina20-␮Lreactionusing50ngoftheRNAtemplate and200UofSuperscriptIIIReverseTranscriptase(Invitrogen, Carlsbad,CA,USA).ThecDNAwasamplifiedwithKlenTaq-LA polymerase(Clontech,MountainView,CA,USA).Theentire IBSP4ncpORFwasPCR-amplifiedinthreefragments.Thefirst PCRfragment(4081bp)wasamplifiedwitholigonucleotides1 and2,thesecondfragment(4466bp)wasamplifiedwitholigos 3and4,andthethirdfragment(3360bp)wasamplifiedwith oligos5and6(Table1).ThePCRconditionswereasfollows: initialdenaturationat95◦Cfor5min,35cycles(denaturation at95◦Cfor30s,annealingat52◦Cfor30s,andextensionat 72◦Cfor1minforeach1000bp),andfinalextensionat72◦C for10min.

Yeasttransformationandidentificationofrecombinant clones

TheplasmidpBSCNADLHDRwasdigestedwithBamHI(New

EnglandBioLabs,Ipswich,MA,USA)toremovetheNADLORF, leavingintactthe5and3UTRs(Fig.1),followedby dephos-phorylationwith5Uofcalf-intestinalalkalinephosphatase (NewEnglandBioLabs).ThethreePCRproducts correspond-ingtothewholeIBSP4ncpORFplustheBamHI-digestedvector

pBSC-NADL-HDR were introduced simultaneously into the

yeast S. cerevisiae strain RFY206 using the lithium acetate method.24 _{Aftertransformation,yeastcellswere inoculated} inYNBsolidmedium(YeastNitrogenBase–Sigma–Aldrich) withouttryptophanandmaintainedat30◦Cforuptothree days. Five recombinant yeastcolonieswere chosen,picked

andamplifiedinYNBmedium.PlasmidDNAwasextracted

fromyeastspheroplastpelletsusingQIAprepSpinMiniPrep kit(Qiagen).Thepresenceofclonedfragmentswasconfirmed byPCR,followedbynucleotidesequencing.Afterthe confirma-tion,twointhreerecombinantclones(pBSCIBSP4ncp#2and pBSCIBSP4ncp#3)werefurtheramplifiedandcharacterized.

Nucleotidesequencing

TheconstructedcDNAclones,pBSCIBSP4ncp#2and#3,and full-length PCR products obtained from both clones were submittedforcompletenucleotidesequencing.Theparental virus IBSP4ncpandachimeric virus,chi-NADL/IBSP4ncp#2, derivedfromthecorrespondingpBSCIBSP4ncp#2cDNAclone

and recovered at passages 0 (p0, rescued virus) and p5

were submitted for nucleotide sequencing of the whole

genomes. All viral sequences were amplified from

super-natantofinfectedcells.Sequencingreactionswereperformed usingthemixBigDye®_{Terminatorv3.1CycleSequencingKit} (AppliedBiosystems)accordingtothemanufacturer.Products ofsequencingwereresolvedinanABI3100GeneticAnalyzer sequencer(AppliedBiosystems).Sequenceanalyseswere con-ductedbyusingthepackageLasergene®_{(DNAstarInc.)anda} consensussequenceofeachviruswasgeneratedbythe pro-gramSeqManII(Lasergene®_{,DNAstarInc.).}

Full-genomePCRamplificationandinvitrotranscription

A PCR was performed to amplify and linearize the

entire genomes of the chimeric viruses including the

A

B

C

5’UTR 5’UTR 25 nt 325 nt

Sequences remaining in vector after digestion

Homologous sequences overlapping fragments Sequences of the IBSP4ncp genome

pBSC_NADL_HDR digested with BamHI

74 nt 251 nt 25 nt Npro Fragment 1 4.081 nt Fragment 2 4.466 nt Fragment 3 3.360 nt 836 nt c Erns E1 E2 p7 NS2-3 IBSP4ncp

NS4A NS4B NS5A NS5B 3’UTR

3’UTR

Fig.1–RepresentationofthestrategyofconstructionofthechimericcDNAclonesofBVDVstrainIBSP4ncp.(A).Genome organizationofBVDVIBSP4ncp.(B).HomologousrecombinationinyeastofthreePCRproducts(fragments1,2and3)and

sequence of the bacteriophage T7 RNA polymerase

promoter for in vitro transcription. For PCR

amplifica-tion, the reaction mixture contained 100ng of DNA of

a recombinant clone (pBSCIBSP4ncp#2 or #3), 1U of

KlenTaq-LA DNA polymerase mix (Clontech), 0.5U ofVent

DNA polymerase (Sigma–Aldrich), and oligonucleotides

(pBSC-T7-NADL-F and NADL-3UTR-R, Table 1). The PCR

products were phenol-chloroform-extracted and

ethanol-precipitated prior to performing in vitro transcription using the MEGAscript® _{T7 kit (Ambion, Foster City, CA,} USA).

Transfectionandmonitoringofinfectivityof

invitro-transcribedRNAs

For transfection, MDBK cells were trypsinized, washed

with serum-free MEM, then washed twice with PBS,

pH 7.2/diethylpyrocarbonate at 4◦C, and resuspended at 8×106_{cells/mL in400␮LofCytomix(120mMKCl,0.15mM} CaCl2, 10mMK2HPO4/KH2PO4, 25mM HEPES, 2mM EGTA, 5mMMgCl2,2mMATP,5mMglutathione,andpHadjusted to7.6withKOH).Electroporationwasperformedusing5␮g oftranscribedRNAasdescribed byQuetal.25 _Cellcultures weremaintainedforthreedaysandmonitoreddailyforBVDV antigensbytheIFAassay.Thepresenceofinfectiousvirusin thesupernatantsofthetransfectedMDBKcultureswas con-firmedbyinoculatingfreshMDBKcellcultureswithaliquotsof thesupernatants,followedbytheIFAassay72hlater.Viruses recoveredfromthesupernatantsofthetransfectedcellswere designatedchi-NADL/IBSP4ncp#2andchi-NADL/IBSP4ncp#3, passage0(p0).

CharacterizationofvirusesderivedfromcDNAclones

Infectivityandstabilityincellculture

The chimeric viruses (chi-NADL/IBSP4ncp#2 and

chi-NADL/IBSP4ncp#3) were subjected to10 passages inMDBK

cells.Cellswereinoculatedwiththevirusesatamultiplicityof infection(MOI)of0.5foreachpassageandincubatedat37◦C, 5%CO2for72h.Attheendofeachpassage,thesupernatants were tested bythe IFAassay, and virus quantificationwas performedbyaplaqueassay.

Kineticsofvirusreplicationandfocusassays

The viruses chi-NADL/IBSP4ncp#2 and #3 at p5 and the

parental virus IBSP4ncp were individually inoculated into

MDBK cell cultures at an MOI of 0.3 each. The inoculum

was removed, and the cell monolayers were washed with

MEM,followedbytheadditionoffreshculturemediumand incubationat 37◦C,5%CO2. Aliquots ofthe culture super-natantswereharvestedatdifferenttimes(0,8,16,24,32,40, 48,56,64,and72h),andviruseswerequantifiedbylimiting dilution.Toexaminethesizeandmorphologyofinfectious fociproduced bythe recombinantvirusesatp5andbythe parentalvirusIBSP4ncp,90%confluentMDBKcellmonolayers growninsix-wellplateswereinoculatedwithviraldilutions (10−1to10−7).After1hadsorptionat37◦C,theviral

inocu-lum was removed, and the cell monolayers were washed,

overlaid with 1% agarose in MEM plus 5% equine serum,

and incubated at 37◦C, 5% CO2. After 72h, the monolay-ers were fixed and subjected to IPX staining as described above.

StabilityofrecombinantyeastcDNAclonesinE.coli

The recombinant plasmids pBSCIBSP4ncp#2 and #3 were

electroporated into E.coliDH10B(Invitrogen)using anECM 830BTXelectroporator.Theelectroporationconditionswere as follows:2.75kV,99␮s, 5pulses, and 1s betweenpulses. Colonies weregrownat37◦CinLuria-Bertanimediumwith chloramphenicol(20␮g/mL)at37◦Cfor18–20h.PlasmidDNA wasthenpurifiedwiththeQIAGENPlasmidMidiKit(Qiagen) andusedasatemplateforinvitrotranscription.MDBKcells wereelectroporatedwiththeRNAasdescribedabove. Infec-tivity ofthe RNAwasaccessedbytheIFAassayperformed at 24, 48, and 72h after the transfection. Progeny viruses wereharvestedandsubjectedtofivepassagesinMDBKcells, with the infectivity monitored ateach passageby the IFA assay.

Results

ConstructionofchimericNADL-IBSP4ncpcDNAclones

RecombinantcDNAclonescontainingtheentireORFofthe Brazilian BVDVstrain IBSP4ncpand theUTRs ofthe NADL strainweresuccessfullyconstructedbyhomologous recombi-nationinyeast.ThecorrectrecombinationoftheNADLUTRs andtheIBSP4ncpORFwasconfirmedbynucleotide sequenc-ingoftheUTR-ORFjunctions.Tworecombinantviruseswere recoveredforfurthercharacterization(chi-NADL/IBSP4ncp#2 andchi-NADL/IBSP4ncp#3).

InfectivityandstabilityofRNAtranscribedinvitrofrom

recombinantcDNAclones

ToaccesstheinfectivityofRNAderivedfromtheclonedBVDV

genome, the plasmid DNA of the clones pBSCIBSP4ncp#2

and #3,isolated from yeastcells,wasinitially subjectedto

a PCR encompassing the entire viral genome. The

ampli-cons were used astemplatesforin vitrotranscription, and the transcribed RNAs were then used for transfection of MDBKcells.ViralantigensweredetectedintheMDBKcells transfected withthetranscribedRNAofbothclonesat24h post-transfectionbytheIFAassay.Thetransfectionresulted in efficientvirus recoveryforbothclones, witha meanP0 titerof104.5_{plaque-formingunits(PFU)/mLat72hafterthe} transfection(datanotshown).Thevirusesrecoveredfromthe transfected cell cultureswere namedchi-NADL/IBSP4ncp#2 andchi-NADL/IBSP4ncp#3.Toconfirmthepresenceof infec-tiousprogenyviruses,thesupernatantscollectedfromthese cultures were inoculated to fresh MDBK cells, followed by the IFAassayforviralantigens72hlater(datanotshown). Thestabilityofthechi-NADL/IBSP4ncp#2and #3virusesin MDBK cells wasconfirmed during10passages (Fig. 2A).At all tested passages, the viruses reached titers of approx-imately 105.5–6.0 _{PFU/mL at 72h post-infection (data not} shown).

A

_{Negative control}

B

C

Chi-NADL/IBSP4ncp#2 Chi-NADL/IBSP4ncp#3 Chi-NADL/IBSP4ncp#2 Chi-NADL/IBSP4ncp#3 Chi-NADL/IBSP4ncp#2 Chi-NADL/IBSP4ncp#3 IBSP4ncp p5 p5 p10 p5 coli p5 Negative control 7 IBSP4ncp 6 5 Vir

us titer PFU/mL (log10)

4 3 2 1 0 0 8 16 24 32

Hours post inoculation IBSP4ncp

40 48 56 64 72

Fig.2–Infectivityofrecombinantviruses.(A)IFAforviralantigens.Negativecontrol:mock-infectedMDBKcells;positive control:MDBKcellsinoculatedwiththeparentalstrain;chi-NADL/IBSP4ncp#2and#3virus.Andthesameviruses(#2and 3)recoveredfromplasmidDNAamplifiedinE.coli(p5).(B)IPXforviralantigens.Morphologyofinfectiousfociproducedin MDBKcellsbyvirusesrescuedatp5.(C)Replicationcurveofvirusesrecoveredatp5andparentalvirus.MDBKcellswere inoculatedwiththerespectivevirusesatanMOIof0.3andsupernatantscollectedatdifferentintervalsweresubmittedto virusquantificationbyIPX.Eachvaluerepresentsthemeanoftwoindependentexperiments.

Replicationkinetics,focussize,andmorphology

ConfluentMDBKmonolayerswereinoculatedwitheachvirus at an MOI of 0.3, and the culture supernatants collected at different intervals were titrated for viruses. Both chi-NADL/IBSP4ncp#2 and #3 replicated to similar levels, and thekineticswasindistinguishablefromthatoftheparental strain (Fig. 2C). Then, we investigated using IPX staining the morphology and size of the plaques/foci produced on MDBKcellsbychi-NADL/IBSP4ncp#2and#3atp5and com-pared the features with those of the parental strain. The sizeandmorphologyoftheinfectiousfociproducedby chi-NADL/IBSP4ncp#2and#3wereindistinguishablefromthose ofthefociproducedbytheparentalvirus(Fig.2B).

Genomesequencing

We chose one clone (chi-NADL/IBSP4ncp#2) to investigate forpossiblemutationsintheviralgenome.Atp0,onesilent

mutationwasdetectedintheNpro_{gene,andanother} muta-tionwasdetectedintheE1gene,butthelatterrevertedtothe originalnucleotideatp5.Atp5,afewadditionalmutations weredetectedinthefirstORF-encodedprotein,Npro_.Among those,threesingle-nucleotidechanges(nt463,499,and514) didnotresultinaminoacidchanges,whileoneresultedin

a substitution by an amino acid from the same chemical

group(Glu550Asp).Besides,onemutation(Ser12079Asn)was detectedintheNS5Bgene,resultinginasubstitutionbyan aminoacidfromadifferentchemicalgroup.Themutations areshowninTable2.

StabilityofcDNAclonesinE.coli

To accessthe stability of the recombinant cDNA clonesin bacteria,theplasmidspBSCIBSP4ncp#2and#3were ampli-fiedinE.coli.PlasmidDNAwasextractedfromthebacterium, amplifiedbyPCR, and usedasa templateforinvitro tran-scription.TheRNAstranscribedfromtheseampliconswere

Table2–AnalysisofsequencesofthevirusrecoveredfromthecDNAcloneandparentalvirus.

Localization Nucleotidea _{IBSP4ncp Chi-NADL/IBSP4ncp#2passage0 Chi-NADL/IBSP4ncp#2passage5 Nameortypeofmutation}

Npro _{439 T C C Silent} Npro _{463 C C T Silent} Npro _{499 G G A Silent} Npro _{514 A A G Silent} Npro _{550 A A T Glu550Asp} E1 2295 A Cb _{A –} NS5B 12,079 G G A Ser3898Asn

a _{NucleotideandaminoacidspositionscorrespondtothoseofthepublishedIBSP4ncpgenome/polyprotein.} b _{Thisnucleotidechangerevertedtotheoriginalatpassage5.}

infectious upon transfection into MDBKcells. Theprogeny viruseswere subjected to fivepassages inMDBK cells and maintainedtheirinfectivityandreplicationefficiency(Fig.2A). Atalltestedpassages,thevirusesreachedtitersof approx-imately 105.5–6.0 _{PFU/mL at 72h post-infection (data not} shown).ThestabilityofviralcDNA inprokaryotesystemis importantduetohigheramountandqualityofrecombinant DNAextractedfrombacteriainrelationtoyeast,facilitatingits futureuseinsomeexperiments,assuchgenomesequencing andinvitrotranscription.

Discussion

Full-length,chimericcDNAclonesofBVDV-1wereconstructed usingthestrategyofhomologousrecombinationinyeast.The assembledclonescontainedtheNADL5and3UTRsflanking theORFofarepresentativeBrazilianBVDV-1strain(IBSP4ncp). Thevirusesrecoveredfromthecellstransfectedwithin

vitro-transcribedRNAwereviable,maintainedthemainreplicative propertiesoftheparentalvirus,andwerestableover10 pas-sagesincellculture.Afewmutationsintheviralgenomewere identified;nevertheless,therewerenophenotypicchangesin theinfectious cloneinvitro.Moreover,the yeast-assembled plasmidswereamplifiedinE.coli,theRNAtranscribedinvitro

fromthebacterialplasmidDNAgeneratedinfectiousviruses upontransfection,andtheresultingviruseswerestableincell culture.Thus,thestrategyofhomologousrecombinationin yeastseemssuitablefortheconstructionandmanipulation ofpestivirusrecombinantcDNAclones.

Homologous recombination in the yeast S. cerevisiae

has arisen as molecular methodology for many

applica-tions,includingtheconstructionofcDNAclonesofvarious viruses.Inthisstrategy,DNAfragmentscontainingnucleotide sequences homologous tothe vector attheir ends may be directlyclonedbyinvivorecombinationinthehost.Inthis way,multipleDNAfragmentscanbecorrectlyassembledina uniquemolecule.26_{Thisstrategyhasseveralapplicationsand} showsadvantagesovertraditionalcloningmethods.In partic-ular,itdoesnotdependonmultiplerestrictionsitesortheuse ofmanyamplificationproducts,unliketraditionalcloningin bacteria,asreportedbyParketal.16_{toconstructaCSFVcDNA} clone.16_{Thesepropertiesmakethisapproacheasier,less} labo-rious,andmoreefficientthantraditionalcloningstrategies.26 Alongwithotherapplicationsusingviruses,24 _this method-ologyhassuccessfullybeenusedtoovercometheproblems ofinstabilityofsomeflavivirusrecombinantcDNAgenomes inE. coli.20,27 _{Aprerequisite forhomologousrecombination} inyeast is the presence ofnucleotide sequence homology (15–50bp)attheendsoftheDNAfragmentsandthevector toberecombined,necessarytorecruittheyeast recombina-tionmachinery.28,29_{Inoursystem,homologoussequencesof} 25bpinboth UTRsattheends offragments1and 3were shown tosufficetodirect recombinationinthe yeast. The flexibilityintermsofthesizeofclonedfragments,thelength ofhomologoussequences,andtheuseofintrinsicrestriction sitespresentsobviousadvantagesofthiscloningstrategy, sim-plifyingtheprocedures.Also,thereisno needtopurchase cloningkitsusedforconventionalcloningstrategies.

Therescuedviruseswere stableover10 passagesincell culture,maintaining thereplication efficiencyand kinetics, focussize,andmorphologyoftheparentalvirus.Thestability incellcultureachievedbyoursystemwasbetterthanBAC system forsome pestiviruses,although this techniquehas been usedtoreducetheinstabilityofcDNA clones.17 _Thus, neitherthegeneticmanipulationnorthepresenceofthe het-erologousUTRsadverselyaffectedthereplicationproperties oftherescuedvirusesoralteredtheirmainphenotypic prop-erties in vitro. TheNADL UTRsequences were usedin the constructsincethecorrespondingIBSP4ncpsequenceswere

unknownatthetimewhenweassembledthecDNAclones.

Asimilarstrategywasdescribedearlier,inonestudypartsof theCP75and3UTRswerereplacedbypartsofthe respec-tive NADL UTRs10 _{and Hoffmann}30 _{createsan interspecies}

cDNA clonewith BVDV UTRs plus CSFVORF, both viruses

replicated efficiently.10,30 _{Theheterologous UTRspresentin} ourconstructmayserveasgeneticmarkers,confirmingthe recombinant origin and helping avoid potential contami-nations with the parental virus. Anyway, the viability and replicationefficiencyoftherescuedvirusesshowthepromise ofthismethodologyforgeneratingrecombinantBVDV.

Nucleotide sequencing of the rescued virus

chi-NADL/IBSP4ncp#2 at p0, revealed one silent mutation in

the Npro _{gene, which was still detected at p5. Another} mutation was detected in the E1 gene at p0 but reverted to theoriginal nucleotideatp5 (Table2).Atp5,mutations were mainly found in the first ORF-encoded protein, Npro

(Table 2). Three nucleotide changes (nt 463, 499, and 514)

did not result in amino acid substitutions. However, two

nucleotides changes in the NS5B gene resulted in amino

acids substitution: one by an amino acid from the same

chemicalgroup(Glu550Asp)andotherbyanaminoacidfrom adifferentchemicalgroup(Ser12079Asn)(Table2).However,

as mentioned above, no obvious phenotypic changes were

observed in the rescued viruses in cell culture, compared to the parental strain. Parket al.16 _{recentlyconstructed, a} cDNAcloneusingstandardtechniquesandobtainedsimilar mutationsrates,neverthelessthephenotypeoftherescued virus showed lower levels ofreplication. No mutationwas foundintheoriginalplasmidsorinthePCRproductsusedas templatesforinvitrotranscriptioninourstudy.Sincecertain

DNA manipulationswould requirehigheramountsofDNA,

we also investigated the stability of the yeast-assembled DNAplasmidsinE.coli.Thesuccessfulrecoveryofinfectious virusesthatwerestableincellcultureoverfivepassagesfrom the RNAtranscribedfrom theplasmids replicated inE.coli

demonstratedthattheseplasmidswerestableand,therefore, maybeamplifiedandmaintainedinthishost.

In summary, the recombinant viruses reported herein, chimericintheirUTRs,containtheentireORFofthe Brazil-ianBVDVstrainIBSP4ncpandretainitsproteinandantigenic properties. IBSP4ncp was demonstrated to display a high degree of serological cross-reactivity with a wide range of BrazilianBVDVisolates21_{and,therefore,isconsideredtobe} a representative strain and a candidate for vaccine devel-opment. Thus, in the future, a recombinant derivative of IBSP4ncpcanbeusedasabackbonetogeneraterecombinant vaccineclones,duetoitshighantigenicsimilaritytoBrazilian fieldisolates.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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