Construction of Recombinant Feline HerpesvirusType 1 Expressing Toxoplasma gondii SRS1

著者（英） Mishima Masayuki, Xuan Xuenan, Takeiri Akira,Makala Levi H. C, Igarashi Ikuo, FujisakiKozo, Nagasawa Hideyuki, Mikami Takeshi

journal orpublication title

The journal of protozoology research

volume 10number 3page range 166-172year 2000-07URL http://id.nii.ac.jp/1588/00001538/

J．Pr〃Jo乙00／．鮎∫．．川，／d∂－／72（20（叫

CのP）・璃ムJ◎2α抑，∧加の〃d尺g∫gd几・ムCど〃Jどり0′P叩Joz¢御βi∫ed∫g∫

ConstructionofRecombinantFelineHerpesviruSTypel

ExpressimgTb‡叩Jα5m〟gO〃dffSRSI

MASAYUKIMTSH7MAII2，XUENANXUANl，AKIRATAKETRI2′

LEVIMAKALA］′lKUOICARASHIl′KOZOFUJISAKI】′

HrDEYUKINAGASAWAIANDTAKESHTMIKAMIl

ノ〃α血〝α川e∫edrぐんCe〃fer♪rP和わヱ0α〃β血α∫g∫，（旭オ揖m抽ル打∫恒・，〃0比αf‘れノ叩β〃β〝d

プF叫／～GoJgmわα伽．†gαrC力⊥βk，C加gαJP厄r〝lαCビ血cαJC仇，山d・，∫兢〟0れカグd〃

Recciv軋りuly25．2（珊】訓dAccepledOc【ot忙ーほ．2000

Keyll・Ords：乃∫叩Jα∫〝∽，、－aCCine，SRSI，FHV

MqjorfactorsintransmissionofTbxqpLasmagondiiintohumansareoocysts infoodorwaterandtissuecystsinmeat（RuizandFrenkel1980；Dubeyetal・1990；

Buff01ano etal．1996；Barilet al．1999）．Previous studies have demonstrated a

positiverelationbetweenenvironmentalcontaminationwith00CyStSfromcatfeces andexposureoffarmanimalstotheparasite（Frenkeletal・1975；FrenkelandRuiz

1981）．Cats shed oocysts after the primaryinfection，Whereasinducedimmunity

preventscatsfromoocystproductionfo1lowing subsequentinfection（Dubeyand Frenkel1974；Lappinetal，1989）．Thus，VaCCinationofcatsisasigni鏑cantmeasure

inpublichealth・Live7二gondiisuchasmutantstraJnSOrirradiatedtachyzoiteshave

beenvaccinecandidates（Frenkeletal，1991；FreyreetaJ・1993；Omataetal・1996）・

However，itisnotrealistictoimmunizebothdomesticanduncontrolledcatsoneby onewiththesevaccines．Furlher，thelive7bxpp［asmacellscarryariskofaccidental

infectiontohumans・rIbovercometheseproblems，WeCOnStruCtedanovelcandidate

fbracatvaccinecomposedofanimmunogenicantlgenOfTgondiiandaviralvector・

SRSlofTgondiiisa46kDaantlgendivergentlytranscribedbythebirdirectional SAGIpromoter（Hehlelal・1997）・Despitetheprecisero］eoftheantigenhasnot

beenspeCined，VaCCinationwitharecombinarltSRSlproducedbyE・COLimodifies hostimmune responses afterinfection and partia11y protects mice from Lethal

ノダざ

FHVIEXPRESSJNGTOXOPL4Sル掴SRSl

Challengeof7二gondii（Mishimaetal・‘inpress）．Felineherpesvirustype1（FHVl）isa

WOrld－SPread pathogen causLng Viralrhinotracheitisin cats．Previous studies has

SuggeStedthalFHVIcanbeausefulvectorinveterinaryfield（Yokoyamaetal．1997

reviewed）・WeusedFHVlasadeliveryvectorofSRSlbecauseoftworeasons；1）

HostrangeofFHVlissthctlyresthctedtoFelidae，and2）ArecombinantFHVIcan

automaticalLyspreadamongcatswithoutinoculatlngOnebyone・

The parent FHVIC7301strain（Mochizukietal．1977）and the modified

ViruS Were grOWn On Crandelfeline kidney（CRFK）cells（Crandelet al．1973）

maintajnedinDulbecco’smodinedEagle’sminimumessentialmedium（Sigma，MO）

SupPlemented with8％heatinactlVated fbtalbovine serum and antibiotics．Stock

ViruSeSWereharvestedfromculture supematantofinfectedCRFKcells．Theentire

Openreadingframe（ORF）ofSRSIwasamplinedfrom genomic DNAoftheRH

Strainwiththe polymerasechainreactioJV（PCR）techniqueusingaprimerpairof

SRSl－1（acgaattccGCAAATGGTGAGGA）and SRSト2（acgaattcACCTlnGACGG

CAC）・TheamplifiedSRSlfragmentwasinsertedbetweentheCAGpromoteranda

POlyadenylic acid（poly－A）in the pCAGGS expression vector（Niwa etal．1991）

kindly provided by Dr．Miyazakifrom Osaka University MedicalSchool．The

resulting CAG－SRSl－pOly－A expression unit wasinsertedinto FHVlgenome as

Previouslydescribed（Yokoyamaetal．1996b），

Figurel・IntegratlOnOfCAGpromoter， SRSlORFandpolyademylation別gnal into（he FHV genome．The SmaJ－ EcoRVfragmentwasexchanged．PCR primers and Southern blotting probes arealsoindicated．

◆ sRSト1。。m。 5RSl－2prmler

： ＼い、！い∴

Therecombinamtvirusgenomewasexaminedforlocalizationoftheinserted

expressionunitwithSouthernblottlng．FHV／SRSlgenomicDNAwasextractedfrom

CulturesupernatantOfinfectedCRFKcellsaspreviouslydescribed（1shizawa1991）．

TheDNAwasdigest色dwithEcoRV，electrophoresedandtransferred［oHybondN＋

membrane（Amersham，EngLand）．The membrane was probed withTK and SRSl

Jβ7

FHVIEXPRESSINGTOXOPL4SAL4SRSl

probesshowninFig．1byDIG（RochDiagnostics，Switzerland）systemaccordingto

themanufacture・sspeciLication・TKproberevealedal・3kbpfragmentofwildtype

FHVlanda4・5kbpfragmentofFHV／SRSl・TherecombinationoftheviruSreSulted

inanapproximately3kbpextensionoftheTKfragment（Fig・2A）・Theextension

wasidenticaltotheexpectedlengthforsubtractionofTKdeletion鈷・OmtheSRSl

insertionaf［ertherecombinationprocedures・Electrophoreticmlgrationsofgenomic

DNAftagmentsofSRSlandTKwerealsocomparedinSouthemhybridization・The SRSIprobereactedtoa4・5kbpfraBmentOftherecombinantgenomewhereasit revealednospeCi重creactiontothewildtypegenOme（Fig・2B）・Themigrationofthe

SRSlandTKfragmentoftherecombinantgenomewasidentical（Fig・2）・

A B Figure2・SoutherJlblotanalysISOntheFHV／SRSI DNA．PurifiedDNAfronFHVwi1dtype（lanel） amd the recombinant（1ane2）were digested with EcoRVandprobedforTK（A）andSAG2（B）LThe recombinant c且rried the both sequencesin one rragment．

4．5kbp

1．3kbp

1 2 1 2

Figure3・PCRanalysisfordirectionoftheinserted SRSlu11itinFHV／SRSlgenorne・ApairofTK－1 and SRSl－1prlmerincreased a correspondiT．g fragment（lane2）whereas a pair ofTK－1and SRSト2did not work（1且ne3）．Lan巳Slamd4 contained100bpladder marker andl／hLndlIl marker，reSpeCtlVely・

2．Okbp 1．5kbp

ThedirectionoftheinsertedSRSlunitwasevaluatedwithPCRassayuslng

twopnmerpalrSOfTK－1andSRSl－1，andTK－landSRSl－2rOnlyacombinationof

TK，1andSRSl－1amplinedaDNAfragment（Fig・3）・Tbeamplifiedfragmentshas

thesimi1arsizetothecalculatedlengthforatotaloftheSRSl・pOly－AtailandTK

sequencebetweenTK－1pnmerandEcoRVsiteshowninFig・1・Theseobservations

evidencedthattheSRSlunitwasdivergentlylntegratedintotheTKreglOnOfFHVl genomeasrepresentedinFig・1・TnfectionofFfIVlisof［enletha］fornewbomor

Jβ♂

FrIVIEXPRESSINGTOXOPLAS児4SRSl

debilitated cats（Povey1979r6viewed）．However，TK deficient FHVIshows

remarkably reduced pathogenicityin cats（Yokoyama et a）・1995and1996a）・The

insertionoftheSRSlexpressionunitinthepresentstudyresultedinalargedeletion

OfTKreg10n．These suggestedthatFHV／SRSlisreduceditspathogeniclty bythis

recombination，Whichisadvantageousasavaccineparticularlyねrkittens．

Figure4・TraJISCrlPtlOn Of SRSlORFin FHV／ SRSlinfected CRFK ce］］s．TotaIRNA from the infectedcdlswasex且minedwithRT一打R（lane2） aJld PCR（lane3）．Amarkerwasalsoindicatedirl lanel（100bpladder）．RT－PCRamplifiedaDNA fr且grner［t COrreSPOnding to the entire ORF of SAG2．

rIbassessatranscrlPt10nOfSRSlbytherecombinantviruS，tOtalRNAftom

CRFK cellsinfected with FHV／SRSIwas examinedin reverse transcrlptlOn

polymerase chain reaction（RTPCR）・rIbtalRNAwas extractedfrom FHV／SRSl－

infectedCRFKcellsandwastreatedwithDNase．Th∋RNAwastestedwithRTゼCR

uslnga POly－Treverse－tranSCnPtlOnPnmerand SRSl－1and SRSl－2PCRprimers

ShowninFig・1・ApairofSRSト1andSRSl－2amplinedLheentireORFofSRSl

from（heRNA，WhereasPCRwithoutreversetranscriptiondidnotwork（Fig・4）・This

resultdemonstratedthattheCAGpromptersuccessfu11ydrovethetranscnpt10nOfthe

SRSlgeneintheinfectedce11s・TheCAGpromoterisenglneeredbyconnectlngthe

CytOmegaloviruSimmediate early（CMV－IE）enhancer sequence tothe mod拍ed

Chickenbeta－aCtiTl（AG）promoterandbothsynergisticlyenhancetranscriptionofthe

followingsequenceinvariousmammaliancells（Niwaetal・1991）・TheAGpromoter

has a strong ubiquitous activity（Miyazakiet al．1989），The CMV－IE enhamcer／

promoterinsertedinto a recombinant herpes simplex viruS type1increases

transcnptlOnOfafollowlngSequenCebynve－foldwhenCMVisinoculated，andthe

CMVphosphoproteinpp71exhibitsastimulationofgeneexpression（Homeretal・

1999）．Althoughactivity of CMV－IE enhancerin FHV／SRSl－inftcted cellsis not

evaluated，CO－tranSfectionorco－eXpreSSionofpp77mightenhancetheproductionof

therecombinantSRSlinFHV／SRSl－infectedce11s．

Jざβ

FHVIEXPRESSINGTOXOPLAS仙SRSl

Figure 5. Expression of SRSl in FHV／SRSl－infected CRFK ceLIs．FHV wildtype（A）andFHV／SRSl（B）infected CRFKcellswereexaminedby tFAusing l：200dilutedmouseserumcorrectedfrom mousechronicallyinfectedwithTgondLL The ass且y elicited positiYe reSpOnSe tO FHV／SRSlinfectedcells．

Production ofrecombinant SRSlininfected cells was evaluated withIFA， CRFKcellsinfectedwithFHV／SRSIwerewashedandsuspendedinanappropnate volumeofphosphate－buffered saline（PBS）・ThecellsuspenSion was dropped on

glassslides，air－driedand負xedincoldacetone・Theslideswereincubatedforone

hourat37白Cwith1：100dilutedserumCO11ectedfromamousechronicallyinfected

withTgondiiBeverleystrainafterabsorptlOnWithwildFHVl－infectedCRFKcells・

Then，theslideswerewashedwithPBS，incubatedwithfluoresceinisothiocyanateL

conjugatedantiLmOuSelgM＋G＋Aantibodies（SouthernBiotechnology，AL），WaShed

agalnandobserved underfluorescencelasermicroscopy・Ananti－Serumfrom T

gondiE－infectedmouserecognizedtheFHV／SRSl－infectedce11swhereasitdid－nOt

reacttowiJdFHVl－infectedcells（Fig，5）．TherecombjnantFHV／SRSlproductwas

recognizedbythemouseserumimducedbyTgondiiinfection・Thisresultsuggests

that the recombinant SRSlmaintains someimmunogenic epltOpeS Ofthe native antlgen・Therefore，immrLnization withFI｛Ⅴ／SRSlmightinduceimmunltyagalnSt

nativeSRSlantlgen．

The present study demonstrated that FHV／SRSIcan deliver the SRSl expressionsystemintocatce11sandcanexpressrecombinantSRSlintheinfected cells．FHVlinftctionischaracterizedbythefeatureoflatencyaf［erpnrnaryinfection

andpeTiodicalreactivation・ThelatencypromotestheestablishmentofendemicltyOf

theviruS and the reactivation helps viruS Spread and reLStimulation ofthe host immunity・Bythisviralstrategy，aFHVlbaserecombinantvaccinemightimmunize

motonlydomesticcatsbutalsostrayorwildcatswithoutinoculatlngOnebyone・

Therefore，areCOmbinantFHVlexpresslnga7bxopla∫maimmunogenicantlgenmay

beausefulvaccineforcats．FurtherworkisnowneededtoexaminehowFHV／SRSI

vaccineinfluencesonprotectiveimmunltyOfcatsagalnStTgofuliiinfbction・

ノア♂

FHVIEXPRESSTNGTOXOPLASMASRSl

ACKNOWLEDGMENTS

ThisworkwassupportedbyagrantfromtheMinistryofEducation，Science，

SportsandCulture，Japan・

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