sdlation from⊂attIe ofa Prion Strain Distin⊂tfromThat⊂aus

BENE2006−024
PUISFへi′，！：さミンこ∫
竺EN∂A（∝SS打叫ava眺1e。nline
］sdlation from⊂attIe ofa Prion Strain
Distin⊂tfromThat⊂aus］ngβovineSpongiforn ●
En⊂ePha］opathy
Vin⊂entB6ringuel，AnnaBen⊂5ik2￠pAnni⊂kLeDurle，Fab盲enneReinel．ThanhLanLa首1，Natha−ie⊂henais3，Ga紺eT＝
Anne−Ga釧eBia⊂ab占2．ThierryBaron2，］ean−Lu（Vi］otte3，HubertLaudel＋
1ln頭l瓜NationaldelaRe⊂krdleAg†OnOmique．Ⅵro109ielmmuno】ogieMo姶伽bi旧．止Uyヾn・Jo∼ai．Fran亡e．之A9印⊂e打an甲ise（kS卓印血色虫nね鵬desAiim8nt∼．Unilさ
Agenl∼Tran釘nj∼5ib】esNon⊂仰Ventiome】s．け○へFran⊂e，3ln京江沼Naポ○桐】delaR∝h亡ー仇e入野onomique．G細則qu亡朗∝摘而qu色et⊂〆叩血相qu亀JouyヾnJ05aS．F柑∩⊂e
Todate，bovine叩Ongiformen（ePhalopathy（8SE）andit5human（OunterPart，）ariant⊂reutz：feJdtJakobdisease，have
beenasso（i早tedwithas盲nglepnonstrain・Thisstrajnisdlaraderisedbyaun．queandremarkabtystablebio（heni⊂al
PrOfiIe ofabnormaIprotease−reSistant prion protein（PrPr亡−）is01ated fron brains ofaffeded animaJ50Tlhumans．
H‘＞WeVer・aIternatePrPrtS5ignaturesin（attlehavere⊂ent‡y．beendis（OVeredthroughJarge−S（ales【reening・Totest
Whethertheseal50rePreSeqtSeParatePrIOnStrains．weino⊂u］atedFren（h（attJeisふIitesくharaderisedbyaPrPrモーof
higher apparent mo］e（ular mas5−⊂alJed H−tyPe−into transgen亭⊂
deveJopedneurbTbgi⊂alsymptomsandsu【（umbedtotheseisoLates，ShowingthattheserepreserltanOVeIstrainof
infe⊂tiousprJOnS・（mpoTtantIy．thisa9？nteXhibitedstrain−SPe（jfi⊂featuresdearJydistindfromthatofBSE agent
ino｛ulatedtothesarnemi（e，WhkhwereretainedonfLJrtherpa5Sage．Moreover．italsodifferedfroma］Jsheeps
isoJatespassagedsofarinovjrrePrP−eXPreSSingmi⊂e．OurfindingsthereforeraisethebossibiIitythateitherv
PrJOnStrainsmayexistin（att］e，Orthatthe8SEagenthasundergonedivergentevoJutionin50meanimals．
⊂itdtion＝8‘，ingu亡V．Ben⊂5ikA．LeDuIA，Reln亡F．LiiTLeta］・（2006）lsdatjonfrom⊂attIeofaprionstraindi5thctfroIT．that⊂au扇ngbovine5POngifoTTr［en⊂ePhaJop
PL（ISPa仇og封】0）：ellえDOl：10．1371月OUmat．PPat．0020112
HヤPe Variants to twolines of PrP transgenic n−ice・
Furthermore，WePrOVidecompellhlgeVider）Cethatthisagent
Whi】etransmissiblespongiformen⊂ePhalopathies汀SEs）in
has unlquefeatures compared■tO ePIZOOtic BSEand other
sma］7ruminant5and humans are believed toinvoIve distin⊂t
related agents・We a）so estab）ish thatthereis nolink wit】一
Prionstrains［1，2］，aSingleprionstrainhasbeenaLSSO⊂iatedsoOVineTSEisolates transmittedso hrto thesemodeIs．
lntrodu（tion
faT Wilh bovine spongiform encephalopathy四SE）andits
human（Ountell）art，VariantCreutzfe）dtJakobdi5eaSe（VqD）
f3・−6】・Inparticular，theabnormal，PrOteaSe−reSistanlfol■mOr
Prion pTOtein（PrPr亡’）that accumulatesin the brajns of
inftcledil−dividua］s【7】showsaconsistent】yuniquee）ectro−
Phorelicpro6IeinimmuヮobloIS［8］・Howe、′er71hebiochen一−
i⊂a］leSLIng Of the brains ofs）aughtered and fa17en⊂atl）e，
−Vhi⊂h、へ・aSinl亡】−Si負edsince2000inEuropean⊂Ounlriesasa
mcans LOP】●OteCttheconsumers，hasJedtothcdis⊂OVell・Of
POSitivesample51ha15l−OWeddi5til−CtI）rP一缶pro丘ks・¶ese
atγPICa】profi）es have beel−SO旺edinto t、VO grOuPS SO far，
PrOVisionall）・termed H−tyPe Whenlhe size oflhe protease
resislant f．ragmentsis higher than for BSE，and bovine
anly）ojdoti〔SPOngifomencepha】opathy，OrL−1ype，Whenit
is70Werr9，10］・Thcseobservationsraisetl−ePOSSibi】it）・thatas
yetu11一▲ecOgnjsedpl’ionstl■aillSmayeXistincattleasinother
Resutt5
日−TypeI50JatesAreTran5mi55ibletoMke
Two transgenic mouse］ines were used as rec】Plellt for
transl−−issiol−eXPeI￣iments・The tg540】ineis a−−eWly eslab−
Jished］inelhaLeXpTeSSeSbovinePrP（Protoco）Sl）．resulting
in an enhanced sus⊂ePtibi】ity LO BSE a苫ent C（）mPared to
⊂Om・entionalmj⊂C［6，】2】・metg3381ine，eXPreSSingtheVRQ
（Val”6Arg154GJn17））a一一e−e of ovine PrP，has al−owed an
e掩cienttrans−−−issionorI−aluralscrapleisolate5from sheep
andgoat【13，14］・The ratiol−aleforh−C）uding tg338mi〔ein
thisstudywasthepossibj】itythalcharacterisationofapr】On
Editor：NeilMabbolt．Jnstilute†orAnimaIHealth，UnitedKingdom
Re⊂モivedJune6．2006；A（⊂モPted5モPlember】2−2∝喝Publi5hed Odober20．
SpeCies【1］］．al−d have potelltia］imp】icationsil−1e】1一−50f 200る
Ptlblicl−ea）th・Un）ike bovine amyloidoticspongiform en⊂e−
DOl；10．り71月OUma‡．p匹t．0020112
Pha】opath）・isolates，Whichderivefromanil一一a】s、、・ilhdefil−ed
（opyri9ht‥◎2006B畠ringueetal．Thisi5anOPen−a⊂⊂eSiartidedi5tributedunder
l壷topatl−0logicalabl−Ormalities【10］，Pre⊂iseinforl一一ation
theterrn5Ofthe⊂reative⊂ommon5At（ributionu（enSe，Whi⊂hpeTrnit5UnreStri⊂ted
USe．dislribulion，andreprodu⊂tioninanymedium．pTOVidedtheoT（91nalautho
COrrOboratingapriondiseaseislackingforH−tyPe⊂aSeS・Ⅰ一
WaS therefore c11】⊂jaJ10determine through experilT−enta】
and∼OUr⊂e are⊂rediled．
【ra】1S111i5Siollヽ＼′lletllel−SuC】1Ca5eSl・ef】ecIsO111e a】tel−atio11i11
Abbreviat；ons：BS亡．bovinespongilormen（ePhaIopathy；PrPre5．pTOtea5e−Te∼istanl
PTion protein；TgBov．bovine tg540；TgOv．ovine tg338；TSE，tran5mi5SibFe
5POngi（orTT．en（ePha嘉opathies；VUD．varian†⊂reutzfeldt−）akobdi5e∂5e
Plヤ11−etal）0】ism，】）OSSib】）・i−1ag川ganinlals，Ol−i】11−0卜eal−叫∫
il−f■e⊂tiotlSage11し
●Towhom⊂Or†e叩Onden亡e5houldbeaddre55ed．トmaiトhube「しbude句OUyjnra・†r
1−〕山is sltld）′，、、・e−一ePOl▼t tl−ell▲a】−S】−1issio−10f−a TSE−1ike
dj5eaSe b）・il10Ct】】aLionofFren〔I−CattIeisolatesi（lenlif．ed as￠The5eaUthors⊂0nlribuledequさIly101hi5WOrk．
÷㊧二PしOSPatho9enさtWW叩IospatllOgen5・Org
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NovelPrion5trainliOlatedfrom⊂attle
altack ratein both mouselines・The m甲T”tユTViva7times
ob5erVedwilh⊂aS亡5nO・1andno・2intgBo、imice，−400d，
appear亡d10be‡，rO】onged．⊂Ompared101hose for⊂attle，
sh托P，gOalBSE，弧dhumanvqDino叫
froIT一−250to380d・Su⊂hadiscrepan⊂y⊂Ouldrefk⊂taIesser
i】1托ctiv鉦yorH・lγpe5amp】甲，COn5iste呵Wilhtheircompara−
ti、，elylo、VerPrP’“content【9】．Moreov訂，th仁SurViva】lime
WaSreducedby−100donsubpassage，aPPrOa⊂hingthatfor
BSE丘olr−⊂attkorothersour⊂萬OnSe⊂OndaりrPaSSage，OrOn
Primaryp2LSSagefbrinocu］aofpresumabJyhighertiLre（i．e．，
PrOducing no substanlia7 reduction of surviva）time on
subpass喝e‥BSE¶0・3andARq仏Ialき6Arg闇Gln川】no．】）．
UponlranSmission10tgOYmi〔e●山em亡an
P［OducedbythefburH・t），PeCaSeSWaSStrjking】yhomoge−
neou5（586−612−d），■亡OnSktentwithapotential】γuniqueagenl
汀ableり・niswa5⊂Omp正abletoor亡V亡nShorterthanthe
incubalionperiod5bfepiz：OOticBSEorrehtedino⊂ulaonLhe
5a甲emi⊂e（560−7g愛d）・As叫5tr拍dinFigureいh亡re】atiYe
in⊂ubation periods oやSerVed on tgO†and tgBov mice
appearedtodif払■5igpi鮎antlyalnOhglheHヤpeandB平
t叩eagent5・Iniddition，there血ctioniniれⅢbationp亡バd
Ob乱亡rVeduponseco中d如けan5missidnorH−t沖e（C義eno・2）
、−
a⊂⊂id這n叫pa去5品坤Om一重如iふinan由ヤCa油呵htbe
0ム
ね⊂ilitated on shch Tnice・bァco叩ad＄On W油tb亡OYine
Ob5∈ⅣedforvqDand5heepBSEino亡ula汀abl？1）・0vera】】，
isolates transmittedsoねー．’托540一（ゆOY）a−1dlg338（lgOv）
n−ic亡0V亡ー襟preSSPrPin山ebrainatさimilarIev亡Is（−8−1010−
ag印b・
bld）・BothIinesbaY亡anOma】li長吉pan．血e5amea5PrP仙
th亡Se
HイypePrPre5profiJeI5PreservedinTransgenkMi⊂e
Thebrainsofdiseasedmic亡Wereanalysedbyimmunoblot−
1ingfbrlhea乍Cumulationofabn8ma‖，浮・PrPⅦwasre替dily
mice on which the transgenes were introduced. H-type
i＄OlateさrePreSentatjYeOfaseriesofsevensamp】esidenti鮎d
inFr3n亡eWereino亡ulat亡dintracerebral】y10LgBovandLgOv
mice（Tablel）・TypicalBSEagentsfrom⊂attleandfromother
detecledinal）mic亡teStedsirlCethe丘rs（PaSSage，COnSistent
5pe⊂i亡5WaSinodated to the same mi⊂亡ねー山e5ake or
Withthee餓cienttransmissionobser・VedinbothJines（10nO
COmParison・RemarkabJy，嶽1H−tγPeiso）atesinduced a
and 33J33 positive brains fbr tgBov and tgOv mice，
neurologicaldiseaseonpnmarytransmission．with a）00％ respe亡tive）y）・ThePrPrefmolecularpro51ewasfiir））・uniform
TabJel・Tran5rni5SionofBovineMole⊂UlarVariant⊂ases（H−Type）toTran5geni⊂購⊂eExpTeSSing80VineorOvinePrP
l50Iate
⊂a5e Number
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401王9‡5乃）
612±26（】0／10）
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377±2ユ（6／6）
298±7（9／9）
Fir5t
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FirsI
Se⊂Ond
Firit
First
Fir5t
283±10（5／i）
339±25（5／5）
340±寧け〝】
253±9（6／6】
291±27（5／5】
34ヨ±8（5／5）
29亨±11（6化）
793±2るtO／9）
792±22（即6）
195±9（6用）
835±15（0／6）
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2G3三寧l即6）
5e⊂Ond
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NA
560±朗（5／5）
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590±43†4／可
NA
NA−nOtaVa貼ble：ND．notdone．
ahtra⊂亡柁b帽Iino【ulalionwith2mgbrainli5∼uビequ鵬，e叫n／爪○：di∼ea5ed／ino山8ted−
bE岬erimenlal⊂aSeS
‘FieId亡∂S亡．
DO】：10，1ユ71月OUlnal．ppat．0020112．tOOl
：＠二PLo5Palhogen5lww・Plo5Pathogen5・Org
0957
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BENE2006−024
NoveIPrion St帽inl∼Ol∂tedfrom⊂attte
amongtheisolates・Remarkab］），11ikelhelさSEagentfbrwhich
◎
the typICa］sJgnature WaS CO11Sel−Ved wha（eVer the donor
SPe⊂ies（≧3brain5analy5ed p打COl¶bination），lhe H−t）・Pe
ag帥t e55entia】】y retainedilS biochenlicalphenoり−Pe upOn
5eria】transmissionto唱Bo、・aSWe】Ias10tgOvmi云亡eXpl・eS5ing
◎
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ahelero】ogousPrPC（FiguTe2andbdow）・CornparedtoBSE
PrPrtJ，itwascharacterisedbyasigr）ificanllyhigherapparenl
mo】ecu】ar ma5S（di8七reムc亡meaざured fbr ungり・亡05）・laled
◎
甲亡Cies‥0・9±0・05kDa and O・7±0；06kDainlgBo−・and
tgOv mice，re5peCtiYelγ）aれd the relativ亡prOPOllion50r
gl）・COfbrmswereessential）ysimilar・Afurtherdjfferenc…・aS
◆
◎
◆
theJackofdete⊂tablePrPTCSiT）lhespleenofH−（ypediseased
tgOv】Ⅵic亡仲reetofivespleenst亡St亡dperisolate），ヽVhi】ethis
accumu）atedatsubstantia】）evelsafterBSEor、▼qDinfe⊂tion
100 2鋸 380 400 5DO 600 7抑 800 9抑
（Fi訂Ire2Ⅰさ）・
Meansunlivattime（days±S∈叫
Figリーel■5un′iva仁¶一便inTran5geni（Mi〔e冊e⊂tedwithHイypeand H−TypeandEpizootk85EAgentsExhibitDistinct
NeuropathoIogi⊂aJFeature5
85亡イypeAgent∼
We next・亡Xamined the PrPr亡￡は曙etlngand th亡VaCuOIa−
Mean5Un′ivaltjme5（da鰐±5【叫岬0叩rimaけモーa耶血中Onare血own
わrtg80V（greydrde5いndtgOv囲a亡kdiamond5）mi⊂elnOC山atedw油
1ionin the brain，Which arcrknown to exhibit a s11−ain−
日ヤ匹⊂aSe5．85E．and relatedi∼Olates（5eeTablel）・meinte■V∂l5
dependentvariation［6，15，16】・ThiswaspertbrmedoT）tgBov
mkesince血yexpre5SaPrPChomo−ogous101hatorthe
donors，ind厄ding the number of octarepeaLS［17］，thus
between血ein⊂Ubat盲占云tim亡iOnea⊂hline∂re5igni石田∩吋d肘e−entfo一
日−りPeand8SE8gent5匝＜0・∝旧乙Fi5herte5t）・
D（刈：10．1371乃Oumal．ppat．0020112・900】
PrOViding a relevant⊂OnteXlfor compar＝一g H−tyPe and
epIZOOtic BSEisolates・H・tyPeiso】ates showed a similar
distribution of PrP7Y，deposits on！）OLh pr）marY and
A
tgBov mice
Cat引e
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8＄Eィelatod MI
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FigL，re2・WestemBlotAnalys．sofPrPr亡SintheBrajn5OfTTanSgenkMke］nfe⊂tedvvithH−Typeor8SE−TypeA9entS
（A）Primaryorse⊂OndaTy（lane6）tran5mi55iontotg8ovmi（e・8SE−tyPeino⊂uIainclude（attJe85Eno・3（lane53and7）・5heep85EARQno・1andARRno・
川ane58and9），gOatB5E（lane−0）・andv〔JD（Jane川・TheP（Pr亡ipTOme5Ofboth＝−tyPeand85Ea9entSin⊂attIe（）anes2and3）areesse両州ysimilar
to−ho5eint980Vmi⊂e（，ane54−11）・Braintissueequivalentloaded：2・5m9inlane2；0・15mginJaneヨ；0・5mginlanes4−12・Ml・mO（k−infe〔tedb
MM．mole⊂ularmarker∼．
（B）PrimaTyt（anSmjssiontolgOvnli⊂e・HlypeagenlShowsadistin⊂tP（P，e5patterninthebrain（Br）⊂OmParedtoB5Eagent5（Lane9・85Eno・3；Ianel
goat85E；一anel一．vC｝D）・NotetheIa〔kofPTP−eSsignaJinthe5PIeen（5p）ofHLtyPe−infe⊂tedmke（lane7LunlikethatinBSE−infe〔tedrni〔e（lane8）・Brain
orspleentis5UeeqUIVa−ent．oaded：3rn9．n］ane2；0・一5mglnlane3；0・5mglnJane54−6；2m91nlane57−12・
（CandD】Ratioofhigh−andJovv−mOle⊂U．ar−ma5SPrPre5gly〔OfoTm5inthebrain50ftgBov（⊂拍ndtgOv（D）mkefo＝owing〔ha‖engew仙H−tyPeOrB5E
agent5（dataplotteda5mean5±5EM）・＝−tyPei50late5arereP［e5enteda5triangJe5（no・1・bJue；nO・2・Or∂n9e；nO・3・Pink；andno・5′bJack）a
agent5aS5qUareS（BSEno・3．red；5heepBS亡ARQno・一・grey；5heepB5EARRno・l・ye‖ow；gOat85E・brown；andv⊂JD・）ightblue）・Se（Ondary
lranSmission5arerePre5entedbyunfilled5ymbokofthe5arne（Olour・Notelhe5trikinglydistin［tgly⊂OforrT”a−iobe†ween杵typeandBSEgro＝P5in
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了㊧：pLoSP∂抽叩enりv川佃・Plosp∂lhogens・Org
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NovelPrion5tralnlsolatedfrom⊂a相e
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．て Fi9ure3・Re9ionaIDistr7butionofPrP−−∼andVa⊂UDlaTChangesinthe8rainsofBoJineTra7tS9enkMJtelnfktedwjthHTypeorBSE＾gent5
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5irnila（amOngH−tyPei50lates（A＝B）．anddiffeTentfromthさtOfGattleBS【（qinseveTalareasindi⊂atedbyirrowheads．s∝ha5the亡OrteX．the⊂OrPu5
⊂aIlostJJT”nddoTSaI（Ommissure・alveu5・fimbria・andstratumoriensofthehipp∝aTnPUS・NotethatintensityofPrPdepositioりmarkedIydiffered
betweenH−tyPeand8S【agent5・Jllu5t｛ationofhowthi5aPPeaT5byimmunohi5tO（hernistlyinthestriatum（0）（E）andsubstantianlgra（F）（G）．Hlype−
infe⊂tedmi⊂ebeingles5intensivelylabeJJedthantho5einfe⊂tedwith85Eagent・By⊂Ont｛ast，SPOn9josi5WaSmU⊂hmore5eVereinHヰype−infe⊂ted
brains・8ars：301爪；i鴨e祀7llm・
DOI：10・1371／iournal．ppaLOO20112．gOO3
SeCOndarytransmission，aSaSSeSSedbyhistob）ottingonbrain
intbe⊂aSeOrH−typeVariant（Figure53a】1d4）：al・eaSSu⊂has
COr−Ona）scctions（FigtJre3A−3B）・neStainingdidnoldiffer
theseptllm，hypolhalamus，hippocampus，五
fromthatseenwi山calt］eBSEinse、一era】reglOnS，Su亡hasthe SeVere SPOng）OSis，aCCOmPanied by a pronoun⊂ed reactive
Striatumandsevera）m・CJei6fLhe thalamus，inc）udingthe
g］ia事astr？CytOSisbasedonCFAPslaining（notshown），Whi】e
geni⊂ulale●Ventra】postero−1ateraland−medialaswellasthe
BSE−−indtiとedvacuoIation tqasibodeTate・−in the same areas．
brain stem（Fi訃Ire3A−3C alld data noIShown）．However，
H−TypeAgentIsDi5tin⊂tfromtheOvineTSEI50lates
OLherareassud−aSlhecerebra］⊂OrteX，the⊂OrPuSCa】losum
Transmitted50FartotgOvMi（e
in⊂1udingthe⊂iJlgU］um，the dorsa】com一一一issure，the a）veus，
We占nal】yexan−il−e山vhetheTHヤpei50】atesmayhavean
and 丘mbria oflhe hippocampus were predol−1inantly
OVineTSEorlgin・Themq］Or）tyOft†PICa）andatypICalsheep
S⊂raPJelSO）ateswehavestudiedsofaTlransmilsbefbreayea
StainedwithH−tyPe，WhereasBSEPrPT亡S、VaSratherco■沌ned
in the slralum oriensofthehippocanlPuS（Figul−e3A−3C）．
to tgOvmice（（13，14］and our unptlb】5shed data）・On）y a
Moreo、・er・山eo、・era”inlel−Sltγandaspe⊂10rI）l−Pdel）OSilion
ma一女ed】y di仔eredb亡…・een
groupofshceps⊂raPieiso】atesfron−1tal）・（SSjt）wasfound（O
ill托c【1gOv】1−i⊂eafterapl■0lol−gedsu】1，ivaltin−e、再thil−tl−e
rangeorH−tyPeCaSeS・Il−deedthreeofLthel一一，SSitcaseslno・5，
immunod−emistryrevealedvarioust）・PeSOfPrPdeposits
both cases・thin diffuse PrP depositswere predol而nanlin
H−1ype−il−ftctedbrains，WhereasthelT10Stfreque］一tl）′pel、・aS no・7，andno・8，inducedat），PICal】一eurOdegeneralivedisease
granu）arin BSE−infected mouse brains．In severaIareas，
、Vi11−a】Ⅵeal15urVil′a】timeof6卵±20d（5／5anilllalsaf托cted），
includingthestlうatumandthesubslantianigra（下igure3D−
659±31d（7／7），a∫−d569±37d（4／4），】一e5peCli、・dy．Ca5e−10・5
3G），t】一打eⅥ▼aS a SLrikil−gla⊂k or⊂01−1で】ation between tlle
illCUbatio】11i111ellra55日”】ongel￣llla−1H−t）・Pe CaS＝−0・201−
illte11S呵 Or Plヤ deposiliol− a−−dll−e Se、′e川）・Of− the
Subl）aSSage（4】7±20d，6／6a】−i−−1als af量tcted）．The Plヤ「亡エ
VaCuOlation．01・el・all，llle＼′aCuO†atioIll＼，a5111しIC】1】1101てiIlte11Se
1110】e⊂tllarpron］eobsen・edinthebrai−10rSSil−djseasedlllice
三⑳二PLoSPathogen∼fwww・Plospathogens・Org
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abnorl一一a］Pr−P with a distinclmOlecu）arpaltern，⊂a11ed H−
t）・Pe，COnSistenlly produces a LhLal，TSE−1ike disease tlPOr・
︵≡uS羽u冨呈巴8S
i】一OCtllation to both bovine andovil−e PrPlrarlSgenic mice・
¶l亡S亡I◆e5ulls，亡0汀Oboratlnglher亡Cenltra11S】而ssior】tOl、づId−
type−¶i⊂e【1軋わrmallyestab】ishthat5uCh⊂aSeSil叩Olvea11
aulhentic TSEir虎⊂tious agent・Importantly，We PrOVide
detai】ed eviden⊂e thatthis new！y re⊂Ognised agent diffbrs
frorn ep】ZOOti⊂BSE agent derived fTOm Cattle or other
5peCie5・
Both mo）e⊂ularand biologl⊂alcriteria support thecon−
ClusionthalH−typeandBSEagentsaredistinctpr−OnStrains・
Firsl，lheincubation periods uponll■anSmission tomi⊂e
expresslng either bovine or ovine PrP produced difrerent
patterns・Tl−uS7WhileprlmarytranSmissiontotgOvmi⊂e］ed
tolongersur再valtimesforbothagents，lhein⊂reaSere）ative
10tgtiovmicewassign沌cantlylessforH−tyPethanforBSE−
typeagents（Figurel）1Second．Lhemolecu7arpro別esofthe
Prl）一群rl−a訂−−ent5d亡te⊂tedinlhebrainordi5ea5edmicewere
⊂lear］ydistinguishablein either）ine・Slriking】γ，differences
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essentiallythesameasincatt】ebrain・Third，un］ikethatfor
BSEagents，nOPrPrC）signalcou7dbeseeninthespleenofH−
Brain areas
Figure4．Lesion Pro何e5jn tgBovMi⊂eJnfe⊂ted with H−Type or85E
type−infected tgOv mice，indicative of a stronger neuro−
A9ent5
tropISm at］eastin this host・Fourth．histopatho】ogl⊂a］
Means⊂OreS（±5EM）refIe⊂tingtheinten5ityofva⊂UOlationareshown
examination oftgBovmice revealed a contrast）ng PICture・
fol＝−tyPe（no．1andno．3．triangIes）andBSE（no・1andno・3，Square5）・
Thenineg−ey−matterarea5U5edto⊂On5trU⊂tthepTO触area5fo］low5：
Typi⊂a”y−SeVereSPOnglOSisanddiffusePrPdepositionwere
dorsal汀陀dulla（1）；⊂erebe11ar ⊂0rlex（2）；SuPeriorく0＝kulu5（3】；
hyp（）thalamus（4）；medialthalamu∼（5）；hippo（arnPuS（6）；SePtum（7）； pres亡n【insevemlar亡a50fH−tyPe−ink⊂ledbrain5，Whilethe
medial⊂erebra暮亡OTteX at theIeveIof the thalamu5（8）；and medial sameareasofBSE−infectedbl￣ainsshowedlimitedspongIOSis
⊂erebral⊂OrteXattheIeveJofthe5ePtum（9）．
togetherwi【hmarkedPrPdeposition・Suchdiscrepan⊂iesare
DOl：10．1371乃OUrnat．PPat．0020112．9004
unlike）ytore5uItfromunequa］survivahimessincetheywere
alsoobsen・edonse亡OndaTyPaSSage，Wherethetwoagentshad
COmParab】eincubationduration（unpubJisheddala）・
（rnorethanfourbrainsanalysedperiso】ale）was⊂haracterised
byasignificantlyhigherapparentmolecularmass（differen⊂e
of O．7 j：0．2 kI）a）and a slighl】y higher proportion of
dig】・YCOSyl叫edPrPr亡5ascomparedwithH−tyPe−derivedPrPTeS
（Figure5A−5C）・Thesefeatureswereconsen，edonsecondarY
7l−eisolationfrorncalt】eofapr）On51TaindistiT）Ctfrornthe
oneilT）Pljcatedintl−eBSEepidemicsraisessevera7concerns・
OneiswhetherH−tYPeisolatesmightresu）tfromanexposure
topr10nSOfsma一】ruminantsviaalimentarYOrenvi；onmental
sources，Sin⊂e eatt7e have been shown10be susceptible to
experi・T・enta】infectionbysheep scrapie agent【19】・lnthis
transmission（Figure5A−5C）・Another difference was again
thepronoun⊂edac⊂umulatjo一一OfSSit−aSSOCiatedPrPT亡Sinthe
SP7een，Whi】ethiswasstiJlimpai）−edjnH−tyPe−infe⊂tedmice，
evcn after two passages（thr亡e10舟ve sp）eeJ）S teSted per
regards，thebettercompatibi】ilybetweenpvinePrPsequence
and H−t）・Pe aS COmPared10BSE、VaSintriguing（Figure】）・
Ho、、・eVer，OUrinvestlgationsdonotsupportthis・Amongthe
fivegroupsofnaturaliso】ates wehaveidenli鮎d so farin
COmbination；Figwes2Band5A）・
W’ethen亡OmParedb）lhistobloldistributionandnatureof
tgOvmice（f13，14］and our unpub】ished data），011）y one
PrP7ESdeposiLSWithinthebrainsoflgOvmiceinfecledwitha
group，made upmostlyofSSitisolates・PrOpOSed to be of
SeCOndpassageofH−tyPe（no・2）andSSit（no・5）cases・Both
ma】・kedly dif7tred between the two agents asi11ustratedin
Figure5・Il−deed●】argeplaquesofSSil−aSSOCiatedPrPr亡Swere
presenlinthepreLeCtaJ，一uCleiandinrelatedstruCt11reSOfthe
ialrOgeni⊂ Origin【20】，Sl−OWed anincubation til−つe aS
p】−0】ongedasrorH−tγpeCaSeS・HoⅥ・e、′el一・tl−eP−、Pr亡5molecuIar
PrOf）Ie，natUreOfdeposits，anddislrjbulionwithinthebrain
as、祀．）asthedifferentiaJaccumulalioninthespleenstrongly
】il一一bi⊂SyStel一一 SuChlhe fbrnix・the alveus，負mbria・al−d
distinguish H−t）・PeandSSitisolates・In addition・Lhe】atter
hi】ed50fもr1011てInSmiltotgI主0、・mice・
subk山厄m of t】−ぐhippo〔aローptlS・H−t）・pe−aSSOCialed plヤーm
、＼raS dcleCledinsteadil−1he〔OrPuS Ca】losul−l，COrteX，a】1d
venLro111ediaJthalallli＝lU⊂lej（Figt）re5D and unpublished
data）・Thedepositsseemratherlhinorgranu］ar・S5itcaseno・
8，、、・hi⊂hga、・ell−eSl−011esti−−（t止伯Lion pe−−iodil＝gO、・mice・
、、，aSalsoinoctl】atedb）′iTltraCerebralrol】tetOtgBovmice・Of
nole，IIOdiseasellaSbeenot）Sen・Cdγetin）nicemonitoredup
H−1ype al−d BSE agenLS mlghlbe re）aled despite the汗
dislinguishable phenotypes・Theiso】ation ofan additiona】
5けail−upO】−eXpO5ureOflranSge】−ico‥、・‖dヤpemicetotI−e
epizooLi亡BSEagenthasbeenreported【21いhusquestiol−illg
itsstrai】一homogel−eity・AJso−一一一OJectl】art），P＝一gStudies have
re、一ea】edl】−e P−−eSel−Ce Or a−−−il−Ol・，−−On−BSE−t）′pe PrPr亡∼
lO6（JOdafterillfeclion．11−COnClusion．1hesedatasuggestthalcomp（）nellLin BSE−and、′qD−inftcLed b・−ai・1S r22】−Hel−Ce，
H−1yPeiso】atescoし．］d arisefz・Omthepreferelltia】amp】ifica−
Lhe TJ−t）1PC agentis tl−1reJalCd to the o、′ine TSEisolates
Liol一汗一Cerlajllindi、′iduaJsofastlbco】一l）〕One）llPreSel−LillBSE
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Fi9Ure5．⊂ornparisonofH⊥TypeandS5itIsotateFeaturesuponTransmissiontotgOYMi⊂e
（Aand8）WesternblotaTdysisofPrP−ポinthebrainsandspkensoftgOvmi⊂einfe⊂tedwithHJqrPeC−S5itis0latesat6TSt（LarLeS2→）andse⊂Ond
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thatofH・tyPeO（SheepBSE（Sh85E），a5ShownafterPNGa5etreatment（B）．Notea）∼OthatPrP’E‘isdet∝tedinthespIeen（5p）of5Sft−bLJtnOtOfH−tyPe−
infeaedmi亡e．TissLJeequjvalentI00ded：13mginlanes2→；0．04rngin）ane5；0．5mginlanes6−7；2nginJane8；0・01mginIane9；0・lmginlaneslO−
11．仙川．mole⊂Ula−ma血e汚．
（qRatioofhi9h−andLow−mOJe⊂U］ar−maSSPrPrt＄gly⊂OformsinthebrainoftgOvmiくeinfe⊂tedwithH−tyPeO（55鉦i50late5（datapIottedasmean±
SEMトOnモ甘けPei∽la愉tno．2）短柁PreSenteda；Orangetriangte．SSitisolate58rerePre5enteda5dr⊂k5（SSitno．5．1ed；nO・7．b−OWn；nO・8．咋110W）
5e亡OndarytTanSrnissionsarerepresentedbyunfiJJedsymboLsofthesame⊂Olou（．NotethestabIydjstin⊂tgly⊂Oformratio5betweenH−tyPeandSSit
agent5uPOn；erialpa5Sage．
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mouseb−aiTBat・theleYeIsoftheMppo⊂amPL．SareShown・ThedistrjbutionofH−tyPe−a550⊂iatedPrP．tldepo5itswa5d椚erentfromthatof5Sitin
re9JOnSSLJdlaSthealYe肌Ofthehippo⊂anPUS，the⊂OrPU5⊂aJlosun．theprete亡talnu⊂Iei．the⊂OrteX．andtheventromedia）thalamu；・Notethatthe5ize
OfPrPr6血pos厄markモdtyd沼eredbetweenthetwotype∼0【i501ates．
e
infecLious sources・Compar，ing H−tγPe and BSE−derived
SuCh as thalaPPljed to⊂arCaSS−derived products，has
Variant pnoTISidenti￡edinmicemight beinfbrmativein
invoked as one possible origin fbr the emergel−Ce OfBSE
lhall−eSpeCl・
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A】Lernativdy，SuChunusualcasescouIdref）ectlheexisten⊂e addre5Sthisissueexperime∫lta11y・
1nconc）usion，Ourfindingssupporttheviewthatal）easlレ
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twoandpotenliaIlythree【10】distillCtprionstraiれ5】11aybe
）・eljr5ud−・infkctions maylead to a⊂】inicaIdiseaseil−the
nallJra）host，theyseemtooccurata】owfrequency，WhichisPreSentir）Catt］e・The cuTrenlunCertaintjes regarding the
rer）Tiniscent oflhe sit11ationknown for sporadic qDin Or）g）n．PreValence，andpotentialriskforhumansofastrain
OrTSE agent unre⊂Ognised untilre〔ently should sl】PPOrt
humans（23，24】▼Ofnote．thedisparitiesbetweenintensilyof
continued efTorts10Characteriseitin vivo and uphold the
PrPdepositionandseverityofvacuolationinlhebrainsofH−
1yPe，il−OCUlated tgBov mice have also been observedl、，ithsu11℃il】allC亡eXerted on cattle．
SPOTadicqDbothinhumanormo11Seinfectedbrains（21，25］・
nesedata，however，needtbbeconsolidaledlhroughfbrther Materia）s and Methods
invest】gatiops，irlC7udingepidemi0）0gicajana）ysis・Indeed，an
Isolates．The tl−tyPe【9〕，gOat BSE（CH636⊂aSe t26】），and
il−−pli⊂alio110r山is】altel−SCenariois that su⊂l− boヽ・ine
experimenla】sheepBSEsamp】es［27いvere providedb）′lheF一’en⊂h
TSEReferen亡el・aboratory（＾gen⊂eFTanCai5edeSさnlrilそSanitaire
“atypicalP cases could occurin countries free or BSE
desA）irnents【AFSSALL）・On，F−71nCe）・7l−eSamPles［1’Ol一一Frer）ChBSE
exposure・TheacqulSlt】OnOfnove】】〕一’OPe11iesb）1an endog−
〔aSeSandT．・OmeXPeril一一enu7sheepBSE（ARR【A）aJ36AlinArg17一】
enous，SpOl・adi⊂Catl】eTSEagel−t，aS
ge∫−Ol叩e【2呵）、代代P−▼0、・id亡db）・tl−亡Ⅰ−−5【i11tlNatio−1aIdelaRecl】er⊂l−e
Agro”Omiqtle（lNRA；To＝louse，Fra】l〔e）andlhe7nsli（uteforA・ljnla】
ll甘Ol】gha−−il−ten−−亡diaりり−OStOl−aPI−）′5】COCl−en−ica】treal11−enl
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Health（TAH）NeuropathogenesisUnit（EdiT・burgh，UnitedKingdonl）， des⊂ribedb）rFr7t5erandDkkinsonf15い）yuSing【、、・O10thr亡ebrains
r亡5r，e⊂【il一軒・Tれel・qI）iso】a【亡IYおaヽl′orld H亡allhロー官an血血n
（WI‡0）rer亡−てn⊂eSal叩】erro】−−1heNationalI−−51itlll亡hrBio】∩衰亡a】
Pel￣iso】ale・
Standal’dsandCon（rO］即IBSC；PottersBar．Unit亡dKingdom）・SSit
iso！ates、−亡1−e PrO、∫ided bylheInslillltO Sttperiore diSanita（lSS：
Supporting lnformation
Rom亡，l【叫）・
T、ransg帥icmi⊂eandtransmis5ioI】aおayS・Th亡咤詞Olin亡eXPreSS亡5
1l−⊂b肛in亡PrI）al】d亡−Vi【h60Ctarepeat5under11−e〔l）l−【r＝Iortl−e
ProlocoISl・Des⊂rip（10nO［theBovinePrPTran5geni⊂Mic亡（lgう珊
Line）
亡）・tOn−ega】t汀irus（Cヽ†Ⅴ）promo【亡rOnaFV卑n−Ol■5e】i−−e、ヾ吊−P−・P■抑
Foし1ndalDOI＝】0・137】！i（−11rnal・PPaLOO201】2・SdOO】（92KB‡）OC）．
ba（kgrntlnd（Proto⊂OISり・metg3381ine亡Xp−・eSS亡5【he＼／tご．石R．i，q，7，
al】eI帥rmう眠Prf・a【abomozygou55tate，OnamOllSePげ…Iba⊂kgrotlnd
【29j・T】1＝m‡1Sgel−亡⊂OnS【nlCt厘3）亡OnSi5【Sin a ba（terialalて浦da】 AccessioTINumber5
亡hr一）mOSOme（BAC）inseTtOf】25kbofsh亡亡PDNA（】3j・AIJexperimenLSneGenliank（http＝Il、川▼、、■・n⊂bi・nJm・nil一・gOVIGenbank）ac⊂eSS血1nulll−
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bo、・ineT’げ（N九日81（‖う）and5】−eepPrf−（M3131封．
Pr叩ar亡d亡Ⅹlemporaneol15】yinac】鮎SIImi⊂rObiologlCal〔abin亡tu51−−g
disposabl亡e勺u−PITIenLlndi、・iduallγid亡ntified6−【O10−、、・k−Old mi亡e
l咋T’eiJ10⊂u］atedintra⊂亡rebr3））ywith20Il］ofa】0％（Wt／vo））brain
homogenaLein5％g】u⊂OS亡・Mi⊂eWeremOnitoreddai】ron⊂ei】land
A⊂knowledgments
ki】lぐdin亡Xtr亡mis．
We thank O・Andr亡OJetti（1NRA−ENV，Toulouse，France）forcattle
Ana）ysi50f PrPrCf mole仙hr palle川・AIJpro⊂edures regarding
BSE sample5and he】pIbr mous亡l−i5tOpa山0】ogi⊂alanal）・5is−N・
Pllr沌⊂alion and d亡te⊂lion or】）rP一日行om brain5and spl亡亡nS Or
HLlnterけAH，Edinburgh，United Kingd（】m）for ARR she亡P BSE
infe⊂t亡dmi⊂e、祀reaSd亡S⊂ribedr14］．ICSM】8［30］orSha3ユ【51〕anti−
PrPantibodieswereus亡d・Enzymati⊂deglγ⊂OS）・lation、VaSPerformed SamP）e5，U・Ag一−imi（TSS．Rome，Ita）y），forItaliansheepscrapi亡⊂aSeSJ．
Crassi（Co111】nissariatえl’En亡rSieAtomiqll亡，Sa｛lay，Fran⊂C）h・Sha3l
On d印atllredアrP「【王wilI11，0001Jo†r亡くOmbinanlPNGas亡（New
けmperia）Coll亡ge S⊂hooJo［
ELlgtandBiolabs，Beverly，Massachusetts，Unjt亡dSta［eS）［or2Tlat37 and12F10antibodi亡S．S・Hawke
0
M亡di亡in亡，LolldoJl，UniLed Kingdom）forlCSM18antibod）・，S・
cinl％NonidetP40且ndtheproprielarybufrerasdesc）ibed（30j・
PrllSin打（lnstitlt【ef8r！ヾ亡urOd亡gen亡n【iv亡Di5eaSeS・SanFran亡is（ヤー
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