Ve t. Path ol. 14: 6 7- 72 ( 1()77)
D emyelination in Mice After Two or Three Infections with A virulent Semliki Forest Virus MAY C H EW - L I M .
A . J.
S U CK LIN G
a nd H . E .
W EBB
St . Th om as' Hospi tal Med ica l Sch o ol , Londo n . E ng la nd
Abstract. A d ult m ice give n two or thre e int ra pe rito ne a l inocu lat io ns wit h av iru le nt Se mliki Fo res t Virus sho we d typi ca l lesion s of a vira l e ncep ha lit is s imila r to th o se ca used by a virule nt stra in o f the virus. D em yelinati on a lso wa s se e n in th e medull a a nd in th e fo lia o f th e ce re be lla r white matter. Ne uro no p hag ia was see n o nly in mice th a t had had thre e succes sive infe cti on s . The repe at e d inocul ation s o f aviru lent viru s ex ace rba te s t he e nce p ha litis of a sing le ino culation a nd ca uses de my e linat io n . T he mic e did not have neu ro lo gical clini ca l signs exc e pt fo r a s ho rt-live d weakn ess of th e hind legs . No ce ntra l ne rvou s sys te m lesion s we re see n by th e 7th a nd St h wee k a fte r t he initi al infec tio n a nd a ll m ice recovere d .
Av irule nt Se m liki Fo res t Virus give n intrapcrit on ca lly ca n induce ac ute e nce phalitis in adult mic e which reso lves by 6 we e ks wit ho ut nei rol o gica l signs [8]. O t her research ers ha ve de scribe d e nce pha lo pa t hy in nin e mi ce 26 mo nth s afte r intrace rebral inoc ula tio n with th e avirulen t virus [25] . Thi s paper describ es experim ents which showed the effec t o f a seco nd int rape r iton e al inf ectio n wit h t he sa me vir us eith er 7 or 14 da ys a fte r th e first in oc ulation . M ice in a th ird expe rime nt were inoculated at 7 and 14 d ay s . Th ese e xpe rime nts wer e to asse ss histol ogica lly if repeated inf ections wo uld e vo ke a cent ral nervo us syste m react io n d ifferent fro m th at o f a single in ocu lat io n wit h t he vir us .
Materials and Met hods T he av irule nt stra in o f Se m liki Fo re st Viru s used had undergon e sev e n intrace re bra l pa ssages in 2- 5 da y o ld mice . The stra in wa s a clo na l se lec t io n fro m plaques propaga te d in mon olayer s o f prim ar y chicke n e m b ryo ce lls 141. Spec ific- pa thogen -fr ee Swiss A , G mice. 3-4 we ek s o ld. we re g ive n 0 . 1 ml o f 10- " di lutio n of t he virus in a sus pe ns io n o f ste rile bo vine a lb um in p ho sphat e sa line . pH 7 .0. In ex pe rime nt I . 63 mic e wer e di vid ed into th re e gro ups a nd we re give n a n intraperit on eal inocul ati on o f 0 . 1 ml o f viru s each . G roup I wa s infe cte d aga in a fte r 7 da ys . Grou p 2 was infec te d a seco nd tim e a fte r 14 day s . and gro up 3 mic e we re infected da ys 7 and 14 aft er t he first infectio n . Four co ntro l mice eac h wer e give n a n inj ectio n o f bov ine a lb um in phosp hate sa line . Mic e were kille d by a na es t he tic e the r a t wee kly int er va ls from th e tim e o f th e la st inf ect ion . The e xpe rime nt la ste d 7 wee ks . In expe rime nt 2 . 48 m ice were d ivid ed int o thre e gro u ps . tr eat ed as a bo ve a nd o bse rved fo r 8 we ek s . T he re we re no co ntro l mi ce . In e xpe rime nt 3 . 96 mice were di vid e d in to thre e gro ups a nd tr eated a s above . T he experim e nt lasted 9 wee ks . Tw e nty co ntrols e ac h wer e give n o ne intra pe rito nea l dose o f bovi ne a lb umin pho sph at e sa line a nd a no t he r 24 mice ea ch we re give n o nly o ne ino cula t io n o f virus intra pe rit o ne all y . 67
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C he w-Lim . S uckling a nd Webb
1 Fi~.
1: Sec tion of hipp oca mpu s with as trocy te hyp ert roph y . Caja l's go ld sub limate .
Brains we re fixed in 5 % form alin fo r a t least 7 days . In e xperiments I and 2 b ra ins we re sec tio ne d in th e front al coro na l plan e at two places . The first cut was in front of the hippoca mp us a nd t he seco nd was a t the junctio n of th e midbrain with t he brain ste m . In e xpe rime nt :I eve ry o the r br a in sa m pled was secti on ed sag itta lly. Fro ze n IO-JLm-thick sec tio ns o f hippocampus wer e sta ine d with Ca ja l's mod ified go ld sublima te to show as trocyte s 126]. Brain s we re e mb ed de d in par affin a nd 5- 6- JL m sec tions we re sta ined with haem at oxylin a nd e os in (HE) . T he modi fied We il D a ve n po rt method 11 5. 2 1] was used fo r d em o nstrat ion of microglia a nd o ligoden drocyt es . De mye lina tio n was see n with luxol fas t blu e cou nterstai ne d with cresyl-fas t vio let II I ]. Mall or y's p hospholll ngs tic-ac id- hae matoxy lin sta in was used to show a b no rma l as trocy te s in dem yelin at e d a reas o f pa raffi n sections .
Re sults Perivascu lar cuffs of a viral e nc ep ha litis were see n in sectio ns fro m mi ce kill ed up to 6 we ek s after t he last infection . Maxim u m cuf fing reac tio ns were see n 2 week s afte r th e last inf ection wit h the virus . T he perivasc ular cuff consisted m a inly o f lym p ho cyte s with rare pl asm a cell s . T here wa s a lso in filtra tio n o f t he men inges a nd th e white matter with lympho cyt e s . E ight weeks a fte r ex pe r ime nts 2 a nd 3 were sta rte d th ere wa s minim al peri vascular cuffing in brain s of those mi ce th at had had thre e virus infe cti on s . As trocy te hypert roph y wa s mo st noticeab le in m ice given thr e e inocu la tio ns of virus (f ig . I) . As tro cy te hype rtro ph y persiste d in co nj unctio n wit h the pe rivascu lar cuffing . A stro cyte s a lso were see n within th e foc i of myel in lo ss. A ll the m ice rec eivi ng mo re t han o ne ino cul at ion of virus d e vel o ped d e myeli na ting le sio ns. Dem yel inati on was mo st pro m inent in the me dull a ry su bs ta nce a nd
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D cm yclinat ion in Micc Ta ble I. Sum ma ry of ma in histo logi ca l find ing s Hi st o lo gy o f br ain Icsi o ns
Perivascular cuff ing A st rocyt e hy pert rop hy D em ye lin ati o n o f cere be lla r w hite
Mi cc give n 2 vir us in o culation s m ax imum
max imum
present
ma rk ed
foci o f dcmyc lin a t ion pre sen t
foc i o f d cm yclin ati on more m ark ed hyp ert rophy m ark ed se e n in cere be llum a nd medulla
matt er
Mi cro gli a l rea cti on Nc uro nop hagic nod ule s
Micc give n 3 virus inocul ation s
hyp ert rophy pre sent no ne
folia of th e cerebe llu m 1- 3 wee ks after the la st infection (fig . 2 ) . No e vide nce o f d em ye lination wa s see n in mice at we ek s 7 a nd 8 . In th e ce n tre o f th e foci o f d e mye lin at ion in th e cerebe llum th ere we re few o ligod e ndrocyte s . T hey we re more no tice a ble ar ound t he periph ery . H yp ert rop hied fo rm s of mi crogl ia co uld be see n near b lood vesse ls , in th e mol e cul ar layer of th e ce re be llu m a nd in th e a rea of dem yelin ation . A t 2 a nd 3 week s after the last inocu latio n ne u ron o phag ic nodul es we re see n in th e ce re be llar me dull a of mi ce give n th ree doses of the virus (fig . 3). Ce lls a ro und t he d egen erat ed neuron s were ma inl y m icro g lia . Brain titrati on s 7 d ays afte r th e last infecti on showe d no ev ide nce of virus . T he m ain hist ol ogical findi ngs a re s um m a rize d in tabl e I .
Discussion In a ll gro u ps t here were typi ca l fe ature s of a vira l e nce p ha litis . T he thi ck infi ltra te o f lym ph o cyt e s in th e m eninges and neuronophagi a was sim ilar to th e brain lesio ns d escr ibed for a n a d ult mo use 4 d ays aft er being give n 0 .0 3 ml ino cu la co n ta in ing IOu ; plaq ue forming un its of th e vir ule n t stra in of Se m liki Fo res t Virus int o th e foot pad [ 17 ). The excessive ly hyp ert rophi ed as trocy tes we saw 1- 3 wee ks after t he last infectio n we re no t d issi mi lar to t hose se en by o ne re searc her 72 h a fte r intraperito nea l infe ctio n of m ice wit h 0 .2 m l viru le n t vir us o f titres ran gin g fr om 10- 1 to 10- 7 [26). Two o r three ino cu lati on s of av ir ule n t viru s se e m to exace rb ate the b rain re acti on , a nd histol og icall y th e b ra in le sion s a re sim ila r to th o se of viru len t Se m liki Fo re st V ir us . M ice give n av iru lent vir us s urvive d th e e xpe rime nt , wh ere as 90 % o f mice g ive n intra pe rito ne al infe cti on s with 0. 0 3 m l o f gra de d d ose va ria tio ns fro m 30 0 to 1000 plaque fo rming un its of th e vir ule n t virus di ed afte r 8 days [5]. In o ur ex pe rime nts we sa w demye lina tio n , whic h has not be e n reco rd ed before wit h e ithe r t he av irule n t o r viru le n t st rai ns of Se m lik i Fo res t V ir us . M e ylin sheat hs a re m ultila me llar membran es e nclosi ng axon s . In th e ce n tra l ne rvous sys te m th e y a re ex te nsio ns o f t he plasma membran es o f th e o ligo de ndrocyt es ; in the perip he ra l nervou s syste m th ey a re e xte nsio ns of th e Sc hwa rm ce lls . O t he r ex pe rime n ts ha ve sho wn tha t myel in b re akdo wn in vivo ca n be mech ani ca l [6 ]. tox ic [1 3], a lle rgic [ 12] o r vira l [23 ). Both ac ute a nd chro nic d em yelin a ting disea ses in man a nd a nima ls ca n be ca used by vira l infect ion s .
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C hew-Lim , Suc kling a nd Web b
2
3
Fig. 2: Sec tion o f cere be lla r fo lia wit h foc us of dem yelin ati on . Luxo l-fas t blue a nd cre syl-fas t vio let . Fig. 3 : Sec tion of cere be llar medu llar y substa nce wit h neu ron oph agie nodu le . H E .
The best known a n ima l demyelin at ing di se ase s ca used by vir uses a rc vis na in Ice la nd ic shee p [19 , 20), ca n ine di st em per [16 ] a nd neu ro t ropi c mouse he pat itis [1 ,7 ,1 0 ,1 4 , 22] . With av ir ule nt Se m liki Fo res t V ir us , demyelin at ion was not induce d by a sing le infecti on with a neurot ropic virus de spite an acute e nce phal itis . In all grou ps o f mice give n two or th re e infe cti on s , howev er , dem yelin ati on was consta nt. Repeat ed infections o f virus may d am age th e o ligo de ndrocy te by becom ing sit es fo r virus replica tion . Ma t ure virio ns of Ve nezue la e q ui ne e nce pha lo mye lit is have been seen in th e o ligo de nd rocytes of infect ed ad ult mi ce [9] . Th e me ch ani sm of dem yelinati on in mou se hepa tit is vir us e nce p ha lo mye litis is the a ffin ity of th e vir us for th e o ligo de ndroey tes of th e ad ult mo use, a nd dem yel inati on occurs subse q ue nt ly to th e degenerati on of th e infe ct ed o ligo de nd ro cytes [14] . A no the r ex pla na tio n is th a t dem yelin ati on is an immu nopath ol o gic p hen o m en o n resulting fro m repeate d rath e r th an sing le virus in fecti on . Some, ho weve r , report th at , ch a ngi ng t hc immu nol ogical st at us of th e se m ice by giving intra peritonea l vira l specific a nt ise ru m did not sho w dem ye lin a tion , eve n th ou gh th e y a ltere d th e timin g of th e e ncep ha litic pro cess [18 J. O thers used the tubercl e bacill i and its so lub le pr oducts as a n a ntige n to sho w th at da mage to m yel in in th e periph er al nerves of guinea pigs was a nonspecifi c co nse q ue nce of a spec ific cell -med ia ted immune reactio n in t he vicin ity of a my e lin at e d ne rve [24] . In th ese ex peri me nts th e tw o or three inoc ulat io ns o f t he av ir ulent virus co uld hav e pro du ced de my e lina tion by a sim ila r reactio n, but t he a reas of myel in loss di d not part icul arly surro und cuffe d vesse ls. T he di sap pea rance o f dem ye lin at e d foc i ca n be a ttr ibu ted to the rem yelin atin g ability of th e m ou se bra in [2 , 3 ] . Reso lutio n o f dem ye linat e d foci doe s no t nec essa rily me an demyelin ati on will not re cu r lat e r. In a n ult rastru ctural study re se arch ers sho we d th at demye lin ati on recurred in mic e 16 m o nth s af ter intr a cerebral infecti on with t he mouse hep atiti s vir us [10] .
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Demye lination in Mice
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Although lesions were noticeable microscopicall y in th e central nervou s system no ne of the mice showed neu rol o gical signs except for transient wea k ness of the hind leg s. This cou ld be re lated to th e dem yelination . More infections cou ld ca use e xte ns ive loss o f myelin and ne ur o na l dege ne ra tio n with perman ent cli nical signs . This process o f dem ye lina tio n from repeat ed avirulent Se m liki Fo re st Virus infect ion ma y be usefu l for st ud ying dem yelinating di se ase s of the central nervou s syste m by using a viru s that does not produce clinical signs of an acute e nce pha litis .
Acknowledgements We thank Dr. R. O . Barnard for ad vice and Dr. Sue O a te n fo r the mou se brain virus titres . Supp ort ed by g ra nts from the Ph ilip Fleming T rust a nd Multiple Scle ro sis Soc ie ty.
References Bail ey. 0.'1'.: Pappenheim er . A .M . : C hee ve r. F.S .: Da niels . J.B .: A murine viru s (JHM) ca using disseminated encephalomyelitis with extensive de struction of myelin. II. Pathology . J Exp Mcd 90 : 195- 21 2 . 1949 2 Blak em o re . W.F .: Observations of oligodendrocyte degeneration , the resolution of status spongiosus and remyelination in cup rizone intoxication in mice. J Ne urocyto l 1:41 3- 42 6 . 19 72 3 Blak em or e . W. F. : Remyelination of the superior cerebellar peduncle in the mouse following demyelination induced by feeding cup rlzune , J Neuro l Sci 20: 73- H3. 19 73 4 Bradi sh . C .J .; A llne r, K.; Mab er. H .E .: The virulence of original and derived st ra ins of Semliki forest viru s for mice , guinea pigs and rabb its. J Ge n Viro l 12: 141 -1 60 . 19 71 5 Bradis h. C .J .; A llne r, K .: T he early responses of mice to respiratory and intraperitoneal infection by defined virulent a nd av iru le nt st ra ins of Semliki fore st viru s. J Gc n Virol 15: 205 -2 1X. 1972 6 Bun ge . R .P .; Bu nge . M .B .; Ris , H .: Electron microscopic demyelination in an experimentally induced lesion in adult cat spi nal cord. J Bio phys Bioeh em Cyto l 7: 6 H5- 696 . 196 0 7 C hee ve r. F .S .: Da nie ls. J .B .; I'appe nh e imer . A .M .: Ba iley . 0. '1'.: A murine virus (JHM) causing disseminated encep ha lomyelitis witb extensive destruction of myelin. I. Isolation a nd bio logical propert ies of t he virus. J Ex p Med 90: I H1-1 94 . 1949 H C he w-Lim , May: Mouse encephalitis induced by avirulent Se mliki forest viru s . Vet Pat ho i 12:31'739 3 . 1975 9 Gore lkin . L.: Venezuela n equine encephalomyelitis in an adult a nima l ho st . A m J Path ol 73 :4 25442 . 19 73 10 Hern don , R .M .: Gr iffin . D .E .; McCormi ck , U.; We iner. L.I' .: Mouse hepatitis virus -induced recurrent demyelination . A rch Ne uro l 32 :3 2- 35 . 19 75 II Klu ve r. H .; Barrer a . E .: A method for the combined staining of cells and fibres in the nervous system . J Ne uro pa t ho l Ex p Nc uro l 12:400- 403 . 1953 12 Lampert. 1'. W .: Electron microscopic studies on ordinary and hyperacute experimental allergic encephalomyeliti s. Ac ta Neu rop ath ol 9: 99-1 26 . 196 7 13 Lampert. I' .W .; Schoc hc t , S .S .: Electron microscopic ob servations on experimental spo ngy degeneration of the cere be lla r white ma tt e r . J Ne uropa t hol Exp Ncurol 27: 210-220, 196 H 14 La mpe rt . P.W.; Sims . J .K .; Kn iazcff. A .J .: Mechanism of demyelination in JHM virus encephalitis. Acta Ne uro pa tho l 24 :76- 1'5 . 1973 15 Mar shall. A .I I.E .: A n o utl ine of the cytology a nd pathology of the reticular tissue . pp . 25H-260 ; O liver a nd Boyd . London. 1956 16 McCull ou gh. B .: Krak owk a . S .: Koestn e r. A .: Sha dd uck . J .: Demyelinating acti vity of canine distemper virus isolates in gnotobiotic dogs. J Infect Dis 130 :343- 350 . 19 74 17 Seamer , J .: Ra nd les . W .J .: Fitzgeorge . R. : The course of Se mliki forest virus infection in mice . Br J Ex p Pat hol 4ll: 395-40 2 . 196 7 I H Seam er . J .H .; Boult er . E .A .; Z lot ni k . I.: Delayed on set of encephalitis in mice passively imm unised against Se mliki forest virus. Br J Ex p Pat hoI 52 :40H-41 4 . 1971 19 Sigu rd sson . B .: I'a lsson. I' .A .; G rimso n , H .: Visna, a dem yelinating transmissahle d isease of sheep . J Ne uropat ho l E xp Ne urn l 16: 31'9 -403 . 195 7
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20 Sigurdsso n , B ,; T ho rma r, H ,; Palsso n , P,A. : Cultivatio n of visna virus in tissu e culture , A rch Ges Virusforsc h 10: 36/;- 3 /;1, 1960 2 1 We il, A .; Davenpo rt , H .A .: Sta ining of oligod endroglia a nd microglia in celloidin sectio ns. A rch Ncurol Psychiat (Lo ndo n) 30 : 175- 17/; , 1933 22 Wei ner , P.L. : Pathogen esis of dem yel inati on indu ced hy mou se hepatitis vir us. Ar ch Ne uro l 28: 29 /;- 303 , 1973 23 Wisn iewski, H .M .; Ra ine , C.S. ; Kay , W.J .: Ob serv ati on s on viral dem yelinatin g encephalomyelitis: canine distem per . Lab Invest 26: 5/;9- 599 . 1972 24 Wisn ie wsk i, H .M .; Bloo m , B.R .: Primary demy elination as a non-specific co nse q uence of a cellmed iated immun e reactio n . J Ex p Mcd 141 :346- 359 , 1975 25 Z lotnik, I. ; G rant, 0 .1'. ; Balte r- Halton , D .: Encephalo pathy in mice followi ng inap par ent Semliki fore st infection . Br J Exp Pathoi 53 : 125- 129 , 1972 26 Z lotnik , I.: T he reactio n of astro cytes to acute viru s infection s of the cent ral nervou s system . Br J Ex p Pat ho i 53 :555- 564 , 196 /;
Req uest re prin ts fro m Dr . May G. B. Lim , SI. Th omas' Hospital Medical Sch oo l. London , SE I 7E H (E ngland) .
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D emyelination in Mice After Two or Three Infections with A virulent Semliki Forest Virus MAY C H EW - L I M .
A . J.
S U CK LIN G
a nd H . E .
W EBB
St . Th om as' Hospi tal Med ica l Sch o ol , Londo n . E ng la nd
Abstract. A d ult m ice give n two or thre e int ra pe rito ne a l inocu lat io ns wit h av iru le nt Se mliki Fo res t Virus sho we d typi ca l lesion s of a vira l e ncep ha lit is s imila r to th o se ca used by a virule nt stra in o f the virus. D em yelinati on a lso wa s se e n in th e medull a a nd in th e fo lia o f th e ce re be lla r white matter. Ne uro no p hag ia was see n o nly in mice th a t had had thre e succes sive infe cti on s . The repe at e d inocul ation s o f aviru lent viru s ex ace rba te s t he e nce p ha litis of a sing le ino culation a nd ca uses de my e linat io n . T he mic e did not have neu ro lo gical clini ca l signs exc e pt fo r a s ho rt-live d weakn ess of th e hind legs . No ce ntra l ne rvou s sys te m lesion s we re see n by th e 7th a nd St h wee k a fte r t he initi al infec tio n a nd a ll m ice recovere d .
Av irule nt Se m liki Fo res t Virus give n intrapcrit on ca lly ca n induce ac ute e nce phalitis in adult mic e which reso lves by 6 we e ks wit ho ut nei rol o gica l signs [8]. O t her research ers ha ve de scribe d e nce pha lo pa t hy in nin e mi ce 26 mo nth s afte r intrace rebral inoc ula tio n with th e avirulen t virus [25] . Thi s paper describ es experim ents which showed the effec t o f a seco nd int rape r iton e al inf ectio n wit h t he sa me vir us eith er 7 or 14 da ys a fte r th e first in oc ulation . M ice in a th ird expe rime nt were inoculated at 7 and 14 d ay s . Th ese e xpe rime nts wer e to asse ss histol ogica lly if repeated inf ections wo uld e vo ke a cent ral nervo us syste m react io n d ifferent fro m th at o f a single in ocu lat io n wit h t he vir us .
Materials and Met hods T he av irule nt stra in o f Se m liki Fo re st Viru s used had undergon e sev e n intrace re bra l pa ssages in 2- 5 da y o ld mice . The stra in wa s a clo na l se lec t io n fro m plaques propaga te d in mon olayer s o f prim ar y chicke n e m b ryo ce lls 141. Spec ific- pa thogen -fr ee Swiss A , G mice. 3-4 we ek s o ld. we re g ive n 0 . 1 ml o f 10- " di lutio n of t he virus in a sus pe ns io n o f ste rile bo vine a lb um in p ho sphat e sa line . pH 7 .0. In ex pe rime nt I . 63 mic e wer e di vid ed into th re e gro ups a nd we re give n a n intraperit on eal inocul ati on o f 0 . 1 ml o f viru s each . G roup I wa s infe cte d aga in a fte r 7 da ys . Grou p 2 was infec te d a seco nd tim e a fte r 14 day s . and gro up 3 mic e we re infected da ys 7 and 14 aft er t he first infectio n . Four co ntro l mice eac h wer e give n a n inj ectio n o f bov ine a lb um in phosp hate sa line . Mic e were kille d by a na es t he tic e the r a t wee kly int er va ls from th e tim e o f th e la st inf ect ion . The e xpe rime nt la ste d 7 wee ks . In expe rime nt 2 . 48 m ice were d ivid ed int o thre e gro u ps . tr eat ed as a bo ve a nd o bse rved fo r 8 we ek s . T he re we re no co ntro l mi ce . In e xpe rime nt 3 . 96 mice were di vid e d in to thre e gro ups a nd tr eated a s above . T he experim e nt lasted 9 wee ks . Tw e nty co ntrols e ac h wer e give n o ne intra pe rito nea l dose o f bovi ne a lb umin pho sph at e sa line a nd a no t he r 24 mice ea ch we re give n o nly o ne ino cula t io n o f virus intra pe rit o ne all y . 67
Downloaded from vet.sagepub.com at NORTH CAROLINA STATE UNIV on March 6, 2015
68
C he w-Lim . S uckling a nd Webb
1 Fi~.
1: Sec tion of hipp oca mpu s with as trocy te hyp ert roph y . Caja l's go ld sub limate .
Brains we re fixed in 5 % form alin fo r a t least 7 days . In e xperiments I and 2 b ra ins we re sec tio ne d in th e front al coro na l plan e at two places . The first cut was in front of the hippoca mp us a nd t he seco nd was a t the junctio n of th e midbrain with t he brain ste m . In e xpe rime nt :I eve ry o the r br a in sa m pled was secti on ed sag itta lly. Fro ze n IO-JLm-thick sec tio ns o f hippocampus wer e sta ine d with Ca ja l's mod ified go ld sublima te to show as trocyte s 126]. Brain s we re e mb ed de d in par affin a nd 5- 6- JL m sec tions we re sta ined with haem at oxylin a nd e os in (HE) . T he modi fied We il D a ve n po rt method 11 5. 2 1] was used fo r d em o nstrat ion of microglia a nd o ligoden drocyt es . De mye lina tio n was see n with luxol fas t blu e cou nterstai ne d with cresyl-fas t vio let II I ]. Mall or y's p hospholll ngs tic-ac id- hae matoxy lin sta in was used to show a b no rma l as trocy te s in dem yelin at e d a reas o f pa raffi n sections .
Re sults Perivascu lar cuffs of a viral e nc ep ha litis were see n in sectio ns fro m mi ce kill ed up to 6 we ek s after t he last infection . Maxim u m cuf fing reac tio ns were see n 2 week s afte r th e last inf ection wit h the virus . T he perivasc ular cuff consisted m a inly o f lym p ho cyte s with rare pl asm a cell s . T here wa s a lso in filtra tio n o f t he men inges a nd th e white matter with lympho cyt e s . E ight weeks a fte r ex pe r ime nts 2 a nd 3 were sta rte d th ere wa s minim al peri vascular cuffing in brain s of those mi ce th at had had thre e virus infe cti on s . As trocy te hypert roph y wa s mo st noticeab le in m ice given thr e e inocu la tio ns of virus (f ig . I) . As tro cy te hype rtro ph y persiste d in co nj unctio n wit h the pe rivascu lar cuffing . A stro cyte s a lso were see n within th e foc i of myel in lo ss. A ll the m ice rec eivi ng mo re t han o ne ino cul at ion of virus d e vel o ped d e myeli na ting le sio ns. Dem yel inati on was mo st pro m inent in the me dull a ry su bs ta nce a nd
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D cm yclinat ion in Micc Ta ble I. Sum ma ry of ma in histo logi ca l find ing s Hi st o lo gy o f br ain Icsi o ns
Perivascular cuff ing A st rocyt e hy pert rop hy D em ye lin ati o n o f cere be lla r w hite
Mi cc give n 2 vir us in o culation s m ax imum
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foci o f dcmyc lin a t ion pre sen t
foc i o f d cm yclin ati on more m ark ed hyp ert rophy m ark ed se e n in cere be llum a nd medulla
matt er
Mi cro gli a l rea cti on Nc uro nop hagic nod ule s
Micc give n 3 virus inocul ation s
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folia of th e cerebe llu m 1- 3 wee ks after the la st infection (fig . 2 ) . No e vide nce o f d em ye lination wa s see n in mice at we ek s 7 a nd 8 . In th e ce n tre o f th e foci o f d e mye lin at ion in th e cerebe llum th ere we re few o ligod e ndrocyte s . T hey we re more no tice a ble ar ound t he periph ery . H yp ert rop hied fo rm s of mi crogl ia co uld be see n near b lood vesse ls , in th e mol e cul ar layer of th e ce re be llu m a nd in th e a rea of dem yelin ation . A t 2 a nd 3 week s after the last inocu latio n ne u ron o phag ic nodul es we re see n in th e ce re be llar me dull a of mi ce give n th ree doses of the virus (fig . 3). Ce lls a ro und t he d egen erat ed neuron s were ma inl y m icro g lia . Brain titrati on s 7 d ays afte r th e last infecti on showe d no ev ide nce of virus . T he m ain hist ol ogical findi ngs a re s um m a rize d in tabl e I .
Discussion In a ll gro u ps t here were typi ca l fe ature s of a vira l e nce p ha litis . T he thi ck infi ltra te o f lym ph o cyt e s in th e m eninges and neuronophagi a was sim ilar to th e brain lesio ns d escr ibed for a n a d ult mo use 4 d ays aft er being give n 0 .0 3 ml ino cu la co n ta in ing IOu ; plaq ue forming un its of th e vir ule n t stra in of Se m liki Fo res t Virus int o th e foot pad [ 17 ). The excessive ly hyp ert rophi ed as trocy tes we saw 1- 3 wee ks after t he last infectio n we re no t d issi mi lar to t hose se en by o ne re searc her 72 h a fte r intraperito nea l infe ctio n of m ice wit h 0 .2 m l viru le n t vir us o f titres ran gin g fr om 10- 1 to 10- 7 [26). Two o r three ino cu lati on s of av ir ule n t viru s se e m to exace rb ate the b rain re acti on , a nd histol og icall y th e b ra in le sion s a re sim ila r to th o se of viru len t Se m liki Fo re st V ir us . M ice give n av iru lent vir us s urvive d th e e xpe rime nt , wh ere as 90 % o f mice g ive n intra pe rito ne al infe cti on s with 0. 0 3 m l o f gra de d d ose va ria tio ns fro m 30 0 to 1000 plaque fo rming un its of th e vir ule n t virus di ed afte r 8 days [5]. In o ur ex pe rime nts we sa w demye lina tio n , whic h has not be e n reco rd ed before wit h e ithe r t he av irule n t o r viru le n t st rai ns of Se m lik i Fo res t V ir us . M e ylin sheat hs a re m ultila me llar membran es e nclosi ng axon s . In th e ce n tra l ne rvous sys te m th e y a re ex te nsio ns o f t he plasma membran es o f th e o ligo de ndrocyt es ; in the perip he ra l nervou s syste m th ey a re e xte nsio ns of th e Sc hwa rm ce lls . O t he r ex pe rime n ts ha ve sho wn tha t myel in b re akdo wn in vivo ca n be mech ani ca l [6 ]. tox ic [1 3], a lle rgic [ 12] o r vira l [23 ). Both ac ute a nd chro nic d em yelin a ting disea ses in man a nd a nima ls ca n be ca used by vira l infect ion s .
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C hew-Lim , Suc kling a nd Web b
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Fig. 2: Sec tion o f cere be lla r fo lia wit h foc us of dem yelin ati on . Luxo l-fas t blue a nd cre syl-fas t vio let . Fig. 3 : Sec tion of cere be llar medu llar y substa nce wit h neu ron oph agie nodu le . H E .
The best known a n ima l demyelin at ing di se ase s ca used by vir uses a rc vis na in Ice la nd ic shee p [19 , 20), ca n ine di st em per [16 ] a nd neu ro t ropi c mouse he pat itis [1 ,7 ,1 0 ,1 4 , 22] . With av ir ule nt Se m liki Fo res t V ir us , demyelin at ion was not induce d by a sing le infecti on with a neurot ropic virus de spite an acute e nce phal itis . In all grou ps o f mice give n two or th re e infe cti on s , howev er , dem yelin ati on was consta nt. Repeat ed infections o f virus may d am age th e o ligo de ndrocy te by becom ing sit es fo r virus replica tion . Ma t ure virio ns of Ve nezue la e q ui ne e nce pha lo mye lit is have been seen in th e o ligo de nd rocytes of infect ed ad ult mi ce [9] . Th e me ch ani sm of dem yelinati on in mou se hepa tit is vir us e nce p ha lo mye litis is the a ffin ity of th e vir us for th e o ligo de ndroey tes of th e ad ult mo use, a nd dem yel inati on occurs subse q ue nt ly to th e degenerati on of th e infe ct ed o ligo de nd ro cytes [14] . A no the r ex pla na tio n is th a t dem yelin ati on is an immu nopath ol o gic p hen o m en o n resulting fro m repeate d rath e r th an sing le virus in fecti on . Some, ho weve r , report th at , ch a ngi ng t hc immu nol ogical st at us of th e se m ice by giving intra peritonea l vira l specific a nt ise ru m did not sho w dem ye lin a tion , eve n th ou gh th e y a ltere d th e timin g of th e e ncep ha litic pro cess [18 J. O thers used the tubercl e bacill i and its so lub le pr oducts as a n a ntige n to sho w th at da mage to m yel in in th e periph er al nerves of guinea pigs was a nonspecifi c co nse q ue nce of a spec ific cell -med ia ted immune reactio n in t he vicin ity of a my e lin at e d ne rve [24] . In th ese ex peri me nts th e tw o or three inoc ulat io ns o f t he av ir ulent virus co uld hav e pro du ced de my e lina tion by a sim ila r reactio n, but t he a reas of myel in loss di d not part icul arly surro und cuffe d vesse ls. T he di sap pea rance o f dem ye lin at e d foc i ca n be a ttr ibu ted to the rem yelin atin g ability of th e m ou se bra in [2 , 3 ] . Reso lutio n o f dem ye linat e d foci doe s no t nec essa rily me an demyelin ati on will not re cu r lat e r. In a n ult rastru ctural study re se arch ers sho we d th at demye lin ati on recurred in mic e 16 m o nth s af ter intr a cerebral infecti on with t he mouse hep atiti s vir us [10] .
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Although lesions were noticeable microscopicall y in th e central nervou s system no ne of the mice showed neu rol o gical signs except for transient wea k ness of the hind leg s. This cou ld be re lated to th e dem yelination . More infections cou ld ca use e xte ns ive loss o f myelin and ne ur o na l dege ne ra tio n with perman ent cli nical signs . This process o f dem ye lina tio n from repeat ed avirulent Se m liki Fo re st Virus infect ion ma y be usefu l for st ud ying dem yelinating di se ase s of the central nervou s syste m by using a viru s that does not produce clinical signs of an acute e nce pha litis .
Acknowledgements We thank Dr. R. O . Barnard for ad vice and Dr. Sue O a te n fo r the mou se brain virus titres . Supp ort ed by g ra nts from the Ph ilip Fleming T rust a nd Multiple Scle ro sis Soc ie ty.
References Bail ey. 0.'1'.: Pappenheim er . A .M . : C hee ve r. F.S .: Da niels . J.B .: A murine viru s (JHM) ca using disseminated encephalomyelitis with extensive de struction of myelin. II. Pathology . J Exp Mcd 90 : 195- 21 2 . 1949 2 Blak em o re . W.F .: Observations of oligodendrocyte degeneration , the resolution of status spongiosus and remyelination in cup rizone intoxication in mice. J Ne urocyto l 1:41 3- 42 6 . 19 72 3 Blak em or e . W. F. : Remyelination of the superior cerebellar peduncle in the mouse following demyelination induced by feeding cup rlzune , J Neuro l Sci 20: 73- H3. 19 73 4 Bradi sh . C .J .; A llne r, K.; Mab er. H .E .: The virulence of original and derived st ra ins of Semliki forest viru s for mice , guinea pigs and rabb its. J Ge n Viro l 12: 141 -1 60 . 19 71 5 Bradis h. C .J .; A llne r, K .: T he early responses of mice to respiratory and intraperitoneal infection by defined virulent a nd av iru le nt st ra ins of Semliki fore st viru s. J Gc n Virol 15: 205 -2 1X. 1972 6 Bun ge . R .P .; Bu nge . M .B .; Ris , H .: Electron microscopic demyelination in an experimentally induced lesion in adult cat spi nal cord. J Bio phys Bioeh em Cyto l 7: 6 H5- 696 . 196 0 7 C hee ve r. F .S .: Da nie ls. J .B .; I'appe nh e imer . A .M .: Ba iley . 0. '1'.: A murine virus (JHM) causing disseminated encep ha lomyelitis witb extensive destruction of myelin. I. Isolation a nd bio logical propert ies of t he virus. J Ex p Med 90: I H1-1 94 . 1949 H C he w-Lim , May: Mouse encephalitis induced by avirulent Se mliki forest viru s . Vet Pat ho i 12:31'739 3 . 1975 9 Gore lkin . L.: Venezuela n equine encephalomyelitis in an adult a nima l ho st . A m J Path ol 73 :4 25442 . 19 73 10 Hern don , R .M .: Gr iffin . D .E .; McCormi ck , U.; We iner. L.I' .: Mouse hepatitis virus -induced recurrent demyelination . A rch Ne uro l 32 :3 2- 35 . 19 75 II Klu ve r. H .; Barrer a . E .: A method for the combined staining of cells and fibres in the nervous system . J Ne uro pa t ho l Ex p Nc uro l 12:400- 403 . 1953 12 Lampert. 1'. W .: Electron microscopic studies on ordinary and hyperacute experimental allergic encephalomyeliti s. Ac ta Neu rop ath ol 9: 99-1 26 . 196 7 13 Lampert. I' .W .; Schoc hc t , S .S .: Electron microscopic ob servations on experimental spo ngy degeneration of the cere be lla r white ma tt e r . J Ne uropa t hol Exp Ncurol 27: 210-220, 196 H 14 La mpe rt . P.W.; Sims . J .K .; Kn iazcff. A .J .: Mechanism of demyelination in JHM virus encephalitis. Acta Ne uro pa tho l 24 :76- 1'5 . 1973 15 Mar shall. A .I I.E .: A n o utl ine of the cytology a nd pathology of the reticular tissue . pp . 25H-260 ; O liver a nd Boyd . London. 1956 16 McCull ou gh. B .: Krak owk a . S .: Koestn e r. A .: Sha dd uck . J .: Demyelinating acti vity of canine distemper virus isolates in gnotobiotic dogs. J Infect Dis 130 :343- 350 . 19 74 17 Seamer , J .: Ra nd les . W .J .: Fitzgeorge . R. : The course of Se mliki forest virus infection in mice . Br J Ex p Pat hol 4ll: 395-40 2 . 196 7 I H Seam er . J .H .; Boult er . E .A .; Z lot ni k . I.: Delayed on set of encephalitis in mice passively imm unised against Se mliki forest virus. Br J Ex p Pat hoI 52 :40H-41 4 . 1971 19 Sigu rd sson . B .: I'a lsson. I' .A .; G rimso n , H .: Visna, a dem yelinating transmissahle d isease of sheep . J Ne uropat ho l E xp Ne urn l 16: 31'9 -403 . 195 7
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20 Sigurdsso n , B ,; T ho rma r, H ,; Palsso n , P,A. : Cultivatio n of visna virus in tissu e culture , A rch Ges Virusforsc h 10: 36/;- 3 /;1, 1960 2 1 We il, A .; Davenpo rt , H .A .: Sta ining of oligod endroglia a nd microglia in celloidin sectio ns. A rch Ncurol Psychiat (Lo ndo n) 30 : 175- 17/; , 1933 22 Wei ner , P.L. : Pathogen esis of dem yel inati on indu ced hy mou se hepatitis vir us. Ar ch Ne uro l 28: 29 /;- 303 , 1973 23 Wisn iewski, H .M .; Ra ine , C.S. ; Kay , W.J .: Ob serv ati on s on viral dem yelinatin g encephalomyelitis: canine distem per . Lab Invest 26: 5/;9- 599 . 1972 24 Wisn ie wsk i, H .M .; Bloo m , B.R .: Primary demy elination as a non-specific co nse q uence of a cellmed iated immun e reactio n . J Ex p Mcd 141 :346- 359 , 1975 25 Z lotnik, I. ; G rant, 0 .1'. ; Balte r- Halton , D .: Encephalo pathy in mice followi ng inap par ent Semliki fore st infection . Br J Exp Pathoi 53 : 125- 129 , 1972 26 Z lotnik , I.: T he reactio n of astro cytes to acute viru s infection s of the cent ral nervou s system . Br J Ex p Pat ho i 53 :555- 564 , 196 /;
Req uest re prin ts fro m Dr . May G. B. Lim , SI. Th omas' Hospital Medical Sch oo l. London , SE I 7E H (E ngland) .
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