key: cord-272981-8gahvdt0
authors: Wege, Helmut; Watanabe, Rihito; ter Meulen, Volker
title: Relapsing subacute demyelinating encephalomyelitis in rats during the course of coronavirus JHM infection
date: 1984-08-31
journal: Journal of Neuroimmunology
DOI: 10.1016/0165-5728(84)90022-5
sha: 
doc_id: 272981
cord_uid: 8gahvdt0

Abstract Temperature-sensitive mutants of the murine coronavirus JHM induced a subacute demyelinating encephalomyelitis (SDE) in young rats. Neurological symptoms were associated with marked lesions of primary demyelination in the white matter of the central nervous system (CNS), and developing after an incubation time of several weeks to months. Many rats survived this infection and recovered completely from this CNS disease. Among 43 survivors of SDE, 9 rats developed a relapse 27–153 days after onset of the first attack. Neuropathological examination of these animals revealed areas of fresh demyelination together with old remyelinated lesions. Viral antigens were detectable in the neighbourhood of fresh lesions and in some cases infectious virus was re-isolated from rats revealing low antibody titers to JHM virus. These results demonstrate that mutants of JHM virus can induce a relapsing demyelinating disease process, associated with a persistent infection, which possesses some similarities to chronic experimental allergic encephalomyelitis.

pathogenesis are unknown. The most prominent example is multiple sclerosis (MS) which is thought to be associated with an immune pathological process p~sibly acting in concert with a virus infection (Waksman 1981) . It has been shown that an autoimmune reaction against CNS tissue can lead to 6hronic relapsing demyelination as seen in chronic experimental allergic encephalomyelitis (EAE) whereas the role of a virus infection in the induction of such a condition has not so far been directly demonstrated (McFarlin et al. 1974; Paine et al. 1978; Lassmann and Wisniewski 1979; Traugott et al. 1982) . It is there.fore desirable to develop animal models of relapsing demyelination associated with a virus infection in order to analyse the mechanisms by which virus-induced neuropathological changes may Occur. Viruses can induce demyelination which is interpreted as either the consequence of cell death in the course of viral replication, or the result of dysfunction in persistendy infected oligodendrogiia cell*, lmmunopathologieal reactions could also play a role in virus-induced demyelination as studies with Theiler's virus infection in mice suggest. Relapsing demyelination associated with a clinical diseac, e as seen in chronic EAE has been postulated in virai infections of the CNS, especially in man, but not studied experimentally. In the following we describe a model in which certain mutants of muri1~e coronavirus JHM cause clinical relapsis of a subacute demyelinating encephalomyelitis (SDE) in rats. After virus infection in rats inflammatory disseminating CNS lesions of marked demyelination develop accompanied by clinical signs of a subacute disease after varying incubation times. A considerable percentage of diseased animals survive and recover from this infection and some come down with a second attack of SDE after a period of weeks to months. Such animals show both, fresh demyelinating lesions with infiltrations, and remyelina~.ed areas in the brain and spinal cord. Infectious JHM viru:, can be reisolated from brain tissue and virus antigens demonstrated in brain cells. These findings demonstrate that a clinically recognizable relapsing CN5 disease can develop in association with exacerbations of demyelination in the. course of a persistent viral infection of brain tissue.

Outbred specific pathogen-free rats (CHBB/Thom) were purchased from "rhomae (Biberach, F.R.G.'. Lewis rats were obtained from the Zentralinstitut ftir Versuchstiere (Hannover, F.R.G.). 10-15 day old rats received 4 x 103 PFU of JHM virus in 30 t~l tissue culture medium into the left brain hemisphere using a dispmser syringe.

The temperature-sen:;ifive (ts) mutants ts6, ts42 and is43, which were selected from the routine coronavirus JHM after growth in present, of fluoroura~.il have been previously described (Wege et ai. 1983) . The virusct; were propagated on monolayers of Sac(-) cells at 340C with Eagle's minimal essential medium (1*.~EM) and 5% fetal calf serum.

Samples of brain and spinal cord were homogenized immediately a:fter dL~,sec',ion and adsorbed for 1 h at 34°C on monolayers of Sac(.) cells in 24 wel~ cluster plates (Wege et al. 1983 ). The number of wells in which syncytia developed was scored after 3 days incubation at 34°C. The supernatar~ts from positive wells were harvested, pooled ~md titrated at 34°C as well as 39.5°C to measure the temperature sensitivity. Cell~ from reisolation attempts wltich yielded only very few or sl~dl syncytia were trypsinised, mixed with normal Sac(-) cells and passaged further.

To measure neutralizing antibodies, the s(:ra were inactivated for 30 rain at 56°C. mixed in serial dilutions with 100 pfu of wild type JHM virus (f'mal volume 200 ~1) and incubated :~or 1 h at 4°C. Neutralization was assayed using Sac(-) cells in 24-well microplates. The cultures were incubated for 20 h at 37°C without agar overtay. Microplaques were counted after staining with May-Grtinwald and Giemsa, the serum dilution resulting in 50~ plaque reduction (NDso/0.1 ml) was thus calculated.

Total 51L~4-virus ~_,'.:ibodies were titrated using a solid phase eI~yme immunoassay (ELISA), performed according to standard procedures ~fVoller et al. 1976). Antigen was prepared from infected or control Sac(-) cells, wl'fich were disrupted by dounce homogenisation in RSB (Tris-HCl 20 mM, pH 7.4; sodium chloride 100 raM, magnesium chloride 5 mM) and 0.2~ Nonidet P40. The homogenate wes centrifuged 5 min at 10000 × g and the antigens were pelleted by t~Ltracentrifugation at 100000 × g for 90 min. Antigens and indicator immunoglobulins (horseradish peroxidase-lab¢lled anti-rat globulin, Dako) were c~dibrated by bh3ck titrations for reproducible sensitivity. The positive anti-JHM virus serum was prelmred by immunisation of rats with density gradient purified virus (Wege et ~d. 1979 ) and h~ a neutralisation titre 1 : 1250. The antigen block titrations were c~dibrated with a serum dilution of 1:409 to give a netto absorbance value (we mean of virus antigen-coated wells minus mean of control antigen-coated wells) of 1.0. Flat bottom microplates (Dynatech) were coated with antigen and control antigen in 0.05 M sodium carbonate buffet, pH 9.6 overnight at 4°C (100/~l/well), and unbound proteins removed by washing with Tris buffer (0.05 M, pH 7.4) containing 0.15 M sodium chloride and 0.1~ Tween 20. Serum dilutions were made in the same buffe~ containing 5~ newborn calf serum and 0.002~ phenol red. After incubation for I h at 37°C the plates were washed again, then incubated with peroxidase-laladled rabbit anti-rat globulin and after a final wash orthophenylene diamine (0.5 ngg/ml) in citric acid (35 mM, dinatrium hydrogen-phosphate 66.6 mM, bydrogene peroxide 0.01~, pH 5) was added as substrat¢. The reaction was stopped after I h at reran temperature by addition of 50 btl sulfuric acid (3 M) and the colour measured with a multichamiel spectrophotometer (Titertek, Flow Labs.) at 492 rim. The endpoLnt of the titration (ELISA fitre) was defined as that serum dilution which yielded a nett,~ absorbance value more than 3-fold higher than the negative control sera at a dilution of 1 : 10. The positive and negative serum pool which was used for the calibration of the assay by block titrations was included in each experiment.

Tissue was fixed ia buffered 10~ formalin and embedded in paraffin for histological examinati,~n. Sections were stained with hematoxylin-eosin and luxol fast blue. For upon embedding, animals were perfused wi~h 2% g)utaraldehyde and 2% paraformaldehyde, 0.5% sucrose in 0.1 M phosphate buffer solution. The tissue was postfixed by osmi~Jm, dehydrated, embedded in upon and 1/zm sections were stain~ with toluidine flue for light microscopy. Ultrathin sections were stained with uranylacetate and lead citrate for electron microscopy.

Appropriate tissue blocks were fixed in buffered 2~ paraformaidehyd¢ for 4 h to overnight, washed by graded sucrose, finally by 10~ glycerol and 20~ sucrose in 0.1 M phosphate buffer at the ~.emperature of 4°C, embedded in OCT, frozen by liquid nitrogen and kept at --70°C until 10-#m sections were prcparecl by cryostat. The PAP method (Sternberger et aL 1979) was applied by using rabbit anti-JHM .serum (having a neutralization titre of 1 : 1860) at a dilution of 1 : 1000. The rabbit had been immunised with density gradient purified JHM virus (~Vege et al. 1979 ) and the serum was absorbed with acetone-extracted rat brain powder. For control staining, normal preimmune rabbit serum and an anti-measles virus rabbit serum was used. As a second antibody anti-rabbit IgG (Dako Z147) and PAP complex (Dako Z113) were inc!uded. Counter staining was achieved with methyl green.

Intracerebral inoculation of 10-15 days-old Thomae or Lewis rats with temper~. ture-sensitive (ts) mutants was followed by the developn~cnt of SDE after an incubation period of several weeks (Wege et al. 1983 ). This disease started quite often with a mild to moderate hindleg weakness with ataxic gait which causes the rat considerable difficulties in righting itself up when flipped onto its back. At v~ariable times up to several days these signs were followed by severe hindleg weakness leading to hindleg paraly:~is. At such a stage no voluntary movement of the hindlimbs could be observed although some reflex stiffening occurred. Approximately 60-70% of the disea.,:ed animals died within a few days after onset of disease, whereas the surviving animals recovered completel:/, or with a stabilized disability.

The most pron~inent histologic~ll finding consisted of demyelinating plaques located in the white matter ( Fig. 1) primarily of the oplic nerve, midbrain, ports, cerebellum and spinal cord. Within the demyelinated plaque, axons and neurons were well preserved. In ~,ddition, cell infiltrations were observed consisting; of iymphocytes, plasma cells and macropkages. Infectious virus could be isolate~ by conventional methods, and viral antigens were easily detectable in glia cells in the neighbourhood of demyehnatiag plaques (Nagashima et al. ; SOrensen et al. 1980 SOrensen et al. , 1982 Wege et al. 1983 ).

Fi 8,1, Large demyelinating plaque located in the white matter of the spinal c~rd in a Lewis rat with SDE. Hematoxylin-eosin and luxol fast blue staining, x 28.

Of particular interest is the observation that some of the ar.imals whic~ survived and recovered from SDE revealed later a second attack of this disease. Of 276 rats infected with different ts-mutants c f JHM virus 57% developed clin~ical signs of SDE within an incubation pe~od of 14-.158 days. 36% of the diseased an~-'lals survived this infection. Among the 56 survivors of SDE, 13 rats continuously exhibited clinical signs of hindleg paresis or paralysis. The remaining ,*3 animals were apparently healthy. 1~ ut 9 of these animals developed z~ second attack of SDE 2-;8 weeks after recovery from the first attack. As documented in Table 1 these animals had developed their first attack of SDE after an incubation period of 14-33 da2/s. Clinical symptoms of this i ~ ~ ' • first attack lasted from ~ to 25 days until they recovered. In all cases recc,veD' was complete and no disability w~.s noted. These animals came down with a second attack 41-173 days p.i., however, wi~h an averetge incubation time between onset of the first attack of disease and its exacerbation, of 61 days. The clinical picture of the secor, d attack was similar to the first~ except the course seemed to be more severe.

Since these animals were needed for experimental analysis it is not known whe~.her they would have recovered again. The histopathological investigation of relapsing SDE in these rats revealed the following changes as documented in ]rig. 2. All rats studied showed fresh demyelinating lesions with infiltrations of mononuclear cells primarily ic~:ated in spinal cord and brain stem. In the same animals, old lesions were also 10und in pons, thalamus, cerebellum or spinal cord. These old lesions in the whit,; raatter revealed extended remyelination of the CNS and/or PNS type. In some cases proliferation of astroglia was observed. A very pronounced fresh, demyelinated lesion in ;lt: spinal cord at the same level as a remyelinated area is shown in Fig. 2a . Thi,; rat developed a relapse of SDE 138 days p.i. (No. 8 in Table 1 ). A higher magnification of a thin section from the demyelinated plaque clearly reveals naked ~txons in the presence of macrophages (Fig. 2c ). An extended area of remyelination by Schwann cells (PNS type) was noted at the same level of this spinal cord. Higher mal~;nification and electron microscopy clearly demonstrated the association of Schwann calls with axons and the basal membrane (Figs. 2b,e,f,g). Even in the remyelinated areas infiltrating macrophages were detectable. Furthermore, remyelinated areas of the CNS type were found at different leJels of this spinal cord (Fig. ld) . In addition, remyelinated areas of the PNS type and macrophage infiltrations with myelin debris were encountered in the pons.

Attempts to re-isolate JHM virus from these ammals yielded different results (Table 2 ). Despite the fact that all animals tested revealed viral antigens ~n gila cells Fig. 2 . Relapse of SDE 138 days p.;. following inoculation of ts43. The Lewis. rat was infected at an age of 10 days. a: Large demyelinated plaque in the anterior and lateral area of white matter (;~'row heads). "[he anterior white matter is unusually swollen by edema which leads to deformation oF the grey matter. However, in this area neurons are well preserved. In the posterior column (surrounded by arrows) PNS type remyelination was observed. Hematoxylin-eosin and luxol fast blue staining of paraffin-embed~k,d section. The rat was perfused with glutaraldehyde-paraformaldehyde solution. × 64. b: Same level as a, higher magnification from remyelinated area. Numerous nu,cle:i of Schwaxm oils are detectable indicating a PNS type remyelination. Note infiltration by macroph~ges (arrow heads) even in this area. x420. c: Sa nc level of spinal cord with demyelinating plaque as a, l-p.m section after embedding in epon. Naked axons (arrow heads) and infiltration by macropha-es (arrows) with myelin debris indi*:ate fr~,h demyelinafion. Stained with toluidine blue. x 625. d: Thoracic spinal cord, upper level. Remyelination of the CNS type is indica,led by thinly myelinated ~xoas (arrow heads), l-pm section after embedding in epoe., stained with toluidi~e blue. ×550. e: 1-p m section after embedding in epon from same level with remyelination of PN S type as a am~. b. Nu, lei of Schwann cells associated with axons. Macrophage infiltration (arrow heads), x 324.

• md g: Electron microscopy of remyelinated area (,.ame level as a and b). Schwann cells in asso.lation with axons arc surrounded by a basal memarane. × 4400 and × 26 0(.~:. in the neighbourhood of demyelinating plaques (Fig. 3) , onty fro:~ 2 animals (animal Nos. 6 and 7) was infectious virus obtained after homogenization of spinal cord and brain specimens. Only non-infectious virus was recovel,ed from the other animals. Sac(-) cells which were used for those isolation attempts, develotw.d cy~p~thic effects consisting of giant ~ell formation, a typical CPE of corom,v;rus irffection, in which v~Tal antigens were present. No infectious virus was detectable in these cultm'es, however, either in the supernatant or in a cell-associated form. A non-~elding persistent infection was apparently established in these tissue cu!t*~,re cells. With regard to these findings, the immune response of these animals against'. JHM virus is of interest. All animals tested except ?, revealed high antibody titers against JHM virus detectable hy ELISA and neutraliTJng assay. Only serum samples from rat Nos. 6 and 7 showed a low reaction in the ELISA and no detectal,01,: neutralizing antibodies. Yet, i~ fectious virus was isolated only from these 2 animals which lacked neutralizing antibodies. This suggests that the failure to recover infectious virus from the other rats might ,be related to the pr,sence of neutralizing, antibodies which inactivate free infectious virus.

Diseussio.

Intracerebral inoculation of 10-15-days-old Thomae or Lewis rats with certain neurotropic ts.m~ttants of murine coronavirus JHM leads to the d,velopment of a suba~te demyel~rating enc, phalomyelitis which may or may not b, f~LtM./ufimals surviving this infection can recover completely and reveal no clinical signs of disability, but under conditions which are presently not understood a ~concl attack of this CNS disease can develop wlfich is very similar to the tirst or~e.

These observetions demon.,;trate that JHM-persistent infection in tat. CN:~ tissue can remit in demye.linating diseases, with remission and relaF;e of cli mica] symptoms, in connection with exacerbations of neuropaihological changes. Evidence for chronic, recunent demyelination by coronavirus JHM has been obtaine~l hi infections of mice I but no significant clinical changes were observed m this animal (Hemdon et a~. 1975; Haspel et al. 1978; Knobler et aL 1981 Knobler et aL , 1982 Stohlman.and Weiner 1981) .~ A biphasic dkease course with clinical symptoms and recurrent demyelination has been described in Theiler's virus infection of mice (Lipton and Dal Canto 1976; Dal Canto and Lipton 1979; Dal Canto 1982) . In thJ,s infection, young adult n~e develop a flaccid paralysis from which the majority of animals recover. Survi,dn 8 mice subsequently come down after weeks with a spastic, paralyric gait ditovder. In the early dim the grey matter is predominantly involved, resembling human poliomyelitis, whereas in ',he later disease primary demyelination it found relawd to perivascular mononuclear cell infiltrations. In this virus infection the early aqd late disease are neuropathologically quite differew,, whereas in JHM viru~induced recurrent demyelinafion the first and second diseas,~ are cliuically and netx-opathologically very similar. Some evklence has been pro'~ided that Theiler virus induces an immune-lmthologtcal reaction which could contribute to demyelinafion.

immunmuppreudon decreases or prevents de~2ielination, bm the exact pathogenetic raechanitm is unknown.

The induction of a progressive demyelinating, or relapsing demyelinating, disease process, in JHM virus-infected rats has ~me parallels to chronic EAE, an animal model based on sensitation ~8ainst CNS tissue extracts or myelin basic protein (McFarlin et al. 1974; Raine et al. 1978; Lassmanll and Wisniewski 1979; Traugott et al. 1982 . al. 1981; Vandevelde et al. 1982) . These data suggest that different virus infections of the CNS can be associated with an autoimmune response against brain tissue.

On the other hand, it is co~tceivable that the virus infection of CNS ~lls also contributes co the relapsing disease course. It has been shown in visna infection in sheep that the antivirai immune response is not able to control fl~ CN$ infection, In the presence of antibodies to visna virus, mutants continuously develop which cannot be neutralized by the ~ntibodies directed against the parenUD1 virus (N~rayan e~ al. 1978). The emergence, of each new mutant permits the infection of n~v ar~ts of the brain lead!',~g to inflammatory reactions and CNS damage. In coronavir~s infection of rats, JHM virus can be re-isolated after several months of persistence, So far, the antigenicity and biological properties of such re-isolated virus have not been compared to those of the virus used for inoculation. In relation to other i~,,rsistent infections of the CNS such as subacute selerosing panencephalitis, it is rem~kablc tha~ ir fectious JHM virus can be isolated: this suggests that antigenic change~ may occur during persistency.

The occurrence of chronic viral infection, the relapsing clinical course witil the presence of old and fresh dernyetinating plaques and the evidence of a cell.media£ed immune reaction to myelin basic protein during the fi~'st attack of SDE (Wal~nabe et al. 1983) , suggest that this dis~as~ process is a result of a very complex virus.host interaction, in which host factors play an important role. It is conceivable tha~ the autoimmune reaction against elyelin basic protein may also perpetuate the d~sease, even when the virus is no longer demonstrab!, in CNS tissue. The features ol thi~ coronavirus JHM-asscciated CNS disease in rats reveal tome dmilarities to multiple sclerosis in man and ~t is hoped fllat the analysis of the events which lead to thi~ animal disease will be of relevance in our understanding of other relapsin,~, de~ myelinating processes.

Uncoupl~ relationship between demyelination and primary infection of myelinating cells in Theiler's virus encephalomyelitis

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Adoptive trar~fer of EAE-like iedo~ by BM~P s~Jnmlar~ed lymphocytes from ran witL corcnavirus-induced demyelinafing encephalomyelitis

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We thank Margarete Sturm, Hanna Wege and Renate Abt for excellent technical assistance and Helga Kriesinger for typing the manuscript.