- Email: [email protected]
Virus-associated demyelination
Journal of the Neurological Sciences, 1978, 39: 147-154 © Elsevier/North-Holland Biomedical Press
147
VIRUS-ASSOCIATED D E M Y E L I N A T I O N A Model Using Avirulent Semliki Forest Virus Infection of Mice
ANTHONY J. SUCKLING, SNEHLATA PATHAK, SUSAN JAGELMAN and HUGH E. WEBB Department of Neurology, Neurovirology Unit, The Rayne b~stitute, St. Thomas' Hospital, London SE1 7EH (Great BritaihJ
(Received 8 May, 1978) (Accepted 26 June, 1978)
SUMMARY Lesions produced by the infection of Swiss/A2G mice with a single inoculation of an avirulent strain of Semliki forest virus have been studied by light and electron microscopy. Whilst a mild encephalitis was detected in the great majority of mice infected, focal areas of myelin loss were observed in the cerebellar white matter of only 25 ~ of cases. The incidence of myelin loss in other strains of mice ranged from 21 ~ in BSVS mice to 8 ~ in SJL/J mice. The possible mechanisms involved in the pathogenesis of the myelin loss are discussed.
INTRODUCTION Recent studies have described the pathological changes which followed infection of Swiss/A2G mice with an avirulent strain of Semliki forest virus (Chew-Lim 1975; Mackenzie, Suckling, Jagelman and Wilson 1978). In these studies, changes typical o f a mild encephalitis occurred consistently after inoculation of virus, together with focal microcystic lesions located mainly in the cerebellar white matter, mid-brain and lateral tracts of the spinal cord. In addition to the encephalitic and microcystic lesions, foci of demyelination have been observed in the cerebellar white matter after two or three inoculations of virus (Chew-Lim, Suckling and Webb 1977a). During subsequent studies it was found that a loss of myelin could occur in some mice following a single inoculation of virus (Suckling, Jagelman and Webb 1978). The studies reported in the The authors thank the Multiple Sclerosis Society of Great Britain and Northern Ireland, St. Thomas' Hospital Endowment funds and the Philip Fleming Trust for the financial support of this work.
148 present paper were designed to investigate the extent and nature of the myelin loss in Swiss/A2G mice following a single inoculation of avirulent Semliki forest virus. In addition we have compared the general pathological changes and incidence of myelin loss in a number of other strains of mice. MATERIALSAND METHODS
Infection of mice The virus used throughout this work was an avirulent strain of Semliki forest virus, designated A7174]C1SC2 to denote its passage history and referred to subsequently as SFV A7174]. Supplies were kindly provided by Dr. C. J. Bradish, Microbiological Research Establishment, Porton, Wiltshire. The virus had originally been obtained from mosquito squashes, passaged seven times intracerebrally in suckling mice (A7) and clonally selected for extreme avirulence in adult mice from plaques in monolayers of primary chick embryo cells ([74]). C1 denotes a further passage in primary chick embryo cells followed by two further subcultures (SC2; Bradish, Allner and Maber 1971). Lyophilised virus was diluted to 10-3 in 0.75 % bovine serum albumen in phosphate buffered saline (BAPS)at pH 7.3 and stored in aliquots at --70 °C. For the standard model of infection 4-week-old white Swiss/AzG mice (weighing approximately 20 g) and of either sex were used. The mice were taken from a specific pathogen-free colony of random-bred animals and have not been characterised genetically. Virus was administered intraperitoneally as a single dose of 0.1 ml of a 10-4 dilution of lyophilised virus in BAPS which contained approximately 104.0 suckling mouse intracerebral LD~0. In addition to random outbred Swiss/A2G mice, outbred BSVS and inbred BALB/c, CBA and SJL/J mice were used at ages between 4-7 weeks.
Tissue processing Infected animals and those inoculated with BAPS alone were sampled at 5, 7, 10, 14, 18, 21 and 28 days after infection in groups of 3 to 6 animals. Mice were etheranaesthetised, exsanguinated, the brain removed and placed in 10 ~ formol-saline prior to processing by standard histological techniques and staining with haematoxylin and eosin or luxol fast blue and cresyl violet (LFB: the Klfiver-Barrera technique). Areas of paler staining with LFB are not in themselves definitive for myelin loss but these areas often coincided with an easily visible disruption in the continuity of myelin tracts and a degree of "hypercellularity" or histiocytic proliferation. Mice were sampled for ultrastructural studies at 6, 12 and 16 days after infection, were anaesthetised with pentobarbitone sodium and perfused by left ventricular puncture with at least 50 ml 3.5 ~ glutaraldehyde, phosphate-buffered to pH 7.4. The brain was then removed, cubes of 1-2 mm 8 cut from cerebellum and brain stem and immersed in glutaraldehyde at 4 °C for at least 4 hr. The material was prepared as reported earlier (Pathak and Webb 1974). Ultrathin sections were cut, stained with uranyl acetate and lead citrate and examined using an AEI Corinth 500 electron microscope.
149 RESULTS
Clinical findings Clinical signs of disease were not observed in the majority of infected mice of any of the strains used but occasionally hind limb paralysis and ruffling of the fur was noted.
Light microscopy Light-microscopical examination of infected brains from Swiss/A2G mice showed that in almost all infected animals a lymphocytic perivascular infiltration first occurred in samples taken at 5 days after infection and microgliosis in samples taken 7 days after infection. Focal microcystic lesions were first apparent in material taken 10 days after infection and hypertrophied astrocytes were also present in these focal areas. These results confirm the earlier reports of Chew-Lim (1975) and Mackenzie et al. (1978). A loss of myelin was observed in 25 ~ of brains from infected animals taken 14-28 days after inoculation. The myelin loss occurred in focal areas sparsely distributed within the white matter of the cerebellum, brain stem and spinal cord (Fig. 1). There was no particular relationship between these areas and the vessels which showed perivascular cuffing. Infiltrating mononuclear cells were not confined to the perivascular cuffs but were also distributed throughout white matter particularly in the focal areas mentioned above. Where myelin loss was recorded, it occurred frequently in the same anatomical areas as those in which the focal microcystic vacuolation was seen. Earlier work has shown that a resolution of the encephalitis and apparent
Fig. 1. Focus of myelin loss in cerebral peduncle. Disruption of nerve tracts and microcystic vacuoles can be seen. Twenty-one days after inoculation. LFB, × 100.
150 remyelination has occurred by eight weeks after inoculation (Chew-Lira et al. 1977a). Although most of the Swiss/A2G mice infected at 4 weeks old developed inflammatory and microcystic lesions, only 25 ~ developed histologically observable loss of myelin. In several brains from infected mice where no myelin loss could be seen on primary sectioning, serial sections were cut throughout the tissue. Examination of these sections did not reveal any additional microcystic foci or demyelination. Repeated inoculations of virus at 7 or 14, or 7 and 14 days after infection produced no increase in the incidence of demyelination. In the other strains of mice tested, the infection produced a range of values for lesion incidence (Table 1) but when lesions did occur they were identical with those described for the Swiss/A2G mice. Sections from the brains of mice inoculated with virus diluent alone never displayed any histological abnormalities.
Electron microscopy Ultrastructural observations on material from Swiss/A2G mice infected with SFV A7174] showed that virions could not be seen at post-inoculation day (PID) 6 even though an infectivity of greater than 107.° suckling mouse intracerebral LDs0 per ml of 1 0 ~ (w/v) brain suspension is present in brain at this time. This finding is in line with the report of Pathak, Webb, Oaten and Bateman (1976) for mice sampled 3 or 4 days after infection and Pathak and Webb (1978) up to 5 days after infection and is usual for adult mice inoculated intraperitoneally with SFV A7174]. On PID 6, however, some infiltrating mononuclear cells, lymphoblastic-type in appearance, were seen in infected but not control tissue and some mononuclear ceils were seen encircling myelinated axons (Fig. 2). Later, on P I D 16, the extracellular spaces were enlarged, more infiltrating mononuclear cells were seen around blood vessels and deeper in the tissue. Processes from these cells were often in close contact with myelinated axons, in some cases they appeared to penetrate the lamellae of the myelin sheath. Some of the
TABLE 1 INCIDENCE OF INFLAMMATORY PATHOLOGY AND DEMYELINATION IN MICE INFECTED WITH AVIRULENT SEMLIKI FOREST VIRUS Strain
Swiss/AzG BALB/c CBA BSVS SYL/J
Incidence (%) general and a inflammatory pathology
demyelination b
95 (43/45) 100 (ll/ll) 87 (13/15) 85 (12/14) 40 (6/15)
25 (11/45) 18 (2/11) 13 (2/15) 21 (3/14) 8 (1/12)
a Comprising evidence of perivascular cuffing and microcystic foci. b Observed at light microscope level.
151 mononuclear cells had phagocytic capabilities since they showed phagosomes containing myelin debris (Fig. 3). Oligodendrocytes appeared normal in the infected tissue but astrocytes showed some hypertrophy.
Fig. 2. A~mononuclear cell (M) encircling a myelinated axon. A pseudopodial process from this cell (arrow) can be seen in close contact with the myelin. Six days after inoculation, x 24,000.
152
Fig. 3. A process from a mononuclear cell (M), near myelinated axons, showing phagosomes (arrowt containing myelin debris. Lytic bodies (L) can be seen in the cytoplasm. Sixteen days after inoculation. × 36,000. DISCUSSION Infections o f Swiss/A2G mice with S F V A7174] p r o d u c e m a n y varied effects in
153 the CNS of the host (Webb, Chew-Lira, Jageiman, Oaten, Pathak, Suckling and Mackenzie 1978). As well as mild encephalitis and focal microcystic (spongiform) lesions, a single inoculation of SFV A7174], while seldom producing clinical signs of disease, produced a loss of myelin observable at the light microscope level in about a quarter of infected mice. The attack on myelin has been confirmed by ultrastructural investigations and a more detailed ultrastructural study is in progress (Pathak and Webb, in preparation). It may be that myelin disruption does occur in a greater proportion of cases than those observed at the light microscope level. We have found, in addition, that altering some of the components of the model may alter the type of pathology produced or the incidence of myelin loss. Mice which are older than 10 weeks at inoculation tend to show fewer and less severe pathological lesions. Also, if instead of chick embryo cell-derived SFV A7174] being used an inoculum, it is first passaged twice in baby mice and an infected brain suspension used as inoculum, lesions were characterised by more perivascular infiltration and less microcystic vacuolation (Suckling, unpublished observations). It may be the case that the genetic constitution of the host also has a bearing on determining the nature and extent of pathological changes. Several strains display similar lesion incidences but SJL/J mice appear to be much less affected (Table 1) even though susceptibility to neuroinfection is not reduced (Suckling, in preparation). There are many ways in which a loss of myelin may occur following viral infections; these have been reviewed by Wisniewski (1977). A major distinction is drawn between those infections where the cytotoxic effect of virus replication is directly responsible for the destruction of the myelinating cell and subsequent demyelination, and those where the virus infection and the immune response to that infection combined to induce demyelination. This latter type of myelin loss may be termed immunopathological and is associated with a monocytic inflammatory response in the CNS. An example of the cytotoxic type of demyelination is acute murine encephalomyelitis induced by the JHM strain of mouse hepatitis virus (Lampert, Sims and Kniazeff 1973). Immunopathological demyelination has been described in Theiler's murine encephalomyelitis virus infections in mice and may be alleviated by immunosuppressive treatment (Lipton and dal Canto 1976). In SFV A7174] infections in Swiss/AzG mice there appears to be no direct cytotoxic effect due to virus replication. There are no histologically-observable changes in the morphology of infected brains until the appearance of infiltrating cells at 5 days after infection (Mackenzie et al. 1978). At the ultrastructural level only a mild astrocytic hypertrophy has been seen up to 5 days after inoculation. In all other respects the brain tissue appeared normal (Pathak and Webb 1978). Immunosuppression can, however, alter the course of the disease. A single dose of cyclophosphamide one day after virus inoculation, whilst allowing prolongation of the period of high brain virus titres, initially reduced the severity of the encephalitic lesions and microcystic changes. However, when recovery from the initial immunosuppression occurred, as measured by an increase in circulating antibody, clinical illness was more pronounced and pathological changes more severe (Suckling, Jagelman and Webb 1977). Likewise single-dose gamma irradiation caused temporary
154 persistence of virus within the C N S ; recovery from the i m m u n o s u p p r e s s i o n was m a r k e d by a delayed h u m o r a l a n t i b o d y response and more severe demyelination (Chew-Lira, W e b b a n d J a g e l m a n 1977b). Recently we have also infected r a n d o m bred BALB/c-CBA a t h y m i c n u d e mice and their heterozygous ( n u / + ) littermates with SFV A7174]. Myelin loss a n d microcystic lesions in the a p p a r e n t l y i m m u n o c o m p e t e n t n u / ~ - mice were slightly more extensive t h a n those seen in the Swiss/A2G mice a l t h o u g h their incidence was similar. I n contrast, only mild microcystic lesions were seen in the nude mice a n d no loss of myelin was a p p a r e n t (Jagelman, Suckling, W e b b and Bowen 1978). These findings suggest that the myelin loss has an i m m u n o p a t h o l o g i c a l c o m p o n e n t as well as the effect that mouse strain m a y have on disease pathogenesis. F u r t h e r experiments are i n h a n d at present to investigate the effects of b o t h genetic c o n s t i t u t i o n a n d i m m u n o pathology on the incidence a n d severity of myelin loss observed in this infection.
REFERENCES Bradish, C. J., K. Allner and H. B. Maber (1971) The virulence of original and derived strains of Semliki forest for mice, guinea-pigs and rabbits, J. gen. Virol., 12: 141-150. Chew-Lim, M. (1975) Mouse encephalitis induced by avirulent Semliki forest virus, Vet. Path., 12: 387-393. Chew-Lim, M., A. J. Suckling and H. E. Webb (1977a) Demyelinationin mice after two or three infections with avirulent Semliki forest virus, Vet. Path., 14: 67-72. Chew-Lim, M., H. E. Webb and S. Jagelman (1977b) The effect of irradiation on demyelinationinduced by avirulent Semliki forest virus, Brit. J. exp. Path., 58: 459464. Jagelman, S., A. J. Suckling, H. E. Webb and E. T. W. Bowen (1978) The pathogenesis of avirulent Semliki Forest virus infections in athymic nude mice, J. gem Virol., 41 : in press. Lampert, P. W., K. J. Sims and A. J. Kniazeff (1973) Mechanism of demyelination in JHM virus encephalomyelitis, Acta neuropath. (Berl.), 24: 76-85. Lipton, H. L. and M. C. dal Canto (1976) Theiler's virus-induced demyelination - - Prevention by immunosuppression, Science, 192: 62-64. Mackenzie, A., A. J. Suckling, S. Jagelman and A. M. Wilson (1978) Histopathological and enzyme histochemical changes in experimental Semliki forest virus infection in mice and their relevance to scrapie, J. comp. Path., 88: 335-344. Pathak, S. and H. E. Webb (1974) Possible mechanisms for the transport of Semliki Forest virus into and within mouse brain - - An electron-microscopic study, J. neurol. Sci., 23 : 175-184. Pathak, S. and H. E. Webb (1978) An electron-microscopic study of avirulent and virulent Semliki forest virus in the brains of different ages of mice, J. neurol.Sci:, In press. Pathak, S., H. E. Webb, S. W. Oaten and S. Bateman (1976) An electron-microscopic study of the development of virulent and avirulent strains of Semliki forest virus in mouse brain, J. neurol. Sci., 28 : 289-300. Suckling, A. J., S. Jagelman and H. E. Webb (1977) Brain lysosomal glycosidase activity in immunosuppressed mice infected with avirulent Semliki forest virus, btfect. ImmunoL, 15: 386-391. Suckling, A. J., S. Jagelman and H. E. Webb (1978) A comparison of brain lysosomal enzyme activities in four experimental togavirus encephalitides, J. neuroL Sci.i 35: 355-364. Webb, H. E., M. Chew-Lim, S. Jagelman, S. W. Oaten, S. Pathak, A. J. Suckling and A. Mackenzie (1978) Semliki forest virus infections in mice as a model for studying acute and chronic CNS virus infections in man. In: F. Clifford Rose (Ed.), Clinical Neuroimmunology, Blackwell, Oxford, pp. 369-390. Weiner, L. P. (1973) Pathogenesis of demyelinationinduced by a mouse hepatitis virus (JHM virus), Arch. Neurol. (Chic.), 28:398-303. Wisniewski, H. M. (1977) Immunopathology of demyelination in autoimmune diseases and virus in fections, Brit. reed. Bull., 33: 54-59.
147
VIRUS-ASSOCIATED D E M Y E L I N A T I O N A Model Using Avirulent Semliki Forest Virus Infection of Mice
ANTHONY J. SUCKLING, SNEHLATA PATHAK, SUSAN JAGELMAN and HUGH E. WEBB Department of Neurology, Neurovirology Unit, The Rayne b~stitute, St. Thomas' Hospital, London SE1 7EH (Great BritaihJ
(Received 8 May, 1978) (Accepted 26 June, 1978)
SUMMARY Lesions produced by the infection of Swiss/A2G mice with a single inoculation of an avirulent strain of Semliki forest virus have been studied by light and electron microscopy. Whilst a mild encephalitis was detected in the great majority of mice infected, focal areas of myelin loss were observed in the cerebellar white matter of only 25 ~ of cases. The incidence of myelin loss in other strains of mice ranged from 21 ~ in BSVS mice to 8 ~ in SJL/J mice. The possible mechanisms involved in the pathogenesis of the myelin loss are discussed.
INTRODUCTION Recent studies have described the pathological changes which followed infection of Swiss/A2G mice with an avirulent strain of Semliki forest virus (Chew-Lim 1975; Mackenzie, Suckling, Jagelman and Wilson 1978). In these studies, changes typical o f a mild encephalitis occurred consistently after inoculation of virus, together with focal microcystic lesions located mainly in the cerebellar white matter, mid-brain and lateral tracts of the spinal cord. In addition to the encephalitic and microcystic lesions, foci of demyelination have been observed in the cerebellar white matter after two or three inoculations of virus (Chew-Lim, Suckling and Webb 1977a). During subsequent studies it was found that a loss of myelin could occur in some mice following a single inoculation of virus (Suckling, Jagelman and Webb 1978). The studies reported in the The authors thank the Multiple Sclerosis Society of Great Britain and Northern Ireland, St. Thomas' Hospital Endowment funds and the Philip Fleming Trust for the financial support of this work.
148 present paper were designed to investigate the extent and nature of the myelin loss in Swiss/A2G mice following a single inoculation of avirulent Semliki forest virus. In addition we have compared the general pathological changes and incidence of myelin loss in a number of other strains of mice. MATERIALSAND METHODS
Infection of mice The virus used throughout this work was an avirulent strain of Semliki forest virus, designated A7174]C1SC2 to denote its passage history and referred to subsequently as SFV A7174]. Supplies were kindly provided by Dr. C. J. Bradish, Microbiological Research Establishment, Porton, Wiltshire. The virus had originally been obtained from mosquito squashes, passaged seven times intracerebrally in suckling mice (A7) and clonally selected for extreme avirulence in adult mice from plaques in monolayers of primary chick embryo cells ([74]). C1 denotes a further passage in primary chick embryo cells followed by two further subcultures (SC2; Bradish, Allner and Maber 1971). Lyophilised virus was diluted to 10-3 in 0.75 % bovine serum albumen in phosphate buffered saline (BAPS)at pH 7.3 and stored in aliquots at --70 °C. For the standard model of infection 4-week-old white Swiss/AzG mice (weighing approximately 20 g) and of either sex were used. The mice were taken from a specific pathogen-free colony of random-bred animals and have not been characterised genetically. Virus was administered intraperitoneally as a single dose of 0.1 ml of a 10-4 dilution of lyophilised virus in BAPS which contained approximately 104.0 suckling mouse intracerebral LD~0. In addition to random outbred Swiss/A2G mice, outbred BSVS and inbred BALB/c, CBA and SJL/J mice were used at ages between 4-7 weeks.
Tissue processing Infected animals and those inoculated with BAPS alone were sampled at 5, 7, 10, 14, 18, 21 and 28 days after infection in groups of 3 to 6 animals. Mice were etheranaesthetised, exsanguinated, the brain removed and placed in 10 ~ formol-saline prior to processing by standard histological techniques and staining with haematoxylin and eosin or luxol fast blue and cresyl violet (LFB: the Klfiver-Barrera technique). Areas of paler staining with LFB are not in themselves definitive for myelin loss but these areas often coincided with an easily visible disruption in the continuity of myelin tracts and a degree of "hypercellularity" or histiocytic proliferation. Mice were sampled for ultrastructural studies at 6, 12 and 16 days after infection, were anaesthetised with pentobarbitone sodium and perfused by left ventricular puncture with at least 50 ml 3.5 ~ glutaraldehyde, phosphate-buffered to pH 7.4. The brain was then removed, cubes of 1-2 mm 8 cut from cerebellum and brain stem and immersed in glutaraldehyde at 4 °C for at least 4 hr. The material was prepared as reported earlier (Pathak and Webb 1974). Ultrathin sections were cut, stained with uranyl acetate and lead citrate and examined using an AEI Corinth 500 electron microscope.
149 RESULTS
Clinical findings Clinical signs of disease were not observed in the majority of infected mice of any of the strains used but occasionally hind limb paralysis and ruffling of the fur was noted.
Light microscopy Light-microscopical examination of infected brains from Swiss/A2G mice showed that in almost all infected animals a lymphocytic perivascular infiltration first occurred in samples taken at 5 days after infection and microgliosis in samples taken 7 days after infection. Focal microcystic lesions were first apparent in material taken 10 days after infection and hypertrophied astrocytes were also present in these focal areas. These results confirm the earlier reports of Chew-Lim (1975) and Mackenzie et al. (1978). A loss of myelin was observed in 25 ~ of brains from infected animals taken 14-28 days after inoculation. The myelin loss occurred in focal areas sparsely distributed within the white matter of the cerebellum, brain stem and spinal cord (Fig. 1). There was no particular relationship between these areas and the vessels which showed perivascular cuffing. Infiltrating mononuclear cells were not confined to the perivascular cuffs but were also distributed throughout white matter particularly in the focal areas mentioned above. Where myelin loss was recorded, it occurred frequently in the same anatomical areas as those in which the focal microcystic vacuolation was seen. Earlier work has shown that a resolution of the encephalitis and apparent
Fig. 1. Focus of myelin loss in cerebral peduncle. Disruption of nerve tracts and microcystic vacuoles can be seen. Twenty-one days after inoculation. LFB, × 100.
150 remyelination has occurred by eight weeks after inoculation (Chew-Lira et al. 1977a). Although most of the Swiss/A2G mice infected at 4 weeks old developed inflammatory and microcystic lesions, only 25 ~ developed histologically observable loss of myelin. In several brains from infected mice where no myelin loss could be seen on primary sectioning, serial sections were cut throughout the tissue. Examination of these sections did not reveal any additional microcystic foci or demyelination. Repeated inoculations of virus at 7 or 14, or 7 and 14 days after infection produced no increase in the incidence of demyelination. In the other strains of mice tested, the infection produced a range of values for lesion incidence (Table 1) but when lesions did occur they were identical with those described for the Swiss/A2G mice. Sections from the brains of mice inoculated with virus diluent alone never displayed any histological abnormalities.
Electron microscopy Ultrastructural observations on material from Swiss/A2G mice infected with SFV A7174] showed that virions could not be seen at post-inoculation day (PID) 6 even though an infectivity of greater than 107.° suckling mouse intracerebral LDs0 per ml of 1 0 ~ (w/v) brain suspension is present in brain at this time. This finding is in line with the report of Pathak, Webb, Oaten and Bateman (1976) for mice sampled 3 or 4 days after infection and Pathak and Webb (1978) up to 5 days after infection and is usual for adult mice inoculated intraperitoneally with SFV A7174]. On PID 6, however, some infiltrating mononuclear cells, lymphoblastic-type in appearance, were seen in infected but not control tissue and some mononuclear ceils were seen encircling myelinated axons (Fig. 2). Later, on P I D 16, the extracellular spaces were enlarged, more infiltrating mononuclear cells were seen around blood vessels and deeper in the tissue. Processes from these cells were often in close contact with myelinated axons, in some cases they appeared to penetrate the lamellae of the myelin sheath. Some of the
TABLE 1 INCIDENCE OF INFLAMMATORY PATHOLOGY AND DEMYELINATION IN MICE INFECTED WITH AVIRULENT SEMLIKI FOREST VIRUS Strain
Swiss/AzG BALB/c CBA BSVS SYL/J
Incidence (%) general and a inflammatory pathology
demyelination b
95 (43/45) 100 (ll/ll) 87 (13/15) 85 (12/14) 40 (6/15)
25 (11/45) 18 (2/11) 13 (2/15) 21 (3/14) 8 (1/12)
a Comprising evidence of perivascular cuffing and microcystic foci. b Observed at light microscope level.
151 mononuclear cells had phagocytic capabilities since they showed phagosomes containing myelin debris (Fig. 3). Oligodendrocytes appeared normal in the infected tissue but astrocytes showed some hypertrophy.
Fig. 2. A~mononuclear cell (M) encircling a myelinated axon. A pseudopodial process from this cell (arrow) can be seen in close contact with the myelin. Six days after inoculation, x 24,000.
152
Fig. 3. A process from a mononuclear cell (M), near myelinated axons, showing phagosomes (arrowt containing myelin debris. Lytic bodies (L) can be seen in the cytoplasm. Sixteen days after inoculation. × 36,000. DISCUSSION Infections o f Swiss/A2G mice with S F V A7174] p r o d u c e m a n y varied effects in
153 the CNS of the host (Webb, Chew-Lira, Jageiman, Oaten, Pathak, Suckling and Mackenzie 1978). As well as mild encephalitis and focal microcystic (spongiform) lesions, a single inoculation of SFV A7174], while seldom producing clinical signs of disease, produced a loss of myelin observable at the light microscope level in about a quarter of infected mice. The attack on myelin has been confirmed by ultrastructural investigations and a more detailed ultrastructural study is in progress (Pathak and Webb, in preparation). It may be that myelin disruption does occur in a greater proportion of cases than those observed at the light microscope level. We have found, in addition, that altering some of the components of the model may alter the type of pathology produced or the incidence of myelin loss. Mice which are older than 10 weeks at inoculation tend to show fewer and less severe pathological lesions. Also, if instead of chick embryo cell-derived SFV A7174] being used an inoculum, it is first passaged twice in baby mice and an infected brain suspension used as inoculum, lesions were characterised by more perivascular infiltration and less microcystic vacuolation (Suckling, unpublished observations). It may be the case that the genetic constitution of the host also has a bearing on determining the nature and extent of pathological changes. Several strains display similar lesion incidences but SJL/J mice appear to be much less affected (Table 1) even though susceptibility to neuroinfection is not reduced (Suckling, in preparation). There are many ways in which a loss of myelin may occur following viral infections; these have been reviewed by Wisniewski (1977). A major distinction is drawn between those infections where the cytotoxic effect of virus replication is directly responsible for the destruction of the myelinating cell and subsequent demyelination, and those where the virus infection and the immune response to that infection combined to induce demyelination. This latter type of myelin loss may be termed immunopathological and is associated with a monocytic inflammatory response in the CNS. An example of the cytotoxic type of demyelination is acute murine encephalomyelitis induced by the JHM strain of mouse hepatitis virus (Lampert, Sims and Kniazeff 1973). Immunopathological demyelination has been described in Theiler's murine encephalomyelitis virus infections in mice and may be alleviated by immunosuppressive treatment (Lipton and dal Canto 1976). In SFV A7174] infections in Swiss/AzG mice there appears to be no direct cytotoxic effect due to virus replication. There are no histologically-observable changes in the morphology of infected brains until the appearance of infiltrating cells at 5 days after infection (Mackenzie et al. 1978). At the ultrastructural level only a mild astrocytic hypertrophy has been seen up to 5 days after inoculation. In all other respects the brain tissue appeared normal (Pathak and Webb 1978). Immunosuppression can, however, alter the course of the disease. A single dose of cyclophosphamide one day after virus inoculation, whilst allowing prolongation of the period of high brain virus titres, initially reduced the severity of the encephalitic lesions and microcystic changes. However, when recovery from the initial immunosuppression occurred, as measured by an increase in circulating antibody, clinical illness was more pronounced and pathological changes more severe (Suckling, Jagelman and Webb 1977). Likewise single-dose gamma irradiation caused temporary
154 persistence of virus within the C N S ; recovery from the i m m u n o s u p p r e s s i o n was m a r k e d by a delayed h u m o r a l a n t i b o d y response and more severe demyelination (Chew-Lira, W e b b a n d J a g e l m a n 1977b). Recently we have also infected r a n d o m bred BALB/c-CBA a t h y m i c n u d e mice and their heterozygous ( n u / + ) littermates with SFV A7174]. Myelin loss a n d microcystic lesions in the a p p a r e n t l y i m m u n o c o m p e t e n t n u / ~ - mice were slightly more extensive t h a n those seen in the Swiss/A2G mice a l t h o u g h their incidence was similar. I n contrast, only mild microcystic lesions were seen in the nude mice a n d no loss of myelin was a p p a r e n t (Jagelman, Suckling, W e b b and Bowen 1978). These findings suggest that the myelin loss has an i m m u n o p a t h o l o g i c a l c o m p o n e n t as well as the effect that mouse strain m a y have on disease pathogenesis. F u r t h e r experiments are i n h a n d at present to investigate the effects of b o t h genetic c o n s t i t u t i o n a n d i m m u n o pathology on the incidence a n d severity of myelin loss observed in this infection.
REFERENCES Bradish, C. J., K. Allner and H. B. Maber (1971) The virulence of original and derived strains of Semliki forest for mice, guinea-pigs and rabbits, J. gen. Virol., 12: 141-150. Chew-Lim, M. (1975) Mouse encephalitis induced by avirulent Semliki forest virus, Vet. Path., 12: 387-393. Chew-Lim, M., A. J. Suckling and H. E. Webb (1977a) Demyelinationin mice after two or three infections with avirulent Semliki forest virus, Vet. Path., 14: 67-72. Chew-Lim, M., H. E. Webb and S. Jagelman (1977b) The effect of irradiation on demyelinationinduced by avirulent Semliki forest virus, Brit. J. exp. Path., 58: 459464. Jagelman, S., A. J. Suckling, H. E. Webb and E. T. W. Bowen (1978) The pathogenesis of avirulent Semliki Forest virus infections in athymic nude mice, J. gem Virol., 41 : in press. Lampert, P. W., K. J. Sims and A. J. Kniazeff (1973) Mechanism of demyelination in JHM virus encephalomyelitis, Acta neuropath. (Berl.), 24: 76-85. Lipton, H. L. and M. C. dal Canto (1976) Theiler's virus-induced demyelination - - Prevention by immunosuppression, Science, 192: 62-64. Mackenzie, A., A. J. Suckling, S. Jagelman and A. M. Wilson (1978) Histopathological and enzyme histochemical changes in experimental Semliki forest virus infection in mice and their relevance to scrapie, J. comp. Path., 88: 335-344. Pathak, S. and H. E. Webb (1974) Possible mechanisms for the transport of Semliki Forest virus into and within mouse brain - - An electron-microscopic study, J. neurol. Sci., 23 : 175-184. Pathak, S. and H. E. Webb (1978) An electron-microscopic study of avirulent and virulent Semliki forest virus in the brains of different ages of mice, J. neurol.Sci:, In press. Pathak, S., H. E. Webb, S. W. Oaten and S. Bateman (1976) An electron-microscopic study of the development of virulent and avirulent strains of Semliki forest virus in mouse brain, J. neurol. Sci., 28 : 289-300. Suckling, A. J., S. Jagelman and H. E. Webb (1977) Brain lysosomal glycosidase activity in immunosuppressed mice infected with avirulent Semliki forest virus, btfect. ImmunoL, 15: 386-391. Suckling, A. J., S. Jagelman and H. E. Webb (1978) A comparison of brain lysosomal enzyme activities in four experimental togavirus encephalitides, J. neuroL Sci.i 35: 355-364. Webb, H. E., M. Chew-Lim, S. Jagelman, S. W. Oaten, S. Pathak, A. J. Suckling and A. Mackenzie (1978) Semliki forest virus infections in mice as a model for studying acute and chronic CNS virus infections in man. In: F. Clifford Rose (Ed.), Clinical Neuroimmunology, Blackwell, Oxford, pp. 369-390. Weiner, L. P. (1973) Pathogenesis of demyelinationinduced by a mouse hepatitis virus (JHM virus), Arch. Neurol. (Chic.), 28:398-303. Wisniewski, H. M. (1977) Immunopathology of demyelination in autoimmune diseases and virus in fections, Brit. reed. Bull., 33: 54-59.