Virus tropism, distribution, persistence and pathology in the corpus callosum of the Semliki Forest virus-infected mouse brain: a novel system to study virus-oligodendrocyte interactions

The temporal course of cellular pathology in virus-infected oligodendrocytes in vivo is not well defined. Here we study these events in the mouse brain using a novel system in which large numbers of oligodendrocytes can be reproducibly infected. In the mouse, following extraneural inoculation, the A7(74) strain of the alphavirus Semliki Forest virus (SFV) is efficiently neuroinvasive and central nervous system (CNS) infection leads to predominantly perivascular lesions of immune-mediated demyelination. This study demonstrates that direct intracerebral inoculation with SFV A7(74) or the SFV1 vector results in dramatic, selective and widespread infection of the major white matter tract of the brain, the corpus callosum. Mature oligodendrocytes are the predominant cell type infected. Subsequent events are complex; early virus-induced necrotic death of infected cells is followed by apoptotic death of adjacent apparently uninfected cells. A strong inflammatory response and considerable myelin loss are evident from 10 days and virus-positive cells are not observed after this time. In contrast, in athymic nu/nu mice, in the absence of T-cell responses, no inflammatory infiltrates are observed and virus-infected cells persist for over 30 days with extensive vacuolation but less demyelination. The change from an early destructive to a potentially persistent infection of oligodendrocytes is likely to reflect activation of innate immune responses. Activation of peripheral innate defences by inoculation of poly I : C prior to CNS virus infection abrogates the widespread corpus callosum infection. This widespread infection of the corpus callosum provides a novel in vivo system in which to study virus-oligodendrocyte interactions.     

Cell death mechanisms in the olfactory bulb of rats infected intranasally with Semliki Forest virus

Semliki Forest virus (SFV) infection of mice is used as a model to study pathogenic processes occurring in viral encephalitis. It has previously been shown that avirulent strains of SFV differ from virulent strains in showing restricted multiplication in neurones and in producing localized rather than widespread lesions in the central nervous system (CNS). Restricted neuronal damage is age-dependent and does not occur in neonatal animals. In this study, cell death mechanisms occurring in the CNS of adult rats infected intranasally (i.n.) with a virulent (SFV4) and an avirulent (A7) strain of SFV have been investigated. Although i.n. infection of rats was less efficient than that of mice, SFV4 reached a higher titre in the CNS of infected animals than A7. Neuronal destruction and leucocytic infiltration occurred throughout the forebrain of SFV4-infected rats. A7-infected rats remained clinically normal although degenerate neurons and inflammatory changes were present primarily in the olfactory system. Following infection with either A7-SFV or SFV4, TUNEL-positive nuclei were seen in areas of leucocytic infiltration and among the poorly differentiated cells of the rostral migratory stream. Migrating cells had condensed nuclear chromatin, compacted cytoplasm and intact cellular membranes, characteristic of apoptosis, and were sparsely immunolabelled for viral antigen. In SFV4-infected rats, large numbers of contiguous neurones in forebrain areas exhibited cytoplasmic eosinophilia and karyolysis and were surrounded by phagocytic cells. Such neurones contained dense intracytoplasmic deposits of viral antigen and showed weak cytoplasmic TUNEL staining; electron microscopy showed membrane disruption, organelle disintegration, irregular chromatin condensation and cytoplasmic aggregation of virus particles. Bcl-2 staining was similar in infected and control rats and was most intense in randomly distributed Purkinje cells in the cerebellum; neurons in the olfactory bulbs were unstained. These findings indicate that during SFV encephalitis, infiltrating leucocytes and neural precursor cells undergo apoptosis whilst productively infected neurons undergo necrosis.     

Morphology of oligodendrocytes during demyelination in optic nerves of mice infected with Semliki Forest virus

Multiple sclerosis (MS) is a demyelinating disease which affects oligodendrocytes, the myelinating cells of the CNS. Demyelination is known to occur in the optic nerves of Balb/c mice infected with the avirulent A7(74) strain of Semliki Forest virus (SFV), and many of the changes are similar to those of patients with MS. The aim of the present study was to determine how demyelination proceeds in individual oligodendrocytes in SFV infection, to help in understanding the pathology of demyelination and remyelination in MS. The whole-cell morphology of individual oligodendrocyte units (defined as the oligo-dendrocyte, its processes and the internodal myelin segments of the axons it ensheaths) was characterized using intracellular dye injection in isolated intact optic nerves. In untreated control mice, oligodendrocytes had a relatively uniform morphology and each cell on average provided 20 or so nearby axons with single myelin sheaths with internodal lengths of ～ 150 μm. In SFV infected mice, during the peak of demyelination at post inoculation days 14–21, 55% of oligodendrocytes displayed a range of morphological abnormalities, which most likely represented sequential changes in oligodendrocytes during demyelination. Thus, at the earliest stage of demyelination oligodendrocytes developed swellings or vacuolations along their internodal myelin sheaths, which became gradually attenuated and were completely lost in extreme cases. The results show that whole oligodendrocyte units were affected during SFV-induced demyelination and this is the basis of the focal nature of lesions in this viral model of MS. Individual oligodendrocyte units which had lost their full complement of myelin sheaths had the appearance of immature oligodendrocytes, suggesting they had undergone de-differentiation. We concluded that these cells may not be destroyed during demyelination and it is possible they are capable of remyelination which is a feature of SFV infection in mice and MS in humans.     

Pathogenesis of Semliki Forest virus encephalitis

This article provides a review of the pathogenesis of Semliki Forest virus (SFV) encephalitis. In mice, outcome of infection varies according to age of the mouse and strain of the virus and can include acute encephalitis, subacute demyelinating meningoencephalomyelitis, and persistent subclinical central nervous system (CNS) infection. All strains of virus are virulent in mice infected <12 days of age. The L10 strain is also virulent in mice >14 days age, whereas the A7(74) strain is avirulent. The genetic difference between these strains maps to the nsp3 gene. For A7(74) virus, age-related virulence correlates with ability of CNS neurons to replicate virus and undergo apoptotic cell death. Immature developing neurons support complete virus replication but as neuronal populations and circuits mature in the postnatal brain, virus infection becomes progressively restricted and nonproductive. This restricted replication can be overcome by gold I compounds, which may function by inducing neuronal dedifferentiation to a state permissive for virus replication. Biochemical pathways associated with membrane biogenesis may be an important determinant of this effect. Infection of some developing neuronal populations results in apoptosis, whereas infection of mature neurons results in persistent infection. An active type-I interferon system prevents virus spread in extraneural tissues. An initial high-titer plasma viremia is controlled by immunoglobulin M (IgM) antibodies. Virus enters the brain across cerebral endothelial cells and initiates scattered foci of perivascular infection. The blood-brain barrier is disrupted. Neurons and oligodendrocytes are the cell types most frequently infected. Infectivity in the brain can be eliminated by IgG antibodies, though an active T-cell response is required for virus elimination. Lesions of inflammatory demyelination require the presence of CD8(+) T lymphocytes and probably result from destruction by these cells of virally infected oligodendrocytes.     

Avirulent Semliki Forest virus replication and pathology in the central nervous system is enhanced in IL-12-defective and reduced in IL-4-defective mice: a role for Th1 cells in the protective immunity

Experimental infection of mice with avirulent Semliki Forest virus (SFV) has been used as a model of demyelinating disease in humans. A number of studies have shown that T cells may be important for mediating demyelination, but the role of T cells is still, unclear. Here, we show that neuronal necrosis, but not demyelination, was more severe in interleukin (IL)-12-defective mice compared with wild-type mice and this correlated with higher virus titers in the brain. In contrast, the severity of demyelination and neuronal depletion was reduced in IL-4-defective mice and this correlated with reduced brain virus titers and enhanced SFV-specific IFN-gamma production. The findings indicate that type 1 T cells play a role in the control of SFV replication but not directly in SFV-induced pathology in the CNS.     

A role for alpha4-integrin in the pathology following Semliki Forest virus infection

Migration of cells into the central nervous system (CNS) is a pivotal step in the pathogenesis of immune-mediated diseases such as multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE) and virus-induced demyelinating diseases. Such migration is dependent on expression of adhesion molecules. The expression of adhesion molecules in the CNS was studied in Biozzi ABH mice infected with Semliki Forest virus (SFV) A7(74) - an important demyelinating model of MS. Expression of LFA-1alpha/CD11a, LFA-1beta/CD18 and ICAM-1/CD56 were rapidly elevated and remained high whereas MAC-1, CD44 and VCAM-1/CD106 were less widely expressed. The alpha4-integrin VLA-4/CD49d was more specifically associated with CNS lesions. To identify the importance of VLA-4, CD44, ICAM-1 and MAC-1 in the pathogenesis of SFV infection, monoclonal antibodies that block these adhesion molecules were administered in vivo during infection. Anti-VLA-4 treatment dramatically reduced the cellular infiltrates and demyelination within the CNS but did not affect the clearance of virus while antibodies to CD44, ICAM and MAC-1 antibody treatment had no effect. This study demonstrates that SFV infection induces the expression of adhesion molecules within the CNS and that VLA-4 plays an important role in the development of inflammation and demyelination in the CNS following SFV infection.     

Virulent and avirulent strains of Semliki Forest virus show similar cell tropism for the murine central nervous system but differ in the severity and rate of induction of cytolytic damage

The pathogenicity of the avirulent, demyelinating A7 strain of Semliki Forest virus (SFV) and the virulent SFV4 strain (derived from an infectious clone) for the central nervous system of adult BALB/c mice following intranasal infection was compared. The techniques used included immunocytochemistry using anti-SFV antibody and antibodies to cell markers, in situ hybridization (ISH) using a biotinylated cDNA probe specific for SFV, and immunocytochemistry/ISH double labelling. Whereas SFV4 was lethal at 4 days post-infection, A7-infected mice appeared normal at all times. Neuronal necrosis in the pyriform cortex was present in both infections, but developed sooner and was more severe iollowing miection with SFV4 than with A7. Intact neurons and putative oligodendrocytes contained viral RNA and virus-specific antigen in SFV4 infected mice; viral RNA but not virus-specific antigen was detected in similar cells in A7-infected mice. These results confirm that SFV4 and A7 share similar cell tropisms for the murine central nervous system, but differ in the severity and rate of development of cytolytic damage. Intranasal infection is an efficient monitoring system for studies of the molecular basis of pathogenicity of SFV infection in mice.     

Role of interferon-gamma and nitric oxide in the neuropathogenesis of avirulent Semliki Forest virus infection

Semliki Forest virus (SFV) infection of mice provides a useful model for the analysis of viral neuropathogenesis. In this study, the roles of interferon (IFN)-gamma and nitric oxide (NO) in the pathogenesis of SFV infection were assessed using mice deficient in inducible nitric oxide synthase (iNOS-/-), an enzyme important in the production of NO, and mice deficient in IFN-gamma receptor (IFN-gammaR-/-). Gene-knockout and wildtype mice were infected intranasally with the avirulent A7 strain of SFV and neuropathological lesions were correlated with levels of IFN-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 in the olfactory bulbs and frontal cortex. Lesions in IFN-gammaR-/- mice were characterized by higher levels of neuronal necrosis than in wildtype mice. The higher levels of neuronal necrosis were associated with increased levels of SFV antigen in neurones and increased numbers of macrophages and B cells. Relative differences in the severity of demyelination between IFN-gammaR-/- and wildtype mice were not detected. Similar levels of neuronal necrosis and SFV antigen labelling occurred in iNOS-/- mice and wildtype mice and levels of demyelination and macrophage infiltration in the iNOS-/- mice were lower than those in the wildtype strain. A rapid, but transient increase in the concentration of IFN-gamma was demonstrated in the frontal cortex of all infected mice samples. IL-10 levels in the frontal cortex and olfactory bulbs of SFV-infected iNOS-/- mice exceeded those present in the wildtype mice. This study, taken with our previous reports, provides further evidence that type 1 T cell responses are important in the control of brain viral clearance and the prevention of neuronal necrosis, but not in the development of demyelination.     

Expression of viral antigens in the central nervous system of visna-infected sheep: an immunohistochemical study on experimentsl visna induced by virus strains of increased neurovirulence

Summary Icelandic sheep were infected by intracerebral inoculation with visna virus strains of increased neurovirulence. The character and severity of pathological lesions were studied in brains from four sheep that developed clinical signs 5 to 12 weeks after infection. Viral antigens were identified by immunostaining using mouse monoclonal antibodies against two core proteins and the Avidin-Biotin method of detection. The pathological lesions were in general more severe than observed following infection with the parent strain K1514. Primary demyelination, a late manifestation of infection with K1514, was detected. Thus, in addition to causing more severe pathological lesions, these neurovirulent strains apparently have an increased potential to induce primary demyelination. Viral antigens were detected in lymphocytes, plasma cells, macrophages, endothelial cells, pericytes, fibroblasts and choroidal epithelial cells. Neurons and glial cells were antigen negative. The spectrum of infected cells in the brain was similar to that observed in infections with human immunodeficiency virus. These results do not support the view that the demyelination is caused by immunological damage to infected oligodendrocytes. A perturbation of the function of oligodendrocytes through a non-productive infection could be the underlying pathogenetic mechanism and/or a non-specific demyelination due to the intense inflammatory reaction.     

The relationship between axonal transport of protein and demyelination in the optic nerves of mice infected with Semliki Forest Virus

Fast and slow axonal transport of protein have been studied in the optic nerves of mice infected with Semliki Forest Virus (SFV) that causes patchy demyelination throughout the CNS. Intravitreal injections of [3H]proline were given at regular intervals after virus inoculation, the labelled protein in the superior colliculi was then measured after survival periods of 18 h or 10 days, for fast and slow axonal transport studies, respectively. Fast transport studies showed an enhanced amount of protein arriving at the optic nerve terminals (superior colliculus) of the SFV-infected mice prior to the onset of demyelination. In contrast, the slow transport studies showed an enhanced amount of protein at the superior colliculus of the SFV-infected mice during the demyelination period. There was no concomitant increase in labelled protein in the retina at any time after the SFV infection. It is proposed that alteration in the transport of the protein constituents other than major myelin specific components may cause disruption of myelin maintenance in SFV infection.     

Patterns of oligodendrocyte pathology in coronavirus-induced subacute demyelinating encephalomyelitis in the lewis rat

Intracerebral infection of rats with JHM coronavirus induces a chronic inflammatory demyelinating disease, which in many respects mimicks the pathology of multiple sclerosis. We investigated the patterns of demyelination and oligodendrocyte pathology in this model. In early stages of the disease infection of oligodendrocytes was associated with a downregulation of expression of mRNA for proteolipid protein in the absence of myelin destruction. When demyelinating lesions were formed infected oligodendrocytes were destroyed by necrosis, whereas oligodendrocytes that did not contain detectable virus antigen or RNA were in part dying by apoptosis. At this stage of the disease remyelination of the lesions was pronounced. At later stages after infection virus antigen was nearly completely cleared from the lesions. In spite of the lack of detectable virus, ongoing demyelination and unspecific tissue destruction occurred, and oligodendrocytes were mainly destroyed by apoptosis. These late lesions revealed only minimal central remyelination, but they were frequently repaired by Schwann cells. Our studies suggest that the mechanisms of myelin destruction in this model of virus-induced demyelination are complex and that the patterns of tissue damage may change during the course of the disease. Glia 19:1–7, 1997 © 1997 Wiley-Liss, Inc.     

Growth of the WW strain of Theiler virus in mouse central nervous system organotypic culture

Summary Myelinated organotypic cultures of mouse central nervous system (CNS) provide a favorable milieu for growth of the WW strain of Theiler virus (TV), WW-TV induced a cytopathic effect characterized by neuronal destruction and swelling of myelin sheaths. Tissue culture medium and homogenates of infected cultures produced neurologic disease in mice, and TV. was demonstrated in cultures by indirect immunoflourescence and electron microscopy. Ultrastructurally, severe alterations in neurons and the presence of cytoplasmic inclusions containing paracrystalline arrays of 30-nm virus particles were evident in astrocytes. Demyelination appeared to be secondary to lysis of oligodendrocytes by virus. CNS organotypic cultures provide an environment for the study of TV-induced demyelination.     

Serum cytotoxicity to human and rat oligodendrocytes in culture

Serum from multiple sclerosis (MS) patients can cause demyelination in rat CNS explant cultures and induce cytotoxicity to rat oligodendrocytes in culture. The interpretation of these findings for MS is complicated by the fact that injury to myelin and oligodendrocytes can also be induced with normal human serum. In this study, we confirmed that serum from MS patients and healthy control subjects can cause in vitro toxicity to rat oligodendrocytes, as established by a 51Cr release assay, but we did not detect toxicity to human cultured oligodendrocytes. Morphologic changes after 5-6 h incubation with the sera were also extensive in the rat oligodendrocyte cultures. No morphologic changes or changes in cell numbers could be detected in the human cultures upon examination by light microscopy and by immunofluorescent staining with anti-GalC antibody.     

A rat model for human T lymphocyte virus type I-associated myeloneuropathy. down-regulation of bcl-2 expression and increase in sensitivity to TNF-alpha of the spinal oligodendrocytes

We reported that the tumor necrosis factor-alpha (TNF-alpha) expression and apoptotic death of oligodendrocytes appeared to be a major pathogenesis of the demyelination of spinal cords of Wistar-King-Aptekman-Hokudai (WKAH) rats with human T lymphocyte virus type I (HTLV-I) infection, HAM rats. In the present study, we examined the sensitivity to TNF-alpha-induced cell death of in vitro-separated oligodendrocytes from HTLV-I-infected WKAH rats. Although the number of non-viable oligodendrocytes increased by adding recombinant TNF-alpha, in a dose-dependent manner, in both HTLV-I-infected and uninfected control rats, oligodendrocytes from the infected rats were more susceptible to TNF-alpha. In situ detection of DNA fragmentation showed apoptotic death of oligodendrocytes. The expression of bcl-2, an anti-apoptotic gene, was strongly down-regulated in oligodendrocytes of the infected rats but not in the control rats. We suggest that the down-regulation of bcl-2 expression in the oligodendrocytes of the HTLV-I-infected rats may increase the susceptibility to TNF-alpha-induced apoptosis of oligodendrocytes, the result being development of HTLV-I-induced myeloneuropathy in rats.     

Multiple neural cell types are infected in vitro by border disease virus

Border disease (BD) of sheep results from a congenitally acquired nonarbotogavirus infection which causes a highly selective central nervous system (CNS) pathological lesion consisting of diffuse decreased myelination without inflammation or neuronal destruction. Thus, a selective disruption of oligodendroglial function appears to occur. In order to investigate the in vitro cell tropism of BD virus, primary cultures derived from fetal and adult ovine CNS and peripheral nervous system were inoculated with BD virus. Infected cell types were determined by dual immunofluorescent labeling for viral and cell type specific antigens. Infection of all the major cell types represented in these cultures, including oligodendrocytes, astrocytes, fibroblasts, dorsal root ganglion neurons and Schwann cells was found. Oligodendrocytes were only infected earlier and appeared to remain infected longer than astrocytes and fibroblasts. Infectious virus was produced by all cultures and continued to be produced even after the disappearance of nearly all immunocytochemically detectable viral antigen within cells. These studies suggest that the selective dysfunction of the oligodendrocyte in BD is not based on a selective viral tropism.     

Selective and antigen-dependent effects of myelin degeneration on central nervous system inflammation

Damage to myelin sheath or oligodendrocytes may precede or even provoke inflammation of the central nervous system (CNS), but the extent to which these degenerative changes affect inflammation remains largely undefined. To study these processes in more detail, we used CNS antigen-specific T cells in the presence or absence of anti-myelin antibodies to induce experimental autoimmune encephalomyelitis (EAE) in transgenic Lewis rats with low-grade subclinical myelin degeneration and associated microglia cell activation, and in wild-type Lewis rats with an intact CNS. We found that myelin degeneration affects the localization of inflammatory lesions, the numbers of T cells recruited to these lesions, and the severity of the resulting clinical disease. In addition, myelin degeneration and associated microglia cell activation jointly enhance the susceptibility of the CNS to the action of anti-myelin antibodies. Our data show that even subtle alterations of myelin and oligodendrocytes may massively amplify the extent of demyelination and tissue damage, involving different immune effector mechanisms. A similar causal relationship might also operate in human patients with multiple sclerosis, where T cell-mediated inflammation and antibody-mediated demyelination have been documented, and where genetic factors might determine the susceptibility of the target tissue for immune-mediated injury.     

Multifocal CNS demyelination following peripheral inoculation with herpes simplex virus type 1

The peripheral inoculation of herpes simplex virus type 1 (HSV 1) in experimental animals induces central nervous system (CNS) demyelinating lesions, but the potential relevance of this model to multiple sclerosis is lessened by the unifocal nature of the lesion. In this study, inbred strains of mice were selected on the basis of varying resistance to mortality following lip inoculation with virus. A spectrum of CNS pathology was observed, ranging from focal collections of inflammatory cells at the trigeminal root entry zone in resistant strains (C57BL/6J), to unifocal demyelinating lesions in moderately resistant strains (BALB/cByJ), to multifocal demyelinating lesions throughout the brain in susceptible strains (A/J). Findings from viral titration studies of the CNS support a direct cytolytic effect of virus in the development of demyelinating lesions at the trigeminal root entry zone but cannot exclude an immune-mediated component. Furthermore, 50% tissue-culture-infective doses, immunofluorescence, and electron microscopic studies of primary cultures of oligodendrocytes, derived from the three strains of adult mice, identify differences in resistance to HSV 1 infection in vitro, suggesting that differences at this level may also contribute to the pathological appearance. Multifocal lesions in A/J mice were first observed when the infectious virus could no longer be isolated from the CNS and may be the result of an immune-mediated process "triggered" by the acute CNS infection in susceptible strains of mice.     

Altered neurovirulence of temperature-sensitive vesicular stomatitis virus mutants in a murine model by inoculation of bombesin: a neuropeptide. I. Clinical, virological and pathological observations

Previous work in our laboratory has demonstrated that only certain temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) appear capable of producing central nervous system (CNS) infection in a mouse model system. Considerable effort has been devoted to studies directed at unraveling the mechanisms underlying host virulence with these tsVSV mutants. With the previous demonstration that certain neuropeptides, capable of lowering body temperature, alter avirulent into virulent infection, we explored the role of one of these neuropeptides, bombesin, in CNS infection induced by normally avirulent tsG11 VSV, as well as certain tsVSV mutants derived from persistently infected (pi) carrier cultures. Our observations indicate that bombesin dramatically alters CNS infection with either tsG11 VSV as well as tsVSV mutants derived from persistent carrier cultures. When virus alone was inoculated intracerebrally, no sign of illness was observed and no animal died. When bombesin was injected along with normally avirulent tsG11 VSV, or glioma derived tsG31 VSV, 50% of mice died within 6-8 days after inoculation. Moreover, mice infected with virus and neuropeptide demonstrated striking pathological alterations in the CNS. These studies are in agreement with previously published results from others as well as our own laboratory and strongly suggest a direct correlation between CNS temperature and the capacity of certain tsVSV mutants to induce clinical and pathological disease.     

Demyelinating, non-demyelinating and attenuated canine distemper virus strains induce oligodendroglial cytolysis in vitro

A virulent canine distemper virus (CDV) strain that causes demyelination in vivo has been shown to induce oligodendroglial degeneration in vitro. In order to investigate if this effect on oligodendrocytes is specific for demyelinating strains only, primary brain cell cultures were infected with either virulent demyelinating strains (A75/17 and CH84-CDV), a virulent non-demyelinating strain (SH-CDV) or a non-virulent strain (OP-CDV). All virulent viruses caused a persistent type infection with moderate cytolysis whereas the non-virulent strain was highly cytolytic. All strains induced a similar pattern of oligodendroglial degeneration. It was concluded that the ability to induce oligodendroglial degeneration, which is thought to be the in vitro correlate of demyelination in vivo, is inherent to CDV irrespective of the strain. The discrepancy between biological behaviour of CDV strains in brain cell cultures and in vivo can be explained by the more complex virus-cell interactions in vivo than in vitro.     

Oligodendroglial pathology in canine distemper virus infection in vitro

Summary Dog brain cell cultures were infected with different canine distemper virus (CDV) strains to study the oligodendrocytes, which were characterized with eight different antibodies to cover the whole oligodendroglial population in the culture. A few weeks after infection all oligodendroglial cell types started to degenerate and disappeared from the culture. However, since no CDV protein could be demonstrated in the degenerating oligodendrocytes with extensive double-labelling studies, this lesion can not be explained as being a result of cytolytic infection. This conclusion was further supported in experiments with plaque-forming CDV, in which viral replication is restricted to the cytolytic areas only; oligodendrocytes also degenerated in virus-free areas between the plaques. The hypothesis of toxic factors released by other infected cell types in the culture leading to secondary damage of the oligodendrocyte could not be confirmed by transferring supernatants from infected to normal cultures. Whereas the presence of toxic factors can not be completely excluded, the possibility of an abortive infection of the oligodendrocytes with no or very limited viral protein synthesis is discussed.Supported by the Swiss National Science Foundation (grant no. 3.949.84) and the Swiss Multiple Sclerosis Society