Direct comparison of demyelinating disease induced by the Daniel's strain and BeAn strain of Theiler's murine encephalomyelitis virus

We compared CNS disease following intracerebral injection of SJL mice with Daniel's (DA) and BeAn 8386 (BeAn) strains of Theiler's murine encephalomyelitis virus (TMEV). In tissue culture, DA was more virulent then BeAn. There was a higher incidence of demyelination in the spinal cords of SJL/J mice infected with DA as compared to BeAn. However, the extent of demyelination was similar between virus strains when comparing those mice that developed demyelination. Even though BeAn infection resulted in lower incidence of demyelination in the spinal cord, these mice showed significant brain disease similar to that observed with DA. There was approximately 100 times more virus specific RNA in the CNS of DA infected mice as compared to BeAn infected mice. This was reflected by more virus antigen positive cells (macrophages/microglia and oligodendrocytes) in the spinal cord white matter of DA infected mice as compared to BeAn. There was no difference in the brain infiltrating immune cells of DA or BeAn infected mice. However, BeAn infected mice showed higher titers of TMEV specific antibody. Functional deficits as measured by Rotarod were more severe in DA infected versus BeAn infected mice. These findings indicate that the diseases induced by DA or BeAn are distinct.     

CD40L is critical for protection from demyelinating disease and development of spontaneous remyelination in a mouse model of multiple sclerosis

Theiler's murine encephalomyelitis virus (TMEV) induces acute neuronal disease followed by chronic demyelination in susceptible strains of mice. In this study we examined the role of a limited immune defect (deletion or blocking of CD40 ligand [CD40L]) on the extent of brain disease, susceptibility to demyelination, and the ability of demyelinated mice to spontaneously remyelinate following TMEV infection. We demonstrated that CD40L-dependent immune responses participate in pathogenesis in the cerebellum and the spinal cord white matter but protect the striatum of susceptible SJL/J mice. In mice on a background resistant to TMEV-induced demyelination (C57BL/6), the lack of CD40L resulted in increased striatal disease and meningeal inflammation. In addition, CD40L was required to maintain resistance to demyelination and clinical deficits in H-2 ^b mice. CD40L-mediated interactions were also necessary for development of protective H-2^b-restricted cytotoxic T cell responses directed against the VP2 region of TMEV as well as for spontaneous remyelination of the spinal cord white matter. The data presented here demonstrated the critical role of this molecule in both antibody- and cell-mediated protective immune responses in distinct phases of TMEV-mediated pathology.     

Location and distribution of virus antigen in the central nervous system of mice persistently infected with Theiler's virus.

The present study has shown that virus can be readily detected by immunofluorescent staining in the central nervous system (CNS) of SJL mice persistently infected with Theiler''s murine encephalomyelitis virus (TMEV). Considering the low CNS virus content, large amounts of virus antigen were found in the white matter, the site of demyelinating lesions. Virus antigen was detected in all animals killed after post-infection (PI) Day 21, a time which can be considered as the beginning of the persistent phase of this infection, and the appearance of virus antigen in white matter corresponded closely in time with the onset of demyelination. The pathogensis of this persistent infection can now be reasonably well reconstructed from the temporal observations made in this study. It would appear that between the second and third week PI, virus replication largely shifts from neurons in spinal cord gray matter to other cell types located in white matter. While a lower-grade persistent infection (in terms of the relative number of cells containing virus antigen) is established and maintained in cells in the gray matter and inflammatory and leptomeningeal infiltrates, cells in white matter appear to be mainly responsible for perpetuating the infection. Why these cells should supplant neurons as the most susceptible host cell during the chronic phase of the infection is discussed.     

Location and distribution of virus antigen in the central nervous system of mice persistently infected with Theiler's virus

The present study has shown that virus can be readily detected by immunofluorescent staining in the central nervous system (CNS) of SJL mice persistently infected with Theiler's murine encephalomyelitis virus (TMEV). Considering the low CNS virus content, large amounts of virus antigen were found in the white matter, the site of demyelinating lesions. Virus antigen was detected in all animals killed after post-infection (PI) Day 21, a time which can be considered as the beginning of the persistent phase of this infection, and the appearance of virus antigen in white matter corresponded closely in time with the onset of demyelination. The pathogensis of this persistent infection can now be reasonably well reconstructed from the temporal observations made in this study. It would appear that between the second and third week PI, virus replication largely shifts from neurons in spinal cord gray matter to other cell types located in white matter. While a lower-grade persistent infection (in terms of the relative number of cells containing virus antigen) is established and maintained in cells in the gray matter and inflammatory and leptomeningeal infiltrates, cells in white matter appear to be mainly responsible for perpetuating the infection. Why these cells should supplant neurons as the most susceptible host cell during the chronic phase of the infection is discussed.     

Contrasting effects of immunosuppression on Theiler''s virus infection in mice.

In the present study, cyclophosphamide and rabbit anti-mouse thymocyte serum were used to immunosuppress SJL/J mice infected with Theiler's mouse encephalomyelitis virus (TMEV) in order to delineate the potential mechanism(s) of virus-induced cellular injury in this infection. Whereas both immunosuppressive agents produced a significant increase in mortality, this treatment had differing effects on the pathological involvement of gray and white-matter structures in the central nervous system. The central nervous system of immunosuppressed TMEV-infected mice had increased microglial cell proliferation and neuronal necrosis, longer maintenance of high virus levels and spread of virus antigen to involve the neocortex and hippocampal complex. These observations indicate that TMEV causes a cytolotic infection of neurons and possibly other cells in gray matter. In contrast, immunosuppression produced a dramatic reduction in mononuclear inflammatory cells in the leptomeninges and spinal cord white matter of infected mice and prevented demyelination. Further, virus antigen was not detected in the leptomeninges and white matter of immunosuppressed and infected mice. These findings suggest that demyelination of TMEV infection is immune mediated.     

Theiler's virus-induced demyelination in mice immunosuppressed with anti-IgM and in mice expressing the xid gene

Intracerebral infection with Theiler's murine encephalomyelitis virus produces chronic immune-mediated demyelination in susceptible strains of mice. We examined the role of Ig in the pathogenesis of demyelination. In susceptible SJL/J mice (H-2s), suppression of B cell responses with IgG fraction of goat anti-mu (anti-mu IgG) from birth resulted in increased numbers and severity of demyelinating lesions in the spinal cord 35 days after infection. In contrast, treatment of resistant C57BL/10 (H-2b), C57BL/6 (H-2b), or B10.D2 (H-2d) mice with anti-mu IgG had no apparent effect since these mice did not develop demyelination or inflammation in the spinal cord following infection. Similar results were obtained with certain strains of B-cell deficient mice that exhibit the xid gene mutation. Male CBA/NJ (xid) showed increased meningeal inflammation and demyelination compared to male CBA/J mice. However, B6.CBAN, C3.CBAN, or C.CBAn mice showed no or minimal evidence of demyelination despite the presence of the xid mutation. In the SJL/J mouse, the majority of the humoral immune response to virus antigen was restricted to the IgG2b and IgM isotypes. These data indirectly support the hypothesis that immunoglobulins protect partially against development of virus-induced demyelination in susceptible but not resistant animals. In addition, the data argue strongly against the hypothesis that TMEV-induced demyelination is mediated predominantly by humoral autoimmune or humoral viral immune mechanisms.     

Mechanism of Theiler's virus-induced demyelination in nude mice

In its natural murine host, infection with Theiler's murine encephalomyelitis virus (TMEV) produces a chronic, progressive demyelinating disease. To help elucidate the role of host immune mechanisms involved in demyelination, we studied TMEV infection in Nude mice. These animals demonstrated rising titers of infectious virus within the central nervous system and failed to produce anti-TMEV antibody. Neurologic signs including the development of severe hind limb paralysis were evident approximately 2 weeks postinfection with most animals succumbing within the first month. Immunoperoxidase studies demonstrated viral antigen in the cytoplasm of neurons and glial cells for the entire period of observation. Plaques of demyelination associated with scanty inflammatory infiltrates were present in the spinal cord by 14 days postinfection. Electron microscopic studies of the involved white matter revealed numerous degenerating glial cells, many of which contained paracrystalline arrays of picornavirus within their cytoplasm. Some of the infected glial cells were identified as oligodendrocytes by demonstrating their myelin-plasma membrane connections. The studies indicate that in Nude mice TMEV causes a lytic infection of oligodendrocytes producing demyelination independent of the T lymphocyte immune system.     

Theiler's murine encephalomyelitis virus induces tumour necrosis factor-alpha in murine astrocyte cell cultures.

Cytokines have been postulated to exert an important modulatory and recruiting role in demyelination induced by Theiler's murine encephalomyelitis virus (TMEV) in SJL/J mice. Using a cytolytic bioassay and ELISA, we have detected and quantified a cytokine, tumour necrosis factor-alpha (TNF-alpha), in supernatants from astrocyte cultures infected in vitro with TMEV. TNF was detected only after TMEV-specific infection of astrocyte cultures (approximately 200-400 U/ml). In vitro TNF synthesis appeared in a dose- and time-dependent manner and was produced by both SJL/J (a strain susceptible to TMEV-induced demyelination) and BALB/c (a resistant strain) astrocytes. The precise nature of TNF activity was further assessed by fast protein liquid chromatography (FPLC) and antibody neutralization. These results indicate an active role for astrocytes as accessory immune cells in our experimental model for multiple sclerosis.     

Neutralization of chemokines RANTES and MIG increases virus antigen expression and spinal cord pathology during Theiler's virus infection

The role of chemokines during some viral infections is unpredictable because the inflammatory response regulated by these molecules can have two, contrasting effects-viral immunity and immunopathologic injury to host tissues. Using Theiler's virus infection of SJL mice as a model of this type of disease, we have investigated the roles of two chemokines-regulated on activation, normal T cell-expressed and secreted (RANTES) chemokine and monokine induced by IFN-gamma (MIG)-by treating mice with antisera that block lymphocyte migration. Control, infected mice showed virus persistence, mild inflammation and a small degree of demyelination in the white matter of the spinal cord at 6 weeks post-infection. Treatment of mice with RANTES antiserum starting at 2 weeks post-infection increased both viral antigen expression and the severity of inflammatory demyelination at 6 weeks post-infection. MIG antiserum increased the spread of virus and the proportion of spinal cord white matter with demyelination. Overall, viral antigen levels correlated strongly with the extent of pathology. At the RNA level, high virus expression was associated with low IL-2 and high IL-10 levels, and RANTES antiserum decreased the IL-2/IL-10 ratio. Our results suggest that RANTES and MIG participate in an immune response that attempts to restrict viral expression while limiting immunopathology and that anti-chemokine treatment poses the risk of exacerbating both conditions in the long term.     

Ultrastructural immunohistochemical localization of virus in acute and chronic demyelinating Theiler's virus infection.

Mice experimentally infected with Theiler's murine encephalomyelitis virus (TMEV) develop a persistent infection of the central nervous system (CNS). The most striking feature of this infection is the occurrence of inflammatory primary demyelination in the spinal cord white matter. The pathogenesis of myelin degeneration in this model has not been clarified, but morphologic and immunologic data suggest that the host immune response plays a major role in the production of myelin injury. Because of low virus titers in infected adult mice and of the small size of TMEV, virus particles have never been observed in this demyelinating model. Yet elucidation of the types of cells in the CNS supporting virus replication would be important for a better understanding of both virus persistence and virus-induced demyelinating pathology. The present paper is a sequential study of the localization of TMEV in the spinal cord in infected mice by ultrastructural immunohistochemical techniques. Results indicate that virus replication is mainly in neurons during the acute phase of the disease, while in the chronic phase viral inclusions are mainly found in macrophages in and around demyelinating lesions. Other cells are also infected, but to a lesser degree. In the neuronal system both axoplasmic and dendritic flow appear to facilitate the spread of virus in the CNS. In macrophages, the presence of virus particles and the association of virus with altered components of the cytoskeleton support active virus production rather than simple internalization. The macrophage appears to play an important role in both the establishment of virus persistence and in the process of demyelination in this animal model.     

Pro-inflammatory functions of astrocytes correlate with viral clearance and strain-dependent protection from TMEV-induced demyelinating disease

Intracerebral infection of susceptible strains of mice, e.g. SJL/J, with Theiler's murine encephalomyelitis virus (TMEV) leads to a persistent CNS infection accompanied by development of a chronic-progressive inflammatory CNS autoimmune demyelinating disease which is clinically and pathologically similar to human multiple sclerosis. In contrast, resistant strains of mice, e.g. C57BL/6 (B6), effectively clear TMEV from the CNS and do not develop demyelinating disease. Although CD8(+) T cells are crucial for viral clearance in B6 mice, SJL mice also mount potent CD8(+) T cell responses against virus, thus the reason for the viral persistence in the CNS in these mice is unclear. Here, we examined innate anti-viral responses of CNS-resident astrocytes as a potential determinant of viral persistence and disease susceptibility. We demonstrate that B6 astrocytes produce significantly higher levels of cytokines, chemokines and adhesion molecules in response to TMEV infection, or stimulation with IFN-gamma and TNF-alpha or poly I:C than SJL mice. In addition, TMEV more effectively induces MHC I molecules on B6 astrocytes than SJL, corresponding with an increased ability to activate TMEV-specific CD8(+) T cells directly ex vivo. These results suggest that enhanced anti-viral responses of B6 astrocytes contribute to the ability of these mice to clear TMEV from the CNS and therefore to their resistance to the development of autoimmune demyelinating disease.     

Selected mutants of mouse hepatitis virus type 4 (JHM strain) induce different CNS diseases. Pathobiology of disease induced by wild type and mutants ts8 and ts15 in BALB/c and SJL/J mice.

The model system of central nervous system (CNS) disease induced by mouse hepatitis virus type 4 (MHV-4) is explored by comparison of wild type (wt) MHV-4 and two temperature-sensitive (ts) mutants, designated ts8 and ts15, in BALB/c and SJL/J mice. In BALB/c mice, 3 plaque-forming units (PFU) of wt MHV-4 given intracerebrally caused fatal encephalomyelitis in all mice by 7 days after infection, with spread of virus outside the CNS, especially to liver. In SJL/J mice, 3 PFU of wt virus was cleared within 2-3 days, with little spread, and up tp 100 PFU failed to cause fatal encephalomyelitis. However, larger amounts of virus, like 1000 PFU, caused fatal encephalomyelitis in SJL/J mice. In contrast, 10(4) PFU of MHV-4 ts8 did not cause death in either BALB/c or SJL/J mice, and persisted in the CNS of both strains while retaining its ts phenotype. There was significatnly less spread of virus outside the CNS. BALB/c mice usually showed demyelination, remyelination, and recurrent demyelination with ts8, while SJL/J mice only rarely had lesions. Intracerebral inoculation with 10(4) PFU of MHV-4 ts15 was associated with a persistent infection in CNS and liver of BALB/c mice; however, only occasional demyelination and hepatic lesions occurred. TS15 did not cause death in either BALB/c or SJL/J mice and did not cause histopathologic injury in SJL/J mice.     

Role of T cells in resistance to Theiler's virus infection.

Intracerebral infection of C57BL/10SNJ mice with Theiler's virus results in acute encephalitis with subsequent virus clearance and absence of spinal cord demyelination. In contrast, infection of SJL/J mice results in acute encephalitis, virus persistence, and immune-mediated demyelination. These experiments examined the role of T-cell subsets in the in vivo immune response to Theiler's virus in resistant C57BL/10SNJ mice. Depletion of T-cell subsets with monoclonal antibodies (mAbs) directed at CD3 (pan-T-cell marker), CD4+ (class II-restricted) or CD8+ (class I-restricted) T cells resulted in increased frequency of paralysis and death as a result of acute encephalitis. Neuropathologic studies 10 days after infection demonstrated prominent necrosis, primarily in the pyramidal layer of hippocampus and in the thalamus of mice depleted of T-cell subsets. In immunosuppressed and infected C57BL/10SNJ mice, analysis of spinal cord sections 35 days after infection demonstrated small demyelinated lesions relatively devoid of inflammatory cells even though virus antigen could be detected by immunocytochemistry. Both CD4+ and CD8+ T cells are important in the resistance to infection with Theiler's virus in C57BL/10SNJ mice. However, subsequent spinal cord demyelination, to the extent observed in susceptible mice, depends on the presence of virus antigen persistence and a competent cellular immune response.     

Kallikrein 6 regulates early CNS demyelination in a viral model of multiple sclerosis

Kallikrein 6 (Klk6) is a secreted serine protease that is elevated in active multiple sclerosis lesions and patient sera. To further evaluate the involvement of Klk6 in chronic progressive demyelinating disease, we determined its expression in the brain and spinal cord of SJL mice infected with Theiler's murine encephalomyelitis virus (TMEV) and assessed the effects of Klk6-neutralizing antibodies on disease progression. Klk6 RNA expression was elevated in the brain and spinal cord by 7 days postinfection (dpi). Thereafter, Klk6 expression persisted primarily in the spinal cord reaching a peak of fivefold over controls at mid-chronic stages (60?dpi-120?dpi). Significant elevations in Klk6 RNA were also induced in splenocytes stimulated with viral capsid proteins in vitro and in activated human acute monocytic leukemia cells. Klk6-neutralizing antibodies reduced TMEV-driven brain and spinal cord pathology and delayed-type hypersensitivity (DTH) responses when examined at early chronic time points (40?dpi). Reductions in spinal cord pathology included a decrease in activated monocytes/microglia and reductions in the loss of myelin basic protein (MBP). By 180?dpi, pathology scores no longer differed between groups. These findings point to regulatory activities for Klk6 in the development and progression of central nervous system (CNS) inflammation and demyelination that can be effectively targeted through the early chronic stages with neutralizing antibody.     

Injection of the sciatic nerve with TMEV: a new model for peripheral nerve demyelination

Demyelination of the human peripheral nervous system (PNS) can be caused by diverse mechanisms including viral infection. Despite association of several viruses with the development of peripheral demyelination, animal models of the condition have been limited to disease that is either autoimmune or genetic in origin. We describe here a model of PNS demyelination based on direct injection of sciatic nerves of mice with the cardiovirus, Theiler's murine encephalomyelitis virus (TMEV). Sciatic nerves of FVB mice develop inflammatory cell infiltration following TMEV injection. Schwann cells and macrophages are infected with TMEV. Viral replication is observed initially in the sciatic nerves and subsequently the spinal cord. Sciatic nerves are demyelinated by day 5 post-inoculation (p.i.). Injecting sciatic nerves of scid mice resulted in increased levels of virus recovered from the sciatic nerve and spinal cord relative to FVB mice. Demyelination also occurred in scid mice and by 12 days p.i., hindlimbs were paralyzed. This new model of virus-induced peripheral demyelination may be used to dissect processes involved in protection of the PNS from viral insult and to study the early phases of lesion development.     

Initial capsid-specific CD4(+) T cell responses protect against Theiler's murine encephalomyelitisvirus-induced demyelinating disease

Central nervous system (CNS) infection by Theiler's murine encephalomyelitis virus (TMEV) causes an immune-mediated demyelinating disease similar to human multiple sclerosis in susceptible mice. To understand the pathogenic mechanisms, we analyzed the level, specificity, and function of CD4(+) Th cells in susceptible SJL/J and resistant C57BL/6 mice. Compared to resistant mice, susceptible mice have three- to fourfold higher levels of overall CNS-infiltrating CD4(+) T cells during acute infection. CD4(+) T cells in the CNS of both strains display various activation markers and produce high levels of IFN-gamma upon stimulation with anti-CD3 antibody. However, susceptible mice display significantly fewer (tenfold) IFN-gamma-producing Th1 cells specific for viral capsid epitopes as compared to resistant mice. Furthermore, preimmunization with capsid-epitope peptides significantly increased capsid-specific CD4(+) T cells in the CNS during the early stages of viral infection and delayed the development of demyelinating disease in SJL/J mice. This suggests a protective role of capsid-reactive Th cells during early viral infection. Therefore, a low level of the protective Th1 response to viral capsid proteins, in conjunction with Th1 responses to unknown epitopes may delay viral clearance in susceptible mice leading to pathogenesis of demyelination during acute infection, as compared to resistant mice.     

Theiler''s virus infection in mice: an unusual biphasic disease process leading to demyelination.

An unusual biphasic central nervous system disease developed in 3-week-old Swiss outbred mice after intracerebral inoculation of the DA strain of Theiler's murine encephalomyelitis virus. Nine to 20 days postinfection 86% of mice became paralyzed, and approximately one-half of these animals survived. During this period neuronal necrosis and microglial proliferation were seen in thalamus, brainstem, and spinal cord. There was an initial phase of virus growth in spinal cord followed by persistent infection at a lower concentration. Virus antigen was readily found in the cytoplasm of neurons by immunofluorescent staining early in the course of infection, whereas after 30 days there was a paucity of cells containing virus antigen which were present only in the spinal cord white matter. Between 1 and 5 months, an intense mononuclear inflammatory cell lesion evolved in the spinal cord leptomeninges and white matter, which coincided with a mild gait disturbance in some surviving mice, and patchy demyelination was found in areas of inflammation. The acute gray matter pathology would appear to be the result of direct virus lytic effect. Although the late white matter lesion culminating in demyelination probably represents a cytocidal infection similar to the situation that exists in certain picornavirus carrier culture systems, a virus-induced immunopathological process merits further study.