Theiler's virus-associated antigens on the surfaces of cultured glial cells

Infection of the central nervous system by Theiler's murine encephalomyelitis virus (TMEV), a picornavirus, produces chronic demyelinating disease in susceptible mice. In this immunoelectron microscopic study of TMEV infection of neonatal mouse brain cells in culture, TMEV antigen was found on the surfaces of infected oligodendrocytes and astrocytes by labeling with hyperimmune serum from TMEV-infected mice or with rabbit antiserum to purified inactivated DA strain TMEV. Brain-derived macrophages had no TMEV-specific antigen on their surfaces and were not able to maintain productive TMEV infection, even though TMEV antigen was present in the cytoplasm. The presence of TMEV antigens on the surfaces of oligodendrocytes (myelin-producing cells) was unexpected because picornaviruses are nonenveloped viruses and do not bud from cell surfaces. The finding is consistent with the hypothesis that demyelination follows damage of infected oligodendrocytes by immune cells or immunoglobulins that recognize surface virus antigen.     

Schwann cell remyelination and recurrent demyelination in the central nervous system of mice infected with attenuated Theiler's virus.

Theiler's murine encephalomyelitis virus (TMEV) infection produces a chronic demyelinating disease in mice, and myelin breakdown appears to be immune-mediated. By using an attenuated TMEC strain, WW virus, to infect mice, the course of the disease was slowed and the severity of the inflammatory and glial responses were reduced. In this circumstance, most of the demyelinating lesions showed extensive remyelination, predominantly by Schwann cells. In addition, it was demonstrated that there was recurrent demyelinating activity in the central nervous system (CNS) of infected animals. It is suggested that the rapidity and intensity of demyelinating lesions may influence the potential for remyelination and that Schwann cell participation may be a more important mechanism of myelin repair than it is now thought to be. The fact that there is a recurrent demyelination in TMEV infection increases its relevance as an experimental animal model for multiple sclerosis.     

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.     

Theiler's murine encephalomyelitis: a model of demyelination and persistence of virus

Theiler's murine encephalomyelitis virus (TMEV) causes immune-mediated demyelination in susceptible mice which is similar to human demyelinating disorders such as multiple sclerosis. In addition, the picornavirus persists within the central nervous system throughout the course of the chronic demyelinating disease. This article reviews the neuropathology, virology, immunology, and molecular biology of the model system. We analyze the possible mechanisms by which this virus induces demyelination and persists in the nervous system. Finally, we provide a hypothesis that the specificity of primary white matter destruction in the TMEV model depends on immune-sensitized cells which interact with viral antigen plus major histocompatibility complex (MHC) antigens on the surfaces of oligodendrocytes or myelin sheaths.     

Leader (L) and L* proteins of Theiler's murine encephalomyelitis virus (TMEV) and their regulation of the virus' biological activities

Theiler's murine encephalomyelitis virus (TMEV) is divided into two subgroups on the basis of their different biological activities. GDVII subgroup strains produce fatal poliomyelitis in mice without virus persistence or demyelination. In contrast, TO subgroup strains induce demyelinating disease with virus persistence in the spinal cords of weanling mice. Two proteins, whose open reading frames are located in the N-terminus of the polyprotein, recently have been reported to be important for TMEV biological activities. One is leader (L) protein and is processed from the most N-terminus of the polyprotein; its function is still unknown. Although the homology of capsid proteins between DA (a representative strain of TO subgroup) and GDVII strains is over 94% at the amino acid level, that of L shows only 85%. Therefore, L is thought to be a key protein for the subgroup-specific biological activities of TMEV. Various studies have demonstrated that L plays important roles in the escape of virus from host immune defenses in the early stage of infection. The second protein is a 17–18 kDa protein, L*, which is synthesized out-of-frame with the polyprotein. Only TO subgroup strains produce L* since GDVII subgroup strains have an ACG rather than AUG at the initiation site and therefore do not synthesize L*. 'Loss and gain of function' experiments demonstrate that L* is essential for virus growth in macrophages, a target cell for TMEV persistence. L* also has been demonstrated to be necessary for TMEV persistence and demyelination. Further analysis of L and L* will help elucidate the pathomechanism(s) of TMEV-induced demyelinating disease.     

Clearance of Theiler's virus infection depends on the ability to generate a CD8+ T cell response against a single immunodominant viral peptide

Theiler's murine encephalomyelitis virus (TMEV) induces a chronic demyelinating disease in the central nervous system of susceptible mice. Resistance to persistent TMEV infection maps to he D locus of the major histocompatibility complex suggesting a prominent role of antiviral CTL in the protective immune response. Introduction of the D(b) gene into the FVB strain confers resistance to this otherwise susceptible mouse line. Infection of the FVB/D(b) mouse with TMEV provides a model where antiviral resistance is determined by a response elicited by a single class I molecule. Resistant mice of the H-2(b) haplotype mount a vigorous H-2D(b)-restricted immunodominant response to the VP2 capsid protein. To investigate the extent of the contribution of the immunodominant T cell population in resistance to TMEV, FVB/D(b) mice were depleted of VP2-specific CD8(+) T cells by peptide treatment prior to virus infection. Peptide-treated mice were not able to clear the virus and developed extensive demyelination. These findings demonstrate that the D(b)-restricted CD8(+) T cells specific for a single viral peptide can confer resistance to TMEV infection. Our ability to manipulate this cellular response provides a model for investigating the mechanisms mediating protection against virus infection by CD8(+) T cells.     

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.     

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.     

Analysis of murine hepatitis virus (JHM strain) tropism toward Lewis rat glial cells in vitro. Type I astrocytes and brain macrophages (microglia) as primary glial cell targets

The murine hepatitis virus, JHM strain, causes a relapsing subacute demyelinating encephalomyelitis in Lewis rats after intracranial infection. The disease process involves both virus persistence within glial cells and the induction of autoimmunological attack of myelin, however, the relative importance of these features involved in chronic relapsing demyelination remains to be determined. In this report, we analyze the tropism of JHM virus to various neural cell types present within primary Lewis rat central nervous system cultures. Infection of primary cultures with JHM virus revealed that type I astrocytes and brain macrophages are the initial target cells of infection and that the myelin-forming oligodendrocytes are comparatively resistant, becoming infected only rarely through virus mediated cell fusion with previously infected cells. In addition, infection of cultures after removal of oligodendrocytes by various means had no effect on the tropism of JHM virus for the cultures. Cytopathic effects of JHM virus proceed rapidly by cell fusion within the astrocyte-macrophage monolayer, leaving the oligodendrocyte population largely unaffected. Therefore, the highly selective infection of type I astrocytes and macrophages appears to form the basis of JHM virus neurotropism in Lewis rats. These results indicate that JHM virus infection of astrocytes and brain macrophages may be more important in inducing chronic relapsing demyelinating processes than direct infection of the myelin-forming oligodendrocytes. Other possible pathways leading to chronic demyelination in rats involving type I astrocytes and brain macrophages are 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.     

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.     

Protective and Detrimental Roles for Regulatory T Cells in a Viral Model for Multiple Sclerosis

Multiple sclerosis (MS) has been proposed to be an immune‐mediated disease in the central nervous system (CNS) that can be triggered by virus infections. In Theiler's murine encephalomyelitis virus (TMEV) infection, during the first week (acute stage), mice develop polioencephalomyelitis. After 3 weeks (chronic stage), mice develop immune‐mediated demyelination with virus persistence, which has been used as a viral model for MS. Regulatory T cells (Tregs) can suppress inflammation, and have been suggested to be protective in immune‐mediated diseases, including MS. However, in virus‐induced inflammatory demyelination, although Tregs can suppress inflammation, preventing immune‐mediated pathology, Tregs may also suppress antiviral immune responses, leading to more active viral replication and/or persistence. To determine the role and potential translational usage of Tregs in MS, we treated TMEV‐infected mice with ex vivo generated induced Tregs (iTregs) on day 0 (early) or during the chronic stage (therapeutic). Early treatment worsened clinical signs during acute disease. The exacerbation of acute disease was associated with increased virus titers and decreased immune cell recruitment in the CNS. Therapeutic iTreg treatment reduced inflammatory demyelination during chronic disease. Immunologically, iTreg treatment increased interleukin‐10 production from B cells, CD4⁺ T cells and dendritic cells, which may contribute to the decreased CNS inflammation.     

Class I-deficient resistant mice intracerebrally inoculated with Theiler's virus show an increased T cell response to viral antigens and susceptibility to demyelination

Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination in susceptible mouse strains. The histology of TMEV-induced demyelination is similar to that seen in patients suffering from multiple sclerosis. It was previously shown that the susceptibility of mice to TMEV-induced demyelination in certain strain combinations is closely associated with the major histocompatibility complex (MHC) class I locus. Here we examine disease susceptibility of β2-microglobulin (β2M)-deficient transgenic mice lacking class I expression and functional CD8⁺ T cells. In contrast to TMEV-infected parental C57BL/6 mice, the transgenics develop high levels of virus-specific DTH and T cell proliferation accompanied by an increased frequency of central nervous system (CNS) demyelinating lesions. However, clinical signs of demyelination were not noted. Neither antibody titer nor viral persistance were significantly affected in the β2M-deficient mice. These results suggest that in the absence of functional class I/CD8⁺ cells, the class II-restricted T cell response to TMEV is enhanced and CNS pathogenesis is heightened, although the level is not severe enough to result in clinical disease. When the TMEV-infected mice were subcutaneously immunized with virus, however, the β2M-deficient mice displayed clinical symptoms. Therefore, our results strongly suggest that CD8⁺ T cells do not directly contribute to CNS demyelination. In contrast, such T cells appear to be primarily involved in down-regulation of a potentially damaging CD4⁺ T cell response in resistant animals, although some of the T cells may play a role in clearing viral persistence in the CNS, resulting in the protection of the host from viral demyelination.     

Inhibition of Theiler's virus-induced demyelination in vivo by tumor necrosis factor alpha

Employing a murine model of multiple sclerosis which utilizes intracranial injection of Theiler's virus murine encephalomyelitis (TMEV) into SJL/J mice, we tested the potential role of tumor necrosis factor alpha (TNF-alpha) in ameliorating CNS demyelination. Infection with TMEV caused early grey matter inflammation (7 days post-infection) in the brain and spinal cord followed by chronic demyelination (35 days post-infection) in the spinal cord. Administration of recombinant human or mouse TNF-alpha starting 12 h prior to infection and then three times weekly had minimal effect on development of grey matter inflammation in the spinal cord. In contrast, TNF-alpha dramatically reduced demyelination present in spinal cord on days 14 and 35 after TMEV infection (P less than 0.01) when compared to controls. CNS virus titers of TMEV were not modified by TNF-alpha administration as measured on days 7, 14, and 35 following infection. In vivo administration of TNF-alpha inhibits TMEV-induced demyelination in susceptible SJL/J mice without affecting virus replication in the CNS.     

Oral administration of live virus protects susceptible mice from developing Theiler's virus-induced demyelinating disease

Intracerebral infection of susceptible mouse strains with Theiler's murine encephalomyelitis virus (TMEV) results in an immune-mediated demyelinating disease similar to human multiple sclerosis. TMEV infection is widely spread via fecal-oral routes among wild mouse populations, yet these infected mice rarely develop clinical disease. Oral vaccination has often been used to protect the host against many different infectious agents, although the underlying protective mechanism of prior oral exposure is still unknown. To understand the mechanisms involved in protection from demyelinating disease following previous oral infection, immune parameters and disease progression of mice perorally infected with TMEV were compared with those of mice immunized intraperitoneally following intracerebral infection. Mice infected perorally, but not intraperitoneally, prior to CNS viral infection showed lower chronic viral persistence in the CNS and reduced TMEV-induced demyelinating disease. However, a prolonged period of post-oral infection was necessary for effective protection. Mice orally pre-exposed to the virus displayed markedly elevated levels of antibody response to TMEV in the serum, although T cell responses to TMEV in the periphery were not significantly different between perorally and intraperitoneally immunized mice. In addition, orally vaccinated mice showed higher levels of early CNS-infiltration of B cells producing anti-TMEV antibody as well as virus-specific CD4(+) and CD8(+) T cells in the CNS compared to intraperitoneally immunized mice. Therefore, the generation of a sufficient level of protective immune responses appears to require a prolonged time period to confer protection from TMEV-induced demyelinating disease.     

Immunohistochemical detection of osteopontin in the spinal cords of mice with Theiler's murine encephalomyelitis virus-induced demyelinating disease

The spinal cords of mice that were infected with the BeAn 8386 strain of Theiler's murine encephalomyelitis virus (TMEV) were studied to elucidate the involvement of osteopontin in the course of TMEV-induced demyelination. Immunohistochemistry showed staining for osteopontin in the vessels of the normal spinal cords, and more intense immunoreactivity in the vessels within the demyelinating lesions. Intense osteopontin immunoreactivity was observed in the cell bodies, as well as in the extracellular space of the demyelinating lesions, where some glial cells, which included activated microglia/macrophages, were also immunopositive for osteopontin. These findings suggest that osteopontin is upregulated in the demyelinating spinal cord, and that osteopontin from either microglia or astrocytes may be involved in the chemotaxis of inflammatory cells and astrocytes, which ultimately leads to chronic inflammation and astrogliosis in this model system.     

Viral infection and dissemination through the olfactory pathway and the limbic system by Theiler's virus.

Theiler's murine encephalomyelitis virus (TMEV) infection of mice can produce a biphasic disease of the central nervous system (CNS). Most susceptible strains of mice survive the acute infection and develop a chronic demyelinating disease. In this report, we analyzed the routes of spread of TMEV within the CNS of nude mice and target sites eventually infected in the CNS. Compared to the immunocompetent mouse, in which an antiviral immune response is mounted but virus persists, the nude mouse develops a severe encephalomyelitis due to the lack of functional T lymphocytes and provides a useful model for the study of viral dissemination. We demonstrated, by immunohistochemistry, the presence of viral antigen in defined regions of the CNS, corresponding to various structures of the limbic system. In addition, we found a different time course for viral spread using two different sites of intracerebral inoculation, ie, via the olfactory bulb or the cortex. Limbic structures were rapidly infected following olfactory bulb infection and then showed a decrease in viral load, presumably due to loss of target neurons. Using either route of infection, the virus was able to disseminate to similar regions. These results indicate that limbic structures and their connections are very important for the spread of TMEV in the brain. In the spinal cord, not only neuronal but hematogenous pathways were suspected to be involved in the dissemination of Theiler's virus.     

Immunoglobulins stimulate central nervous system remyelination: electron microscopic and morphometric analysis of proliferating cells

Infection with the Daniel strain of Theiler's murine encephalomyelitis virus results in immunemediated primary demyelination in the spinal cords of susceptible SJL/J mice. Treatment of chronically infected mice (3 to 7 months) with purified immunoglobulins directed against spinal cord homogenate resulted in an increase in the number and average size of lesions that were undergoing remyelination by oligodendrocytes. In vivo autoradiography with [3H]thymidine demonstrated labeling of many lymphocytes in areas of demyelination and remyelination. A direct correlation was found between number of labeled lymphocytes infiltrating the lesion and size of demyelinating lesions. Remyelinated areas contained proliferating cells that resembled immature oligodendrocytes or progenitor glial cells morphologically. The number of labeled presumptive glial cells correlated with the area of remyelination. However, central nervous system remyelination occurred even in the presence of proliferating lymphocytes and astrocytic hypertrophy. In addition, treatment of normal uninfected SJL/J mice with antiserum to spinal cord homogenate resulted in increased numbers of proliferating cells in the spinal cord. These experiments suggest that immunoglobulins to a spinal cord antigen may induce proliferation of cells in the central nervous system to promote remyelination.