A lentiviral expression system demonstrates that L* protein of Theiler's murine encephalomyelitis virus (TMEV) has an anti-apoptotic effect in a macrophage cell line

DA subgroup strains of TMEV persist in the CNS of infected mice and induce demyelination. The mechanism(s) of virus persistence and demyelination remains unknown. DA subgroup strains synthesize a 17-kDa protein, called L*, from an initiation site out-of-frame with the polyprotein. The previous study using a mutant virus, DAL*-1 (in which the L* AUG is substituted by an ACG) showed that L* has an anti-apoptotic effect in a macrophage cell line, P388D1. Therefore, we established P388D1 cells that continuatively express L*, in order to confirm its role in TMEV-induced apoptosis. The anti-apoptotic activity of L* may be important in TMEV pathogenesis.     

A lentiviral expression system demonstrates that L* protein of Theiler's murine encephalomyelitis virus (TMEV) is essential for virus growth in a murine macrophage-like cell line

The DA subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) synthesize L* protein, which is translated out of frame with the polyprotein from an alternative AUG, 13 nucleotides downstream from the authentic polyprotein AUG. By a 'loss of function' experiment using a mutant virus, DAL*-1, in which the L* AUG is mutated to an ACG, L* protein is shown to play an important role in virus persistence, TMEV-induced demyelination, and virus growth in macrophages. In the present study, we established an L* protein-expressed macrophage-like cell line and confirmed the importance of L* protein in virus growth in this cell line.     

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.     

Monocytes/macrophages isolated from the mouse central nervous system contain infectious Theiler's murine encephalomyelitis virus (TMEV)

Knowledge of the cells in which Theiler's murine encephalomyelitis virus (TMEV) persists is crucial to understanding the pathogenesis of TMEV-induced demyelinating disease; however, it is still uncertain whether oligodendrocytes or macrophages are the primary target for persistence. In this study, mononuclear cells (MNC) isolated directly from central nervous system (CNS) inflammatory infiltrates of TMEV-infected mice on discontinuous Percoll gradients were found to contain infectious TMEV. Macrophages appeared to be the principal MNC infected as determined by two-color immunofluorescence. Infectious center assay and double immunostaining together indicated the presence and possible synthesis of TMEV in approximately 1 in 225 to 1 in 1000 CNS macrophages, with 1 to 7 PFU produced per macrophage. On the basis of these findings, limited replication in macrophages is consistent with the total CNS virus content detected at any time during the persistent phase of the infection as well as the slow pace of the infection.     

Association of L* protein of Theiler's murine encephalomyelitis virus with microtubules in infected cells

We used an antibody raised against a synthetic peptide corresponding to amino acid residues 70-88 for characterizing the L* protein of Theiler's murine encephalomyelitis virus (TMEV), which is only synthesized in DA subgroup strains from an alternative AUG and is out of frame with the viral polyprotein; evidence suggests that L* protein is critical to viral persistence, demyelination, and growth in murine macrophage cell lines. It was synthesized with kinetics similar to that of other viral proteins, although less in amount. After synthesis, it remained stable in the cytoplasm and was not incorporated into virions. Immunofluorescent staining and immunoblotting of microtubule preparations demonstrated that it is associated with microtubules. Expression of L* protein also demonstrated that the 5' one third of the coding region may be responsible for the association. The association of L* protein with microtubules may be important in the disease-inducing and in vitro characters of L* protein.     

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.     

Neonatal infection with the Daniels strain of Theiler's murine encephalomyelitis virus

The clinical and pathologic manifestation of Theiler's murine encephalomyelitis are age related. Animals infected during the first week of life die of a fulminant encephalitis analogous to human poliomyelitis. By contrast, animals infected within 2 and 4 weeks of age survive but develop chronic relapsing demyelination and persistent infection of the central nervous system. The neonatal infection results in widespread necrosis beginning with neuronal vacuolar degeneration followed by inflammatory infiltrates. Electron microscopy reveals paracrystalline arrays of 27-nm viral particles characteristic of picornaviruses within neurons and macrophages. In addition, oligodendrocytes show reactive changes and intracytoplasmic vacuoles. Immunoperoxidase studies show viral antigen primarily localized within neurons of the cerebral cortex, basal ganglia, hippocampus, and anterior horn cells. Viral antigen is found within the apical dendrites and axonal projections of hippocampal pyramidal cells suggesting that Theiler's murine encephalomyelitis may travel intraaxonally.     

Is Theiler's murine encephalomyelitis virus infection of mice an autoimmune disease?

Viruses can initiate disease by many different means. Direct viral, immune mediated and host factors all play important parts. Molecular mimicry or having cross-reacting determinants that result in immune responses which have the potential to cause damage can be incorporated into this framework. Here, autoimmune responses generated by virus infection have been presented in relation to these other parameters. The cross-reacting immune response originally generated by virus would have to be directed toward or involve a disease inducing site such as an EAE (encephalitogenic), thyroiditis, or diabetogenic site. If the cross-reaction took place at a nondisease inducing site, the ensuring immune response may result in the production of autoantibodies, however no disease would occur. In other systems autoantibodies can potentiate an ongoing inflammatory response. This may be the case that is described here with Theiler's murine encephalomyelitis virus infection. Lastly, viruses having common determinants with MHC determinants may modify immune responses leading to immunosuppression and allowing virus to persist. In addition, similar determinants may lead to disease by an alternative route. For example, we have described a region of human cytomegalovirus that has a common determinant with HLA DR beta chain. This region is associated with diabetes in humans (Todd et al. 1988). Thus, many factors are involved in the outcome of disease induction by viruses of which autoimmunity is one.     

Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. II. Survey of host immune responses and central nervous system virus titers in inbred mouse strains

Previous studies using mouse strains with limited genetic differences and H-2 haplotypes demonstrated that susceptibility to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease strongly correlated with chronically high levels of TMEV-specific delayed-type hypersensitivity (DTH), but not with TMEV-specific T cell proliferation (Tprlf), serum antibody responses, or with CNS virus titers. To determine if this correlation would be supported by analysis of these parameters in a more thorough genetic survey, ten inbred mouse strains, representing a wide variety of genetic backgrounds and H-2 haplotypes, were inoculated intracerebrally (i.c.) with the BeAn strain of TMEV. Significant TMEV-specific DTH was observed in all highly susceptible strains, but was not detectable in intermediate and resistant strains. TMEV-specific serum antibody titers also appeared to correlate with susceptibility to demyelinating disease, however even resistant strains had high antibody responses. Significant differences in CNS TMEV titers existed between strains, but did not correlate with disease susceptibility. DTH and Tprlf responses were observed in 3/4 resistant strains following peripheral immunization with UV-inactivated TMEV indicating that most resistant strains are genetically capable of mounting virus-specific cell-mediated immune (CMI) responses. The data extends our knowledge of host immune responses and virus titers in many different inbred mouse strains persistently infected with TMEV, supports the hypothesis that the demyelination in highly susceptible mice involves a TMEV-specific DTH response, and suggests that the genetic ability to mount specific DTH responses is necessary, but not sufficient for development of the demyelinating disease.     

Glucocorticoid exposure alters the pathogenesis of Theiler's murine encephalomyelitis virus during acute infection

Previous research has shown that chronic restraint stress exacerbates Theiler's virus infection, a murine model for CNS inflammation and multiple sclerosis. The current set of experiments was designed to evaluate the potential role of glucocorticoids in the deleterious effects of restraint stress on acute CNS inflammatory disease. Exposure to chronic restraint stress resulted in elevated levels of corticosterone as well as increased clinical scores and weight loss (Experiment 1). In addition, corticosterone administration alone exacerbated behavioral signs of TMEV-induced sickness (i.e. decreased body weight, increased symptoms of encephalitis, and increased mortality) and reduced inflammation in the CNS (Experiment 2). Infected subjects receiving exogenous corticosterone showed exacerbation of acute phase measures of sickness and severe mortality as well as decreased viral clearance from CNS (Experiment 3). These findings indicate that corticosterone exposure alone is sufficient to exacerbate acute CNS inflammatory disease.     

Theiler's murine encephalomyelitis virus infection induces early expression of c-fos in astrocytes.

We have determined whether Theiler's murine encephalomyelitis virus (TMEV), a picornavirus that produces demyelination in genetically susceptible strains of mice, induces c-fos in pure quiescent cultures of mouse brain astrocytes. As observed in Northern blots, the expression of this immediate early gene increases in a dose-dependent manner, with its expression peaking at a multiplicity of infection of 100. The expression of c-fos is transient, peaking after 30 min and disappearing 2 h after infection. The virus is quickly internalized at 37 degrees C upon binding to its specific receptor located at the cell surface and is actively replicated in the cytoplasm of the astrocytes, as demonstrated by FACS flow cytometry. Using the same technique, nuclear translation of c-fos mRNA is also shown. The specificity of viral induction is demonstrated by its neutralization with TMEV-specific antibodies and by the fact that only viral particles and not purified protein components VP1, VP2, and VP3 induced proto-oncogene expression. This rapid induction of c-fos in astrocytes could be the first stage in the infection of these central nervous system cell populations by TMEV. The biological relevance of these findings is assessed by the demonstration of c-fos activation after viral infection in vivo.     

Serological evidence that Mus musculus is the natural host of Theiler's murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) infection is maintained in mouse colonies by fecal-oral spread (with no apparent role for persistent central nervous system infection) from an acutely infected animal to another. Therefore, serological methods offer the principal way to assess infection in mice and related rodent populations. Infection of mouse colonies with TMEV appears to be worldwide, yet no systematic serologic studies have been reported. In this study, enzyme-linked immunoassay and neutralization analysis of sera from feral Mus musculus obtained from four locations in the United States and one in Russia revealed antibodies to purified TMEV and two linear viral peptide epitopes in more than 50% of the sera derived from the five different locations. A similar analysis of sera from 26 species of related rodents trapped at multiple locations in North America and Europe indicated the presence of anti-TMEV antibodies only in a small proportion of water and bank voles that belong to a different subfamily. These results indicate that Mus musculus is the natural host of TMEV.     

Split tolerance of Thl and Th2 cells in tolerance to Theiler's murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) produces a chronic, inflammatory demyelinating disease in susceptible mouse strains that is used as a model for multiple sclerosis. Because disease susceptibility correlates temporally with the development of virus-specific delayed-type hypersensitivity (DTH) responses, we studied methods and mechanisms by which virus-specific DTH could be specifically inhibited. The intravenous injection of UV-inactivated TMEV coupled to syngeneic splenocytes via a carbodiimide linkage (TMEV-SP), prior to immunization, induced a significant degree of tolerance in virus-specific helper (T_h) cells as determined by decreased DTH and T cell proliferative responses, and decreased interleukin (IL)-2 and interferon (IFN)-Y protein and mRNA levels. In contrast to the reduced levels of T_hl-specific lymphokine mRNA levels, IL-4-specific mRNA levels in response to virus stimulation were not affected in tolerant mice. Surprisingly, the total anti-TMEV antibody response in DTH tolerant mice was enhanced 20-100-fold over sham-tolerized controls and was composed of reduced levels of anti-virus IgG2a, but dramatically increased levels of anti-virus IgGl. The “split-tolerance” was antigen specific, dependent on the concentrations of TMEV and carbodiimide used in the coupling procedure, and varied with the number of coupled syngeneic splenocy tes administered. The fixative effects of carbodiimide on antigen-presenting function were necessary for the induction of DTH tolerance with TMEV-SP, since intravenous administration of virus coupled to splenocytes via a biotin-avidin linkage led to enhanced virus-specific antibody responses, but was unable to inhibit DTH unless concomitantly fixed with carbodiimide. Collectively, the data indicate that T_hl cells (mediating DTH, IL-2 and IFN-γ production, and helper function for IgG2a production) were specifically anergized, with concomitant stimulation of T_h2 cells (producing IL-4 and mediating helper function for IgGl antibody production).     

Altered cell growth and morphology in a BHK-21 cell mutant that lacks a receptor for Theiler's murine encephalomyelitis virus

The receptor for Theiler's murine encephalomyelitis virus (TMEV) remains unknown. In vitro, BHK-21 cells are permissive to infection by TMEV. Selecting mutants of BHK-21 cells produced a cell line (BHKR-) resistant to infection by TMEV. Viral persistence was ruled out by immunofluorescent staining for viral antigens. BHKR- cells were nonpermissive to infection even at high multiplicities of infection. In contrast, cells were able to support one round of virus replication when transfected with infectious TMEV RNA. Binding studies indicated that TMEV was unable to attach to these cells. These data are consistent with the BHKR- cells lacking a receptor for TMEV. Interestingly, BHKR- cells were larger in size and had a significant lag in growth after subculture versus BHK-21 cells. This suggests that the TMEV receptor on BHK-21 cells could play an important role in cell growth and morphology under physiologic conditions. BHKR- cells should facilitate the search for TMEV receptors.     

Generation and characterization of a polyclonal antibody for the detection of Theiler's murine encephalomyelitis virus by light and electron microscopy

The BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) causes a demyelinating leukomyelitis in mice, which serves as an important animal model for multiple sclerosis in humans. The present report describes the generation and characterization of a TMEV-specific polyclonal antibody by immunization of rabbits with purified TMEV of the BeAn strain. The specificity of the antibody was confirmed by Western blotting and sequence analysis of the recognized antigen by high resolution mass spectrometry. The presence of TMEV-specific polyclonal antibodies in post-immunization sera was tested on TMEV-infected L-cells (murine lung tumor cell line) using an immunofluorescence assay. Additionally, the rabbit serum enabled virus detection in formalin-fixed and paraffin-embedded TMEV-infected BHK₂₁ cell pellets and brain tissue of TMEV-infected mice by immunohistochemistry. Immune electron microscopy revealed colloid gold-labeled picornavirus-typical paracrystalline arrays and non-aggregated viral particles of TMEV-infected BHK₂₁ cells. The present report demonstrates the applicability of the generated marker for investigating TMEV cell tropism and viral spread at a cellular and subcellular level in future studies.     

Biochemical analysis of DA strain of Theiler's murine encephalomyelitis virus obtained directly from acutely infected mouse brain.

Growth and neurovirulence of a number of neurotropic viruses show pronounced differences after passage in cell culture compared with continued in vivo passage in the central nervous system. The DA strain of Theiler's murine encephalomyelitis virus provides a model for studying these issues since DA virus grown in mouse brain produces acute neuronal disease in weanling mice, but tissue culture-passed DA virus does not. In addition, DA virus grown in mouse brain has a greater 50% mouse lethal dose/50% tissue culture infective dose ratio than tissue culture-passed DA virus. Comparison of these viruses required the analysis of virus purified directly from infected mouse brain, without tissue culture passage. Capsid proteins from DA virus grown in mouse brain were resolved on sodium dodecyl sulfate-polyacrylamide gels and shown to have the same profile as tissue culture-passed DA virus. Viral RNAs were the same size, with no evidence of defective interfering particle production. Two-dimensional gels of in vitro-labeled RNase T1-digested RNA showed that virus variants were more apparent during acute in vivo passage. These genomic differences may be critical in determining the biological behavior of the virus.     

Apoptosis of infiltrating T cells in the central nervous system of mice infected with Theiler's murine encephalomyelitis virus

Theiler murine encephalomyelitis virus (TMEV), DA strain, induces in susceptible strain of mice a biphasic disease consisting of early acute disease followed by late chronic demyelinating disease. Both phases of the disease are associated with inflammatory infiltrates of the central nervous system (CNS). Late chronic demyelinating disease induced by TMEV serves as an excellent model to study human demyelinating disease, multiple sclerosis. During early acute disease, the virus is partially cleared from the CNS by CD3(+) T cells. These T cells express Fas, FasL, negligible levels of Bcl-2 proteins and undergo activation-induced cell death as determined by TUNEL assay leading to resolution of the inflammatory response. In contrast, during late chronic demyelinating disease, and despite dense perivascular and leptomeningeal infiltrates, only very few cells undergo apoptosis. Mononuclear cells infiltrating the CNS express Bcl-2. It appears that the lack of apoptosis of T cells during late chronic demyelinating disease leads to the accumulation of these cells in the CNS. These cells may play a role in the pathogenesis of the demyelinating disease.     

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.