THEILERS DEMYELINATION--ROLE OF M0S AND OLIGODENDROCYTES

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The goal of the proposed research is to elucidate the cellular sites and molecular basis of viral persistence in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. TMEV-induced demyelinating disease is a highly relevant model system for multiple sclerosis (MS) in which epidemiologic evidence strongly supports involvement of an environmental factor, most likely a virus infection. The strengths of the Theiler's model are the facts that the virus is a natural pathogen of mice (therefore the model is not contrived), the chronicity of the CNS disease, and the many clinical, pathological, immunological and genetic parallels with MS. In this infection, myelin breakdown appears to be immune-mediated rather than due to a cytolytic effect of the virus on oligodendrocytes; however, we now believe that both mechanisms contribute to the pathology. It has been shown that TMEV persistence is necessary to sustain the pathologic process, and that a predominant viral antigen (Ag) load resides in macrophages (Mphis). Mphis therefore appear to be the preferential site (target) for viral persistence. The fact that TMEV multiplication in Mphis is restricted is consistent with the idea that RNA viruses persist only under restricted conditions of virus growth. In contrast, some oligodendrocytes are also infected (during persistence), but the extent of the infection is disputed. As a result of viral persistence, virus-specific CD4+ T cell responses to viral epitopes are sustained at high levels. TMEV-specific delayed-type hypersensitivity (DTH) mediated by major histocompatibility class (MHC) II-restricted CD4+ Th1 T cells (which are associated with immunity to viruses) plays an immunopathologic role in demyelination. The following studies will determine: (1) The number of TMEV RNA molecules (genomes) synthesized in the CNS of mice over time (acute and persistent phases) by competitive reverse transcription-polymerase chain reaction (cRT-PCR) and real-time quantitative RT-PCR, and the ratio of positive to negative viral RNA strands. (2) The percent of infected Mphis, astrocytes and oligodendrocytes isolated on Percoll gradients from the CNS of mice inoculated ic with BeAn virus positive for infectious centers, viral Ag and viral genomes. (3) The effect of altered viral clearance on the cellular sites of TMEV persistence in resistant strains of immunodeficient mice. (4) The kinetics of BeAn virus growth in three murine oligodendrocyte cell lines and primary murine astrocyte cultures.