Indirect evidence implicates a virus in the etiology of Multiple Sclerosis (MS) and more direct evidence shows that viruses can cause other human demyelinating diseases. Coronavirus, mouse hepatitis virus (MHV), strain A59 infection of weanling mice is a good animal model system for the study of virus induced demyelination. After the initial acute encephalitis, virus becomes impossible to detect during the chronic stages of disease while viral nucleic acids may be detected in white matter by in situ hybridization. Furthermore, MHV-A59 causes a persistent, productive, but non-lytic infection in primary glial cells in culture. The long term goal of this project is to determine the molecular basis for MHV-A59 persistence in glial cells in culture and in the mouse central nervous system (CNS) and to determine the relationship between viral persistence and chronic demyelination. In this proposal we plan to study the molecular basis for MHV-A59 persistence both in vitro in glial cell cultures and in vivo in C57BL/6 mice. More specifically we will: 1) characterize viral RNA expression using Northern blot analysis and in situ hybridization during persistent infection in glial cell cultures and the murine CNS and compare expression with that during lytic infection in fibroblasts; 2) compare the expression of viral structural and non-structural proteins during infection in glial cells and fibroblasts using immunoprecipitation and Western immunoblots using antibodies directed against individual viral proteins; 3) compare variants isolated from persistently infected glial cells (PI variants) with viruses isolated from infected animals and with wild type MHV-A59 as far as cytopathology and viral gene expression in vitro and virus spread and tropism in vivo; 4) further characterize the PI variants that are both attenuated in animals and deficient in the ability to induce cell fusion, by cloning and expression of wild type and variant peplomer S proteins; 5) use polymerase chain reaction methodology to amplify portions of viral sequences present during chronic infection in the mouse to determine if there are specific changes in viral gene expression that are associated with persistence in vivo.
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