The long term goal of this project is to understand the molecular basis of some of the biological properties of the murine coronavirus, mouse hepatitis virus (HMV). Some strains of MHV cause persistent demyelinating disease in the central nervous system (CNS) of mice. This infection has been cited as a model for the human disease Multiple Sclerosis. MHV also readily establishes persistent infections in cultured cells. Thus, MHV presents a model system for the study of viral persistence, tissue tropism, and virus-induced demyelination. We propose to undertake a detailed analysis of the molecular biology of replication of this virus and then use this information to start to ask questions about the molecular basis of persistence and neurotropism. Molecular Studies: The sequences of the intergenic regions flanking the E1 and N genes (at the 3 prime end of the MHV-A59 genome) will be obtained by sequencing of cDNA clones. A comparison of these intergenic sequences with sequences on the 5 prime ends of the corresponding mRNAs will bear on the mechanism of synthesis of the subgenomic mRNAs. Negative strand RNA, presumably an intermediate in the replication of genome and transcription of mRNAs will be characterized as to size, kinetics of accumulation, association with positive strand RNA. The sequences at the termini of negative strand RNA will be compared to the terminal sequences of genome RNA to look for putative RNA polymerase initiation sites for replication. Sequence information will be obtained for regions 5 prime to the N and E1 genes by stepwise cDNA cloning across the genome. As a long term goal we plan to clone the entire genome and generate sequences for the genes involved in tropism and persistence. Biological Studies: We will use the information obtained above as well as the reagents generated to start to address the biological questions. Persistently infected cells as well as CNS samples from persistently infected mice will be analyzed with a focus on changes in viral genome expression associated with persistence. DNA probes homologous to human and murine coronaviruses will be used to probe for viral sequences in human tissues from MS, hepatitis and enteric disease patients.