We are interested in the molecular basis of viral pathogenesis. Reoviruses cause a variety of diseases in neonatal mice and are therefore well-suited for these investigations. Moreover, the segmented nature of the reovirus double-stranded RNA genome allows production of """"""""reassortant viruses"""""""", which are powerful tools for identification of the viral genes that determine a particular phenotype, including specific disease patterns. A reovirus variant, 8B, is efficiently myocarditic in neonatal mice, unlike the two parent viruses is was derived from. Evidence suggests that this myocarditis is not immune-mediated (as has been proposed for coxsackievirus-induced myocarditis), but instead reflects a direct cytopathic effect of the virus on cardiac myocytes. The 8B-M1 gene segment (encoding a core protein not exposed on the virion surface) is required for this phenotype, apparently reflecting a critical mutation(s) in the gene. This represents the first insight into the genetic basis for virally-induced myocarditis, which is an important human disease. We propose to continue studies of 8B and related reovirus strains to address: 1) Parameters which are altered in cardiac myocytes and cardiac fibroblasts infected in vitro with 8B or the parental viruses. The viruses are cytopathic to these cells in a manner which parallels tissue necrosis in the heart. Infected cells will be probed for a) viral parameters (titer, nucleic acid and protein syntheses, virion stability), and b) cell parameters (frequency of infection, cytopathic effect, inhibition of macromolecular synthesis, production of interferon). 2) Relationship between parameters which are altered in infections in vitro, and myocarditis in vivo. Cardiac tissue from infected mice will be probed for a) quantity and quality of viral and cell transcription and translation products, b) relative quantities of virions and viral cores, and potentially c) quantity and location of interferon. 3) Characterization of other myocarditic reovirus strains. Parameters identified as important for 8B cytopathic effect in cultured cardiac myocytes (aim#1) and myocarditis in vivo (aim #2) will be investigated for other myocarditic reovirus strains, to see whether the mechanism for 8B-induced myocarditis is generalizable. 4) Genetic analyses of other myocarditic reovirus strains. Reassortant viruses will be used to determine whether the M1 gene is also critical for these myocarditis. 5) Role of the M1 gene in viral replication in the heart. The M1 gene is associated with viral replication in cardiac myocyte cultures (manuscript in press); does it also determine viral replication in the heart? Note that non-myocarditic reovirus strains can replicate to cardiac titers as high as myocarditic strains, so replication alone cannot explain the disease. 6) How can other reovirus genes modify the myocarditis? Preliminary data suggest a role for other reovirus genes in 8B-induced myocarditis.
