Pathogenesis of coxsackievirus B3 induced myocarditis and systemic disease: Viral localization, direct injury and activation of host cell death machinery

Coxsackievirus B3, a cytopathic virus in the family Picomaviridae , induces degenerative changes in host cell morphology following infection. Work presented in this thesis will examine the nature of these cytopathic changes in vivo in myocarditis susceptible and resistant mice, and in vitro in HeLa cells, a common cell line used to dissect coxsackievirus lifecycle and host interaction. In mice, CVB3 infection caused a widespread systemic disease including infection of the heart, pancreas, liver, brain, spleen, salivary glands, lung and kidney. Adjacent sections stained for viral RNA by in situ hybridisation and tissue injury by TUNEL have demonstrated that infected cells are directly injured prior to activation of the cellular arm of the immune system. In the lymphoid organs, there is increased apoptosis that may be responsible for the lymphoid involution characteristically noted early following infection. The association of virus with lymphocytes may interfere with the normal immune response. There is a depression of IL-2 and IL-4 protein levels in infected mice as compared to sham treated animals. In HeLa cells, multiple caspases are processed and activated following the release of cytochrome c from the intermitochondrial space to the cytosol. Caspase-specific substrates are cleaved, but these events are not solely responsible for the degenerative morphologic changes noted following infection. Using ZVAD.fmk or cells stably transfected with Bcl-2 and BCI-x_L, it was further demonstrated that cytopathic effect is not caused by caspase activation. Activation of caspases, however, plays a role in loss of cell viability and the release of the progeny virus from within the cell. Cytopathic effect is probably due to viral protease mediated degradation of host cell structural proteins including focal adhesion kinase that is cleaved by a protease not inhibited by ZVAD.fmk. cDNA microarrays were used to profile the host gene response in the myocardium at days 3, 9, and 30 post infection to dissect the various stages of disease progression, including, direct viral injury, host specific immune response and viral clearance, and late stage cardiac remodelling. From a limited repertoire of genes, we identified 169 known genes that were significantly up or down regulated at one or more of the three timepoints. This study was one of the first in vivo gene expression models examining cardiac disease progression in mice over time. Direct extensions of this thesis includes interrogation of direct host and viral protein-protein interactions, examination of mitogen activated protein kinase signalling pathways and relationship to cell viability and virus replication, and further dissection of the effect of viral proteases on degradation of various host cellular proteins and effect on host cell homeostasis.