A significant fraction of sudden death in children and young adults is due to myocarditis, an inflammatory disease of the heart, most often caused by viral infection. Non-specific clinical signs make myocarditis difficult to diagnose in children and 83% of pediatric cases are not diagnosed at first presentation. There is an urgent need for new sensitive and non-invasive diagnostic modalities. In this study, supported by our preliminary findings, we will use a mouse model of neonatal viral myocarditis induced by mammalian reoviruses (REOV) to identify cell-free (cf)RNA biomarkers of myocarditis in blood. These studies are aided by the first high-resolution, spatially-resolved transcriptomic atlas of REOV myocarditis that we recently created. A significant finding from our spatial transcriptomic studies was the expression of markers of pyroptosis in infected cardiac endothelial cells. Pyroptosis is a form of programmed cell death that differs from apoptosis in that it induces a strong inflammatory response. We hypothesize that pyroptosis of endothelial cells in the heart plays an important role in the subsequent pathogenesis of reovirus-induced myocarditis disease. In our second Aim, we will use knockout mice and drugs that inhibit pyroptosis to investigate its role in the pathogenesis of reovirus-induced myocarditis. We expect these studies to identify novel cfRNA biomarkers of viral myocarditis and to expand our knowledge of the pathogenesis of REOV-induced myocarditis.