Project Abstract Picornavirus infections are a leading cause of viral encephalitis and myocarditis in humans. These viral infections can cause substantial inflammatory changes in the brain and heart and lead to significant morbidity and mortality. The immune system detects picornavirus infections, and other single-stranded RNA (ssRNA) viruses, via DEAD/H-box (DDX) helicases that sense cytosolic viral ssRNA and initiate the protective interferon (IFN) response. Two DDX helicases critical in this process are retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Both RIG-I and MDA5 are members of the RIG-I-like receptor (RLR) family of RNA-sensing helicases. RIG-I is known to sense negative-sense ssRNA (–ssRNA) viruses (e.g., Sendai virus or vesicular stomatitis virus), while MDA5 is the major sensor for RNA from picornaviruses and other positive-sense ssRNA (+ssRNA) viruses including SARS-CoV2 and other coronaviruses. How MDA5 is activated and regulated is currently not well known, which is in striking contrast to RIG-I for which the activating mechanisms have been elucidated in detail. To study the pathogenesis of myocarditis and encephalitis, researchers have used a prototypical member of the picornavirus family, encephalomyocarditis virus (EMCV). Using EMCV, we demonstrated an important role for the A disintegrin and metalloproteinase protein (ADAM9) in viral pathogenesis. We discovered that mice lacking ADAM9 rapidly succumb to EMCV infection without mounting the characteristic IFN response seen in wild-type mice. Our data indicate a novel role for ADAM9 in viral RNA-induced IFN production through the DDX helicase MDA5. In EMCV infection, MDA5 is the sensor that recognizes viral RNA and initiates a signaling cascade that leads to activation of the mitochondrial antiviral signaling (MAVS) pathway and subsequent IFN production. IFN production is triggered when the viral genome interacts with nucleic acid sensors in the host cell to activate downstream pathways. The host IFN response is crucial to protect the host by limiting virus replication. We hypothesize that ADAM9’s role in viral-induced IFN production is mediated through the MDA5-MAVS pathway. The role of ADAM9 in regulating the IFN response to diverse RNA viruses, including picornaviruses and coronaviruses, will be defined in terms of its effects on the ability of MDA5 to interact with viral RNA and its effect on protein-protein interactions and post-translational modifications of MAVS pathway adapters and effector proteins. Through these experiments, we will define new pathways of IFN activation and better define the pathogenesis of RNA viruses in an animal model system for studying encephalitis and myocarditis.