Final Report Summary - RIG‐I LIVE IMAGING (Imaging influenza nucleic acid recognition by RIG-I in living cells.) Among all pathogens, RNA viruses impose a particular challenge due to their ability to evolve rapidly. As intracellular parasites, they directly interact with the host cell machinery and can develop diverse resistance mechanisms to bypass cellular defenses. Hence, it is of crucial importance for the host to react at early steps of the infection. During infection, viral nucleic acids can be sensed by members of the RIG-I like receptors family, namely RIG-I, MDA5 and LGP2. These receptors trigger a rapid response that acts to limit virus replication. Recently much progress has been made in defining the features of RNA recognized by RIG-I and the project therefore re-focused on the fact that much less information is available on the characteristics of RNA recognized by MDA5 or LGP2. The proposal was therefore redesigned to focus on defining the physiological agonists for MDA5 that promote signalling in infected cells. To do this, we focused on the encephalomyocarditis virus (EMCV) infection model, which is a member of the picornaviridae family such as Polio, Rhino, or Coxsackie viruses. We purify RNA directly from complexes obtained by immunoprecipitation of the MDA5 partner, LGP2 and show that this method enriches for stimulatory RNA corresponding to a short portion of the EMCV antisense RNA. Deletion of this region from the EMCV genome generates viruses that are less potent at producing stimulatory RNA and inducing IFN in infected cells or mice. Thus, a discrete region of the EMCV antisense genome can act as a physiologically-relevant MDA5 agonist in infected cells. Identification of the RNAs responsible for MDA5 activation in infected cells sheds light into the nature of RLR recognition and may help develop new ways to prevent and control picornavirus spread. In sum, the results from the project contribute to a better understanding of the innate antiviral response and, potentially, impact on antiviral treatments and vaccine development.