Project Summary Cell intrinsic innate immune pathways present a barrier that nearly all pathogens must overcome in order to successfully complete their infectious lifecycle. Influenza A virus (IAV) is no different, and engages several different strategies in order to evade or otherwise suppress recognition by the host. This is crucial to viral survival, as the production of interferons, the central signaling molecules of the innate response, results in the propagation of a highly antiviral state to which even IAV is susceptible. Regardless, IAV is still detected in a small subset of infected cells. In order to understand, and potentially manipulate, the host response to IAV, we must first understand the nature of those rare infections that propagate an interferon response. Does the incredible genetic and behavioral diversity exhibited in IAV infections predispose their recognition? And, if so, is there a single stereotyped pattern of immunostimulatory variation, or do multiple points of viral failure exist? Over the course of the two-year funding period, Dr. Russell will quantitatively measure how heterogeneity in IAV populations impacts their recognition by innate immune pathways. This will require sequencing bulk, infected, populations of cells as well as single-cell transcriptomic measurements – both of which have been developed and piloted by Dr. Russell while in Dr. Jesse Bloom's lab. Moving beyond the observational science enabled by these sequencing approaches, Dr. Russell will explore both newly- and previously-identified immunostimulatory IAV variation to establish causality. This will include attempts to ascertain whether all immunostimulatory variation acts through the same, shared, mechanisms. This will be primarily accomplished by recapitulating variation in pure viral populations using a reverse-genetic approach, permitting the independent exploration of individual sources of immunostimulatory heterogeneity. Lastly, Dr. Russell will seek to expand his work beyond IAV to a related virus of human importance, Influenza B virus. Comparisons across distinct viral lineages will provide vital insight into whether viral interactions with innate immunity are static over evolutionary time, or whether they are relatively dynamic and thus intractable to a generalizable therapeutic intervention. Overall, this work will generate a robust framework describing the totality of innate immune recognition of these viruses, serving as a substantial resource to the field moving forward.