One of the major determinants of influenza pathogenesis and tropism is the cleavage of the viral HA by proteases, and the subsequent activation of fusion with the host cell. Modifications to the HA cleavage site, introduced by mutations in the viral genome, can have profound effects on disease outcome. This is typified by highly pathogenic avian influenza, where a typically mono-basic cleavage is exchanged for a poly-basic cleavage site allowing differential activation by proteases and spread within the host. However, our current studies have shown that alternative and more discrete cleavage site changes can also modify the protease cleavage profile for a given HA and can affect viral infection and pathogenesis. This project will characterize the spectrum of HA cleavage site changes, as defined from influenza surveillance data, and will integrate study of these variant viruses and HAs with the repertoire of host cell proteases that can act on the virus. It is known that bacterial co-infections contribute significantly to influenza morbidity and mortality. These co-infecting bacteria can secrete proteases that can also activate influenza HA, providing a positive-feedback loop for infection. We will also assess the impact of bacterial proteases on HA cleavage activation and influenza pathogenesis. These studies will provide a more complete picture of influenza activation in the host, allowing strategic understanding of novel influenza virus in circulation and informing new therapeutic strategies targeted towards HA and its activation.