Influenza A virus (IAV) causes annual seasonal epidemics and occasional pandemics resulting in significant levels of mortality and socio-economic costs worldwide. PB1-F2 is a small non-structural protein encoded by an alternate +1 open reading frame in the PB1 gene. PB1-F2 is considered to play important roles in primary influenza virus infection and post-influenza secondary bacterial pneumonia in mice. It is a multifunctional and enigmatic protein with diverse functions attributed to it and the precise contribution of PB1-F2 to the IAV life cycle in avian and mammalian hosts remains largely unknown. In the triple-reassortant H3N2 (TR H3N2) swine influenza virus (SIV) background, we found that PB1-F2 expression did not affect nasal shedding, lung viral load, immunophenotypes, and lung pathology in pigs. On the other hand, in turkeys, deletion of PB1-F2 resulted in early induction of clinical disease and effective transmission among the turkey poults. Interestingly, the virulence associated 66S mutation in PB1-F2 abolished the ability of the IAV to successfully infect turkeys and transmit to in-contacts. These results highlight the strain- and species-specific role of PB1-F2 protein. We also demonstrated that specific amino acid residues in the C-terminal of PB1-F2 determine the pathogenicity of 2009 swine-origin pandemic H1N1 virus in a mouse model. The C-terminal residues 73K, 75R, and 79R together with 66S increased virus replication, decreased type I interferon response, increased infiltration of neutrophils and myeloperoxidase production in lungs resulting in acute respiratory distress syndrome (ARDS) in mice with characteristic clinical and pathological features of acute lung injury (ALI). Further, we found that PB1-F2 induces mitochondrial superoxide production and mitochondrial damage in a sequence dependent manner in IAV-infected lung epithelial cells. PB1-F2-mediated mitochondrial damage promotes Parkin-mediated mitophagy but suppresses the autophagic degradation of damaged mitochondria in the infected lung epithelial cells. Accumulated dysfunctional mitochondria likely to aggravate host cell death and inflammatory responses. Taken together, the present findings enhance our understanding of PB1-F2 protein as a virulence determinant in IAV infection in a species- and strain-specific manner and provide new insights into the impact of genetic changes in PB1-F2 on the host pathogenesis of virulent IAV strains. / Ph. D.