Influenza is a leading cause of respiratory infections, causing tens of millions of cases of disease annually in the United States alone. A contributing factor to this high incidence is the virus's high mutation rate, which generates considerable genetic diversity and allows it to escape natural and vaccine derived immunity. This high mutation rate is, in part, due to the fast-acting, yet error prone, viral polymerase. In addition to genetic diversity, this error prone polymerase also generates genomic diversity, or diversity in the organization of the viral genome. A key component of this genomic diversity are defective interfering (DI) viruses. Influenza DI viruses harbor large internal deletions in the RNA sequence of at least one gene and therefore must co-infect a cell with wild-type virus in order to be replicated. Importantly, they are thought to activate the host immune system and reduce wild-type virus production from co-infected cells. Because of this, the use of DI viruses as a prophylactic or therapeutic for viral infections is an area of active research. However, the evolution of naturally derived DI viruses within single hosts during an infection and the impact these DI viruses have on population-level viral dynamics is poorly understood. In this proposal I plan to define how influenza DI viruses evolve and interact with wild-type viral dynamics at the within-host, between-host, and host population scales. I plan to do this through the analysis of deep sequencing data from human and ferret infections of influenza virus in three specific aims. In Aim 1 I will characterize how DI viruses evolve within individual hosts over the course of infection. I will analyze data from acute and chronic infections with seasonal and maladapted viral strains to determine how infection characteristics shape DI virus dynamics. In Aim 2 I will determine whether there is evidence for the transmission of DI viruses between infected hosts. In Aim 3 I will conduct phylogenetic and phylodynamic analyses to determine whether the generation of DI viruses alters viral spread at the population level. My results will provide insights into the dynamics of naturally derived DI viruses. This work will help to inform research into the use of exogenous DI viruses as an alternative strategy for treatment or prophylaxis against influenza infections.