PROJECT SUMMARY Influenza A viruses (IAV) significantly burden society by causing millions of illnesses annually and generating significant economic losses. Transmission of IAV between humans is thought to occur via virus-laden droplets generated by infected individuals as they cough, sneeze, talk and breathe. IAV access the respiratory mucosa of a new host by being delivered through direct contact, self-inoculation after touching surfaces containing viable virus or through airborne transmission. The outcome of airborne transmission is dictated by the size of the aerosols, which determines the duration for which they are suspended in air, how far they travel, and their site of deposition in the respiratory tract. While direct, indirect, and airborne transmission are considered to contribute to IAV spreading, their individual relevance is unclear. Moreover, whether the contribution of different mode(s) of exposure changes with context, such as host immunity or environmental conditions, remains unknown. We hypothesize that, in a recipient host, the efficiency of infection is modulated by the mode and circumstances of exposure, which are defined by the site of viral deposition and effects of the environment on host responses. To address this hypothesis, we will focus on the recipient host as this aspect of transmission can be tightly manipulated experimentally. We will perform intranasal and aerosol inoculations to model direct and airborne modes of infection in guinea pigs. To monitor the efficiency of infection, I will use barcoded libraries of influenza A/California/07/2009 (H1N1pdm09) and A/Panama/2007/99 (H3N2) viruses to assess the number of unique viruses establishing infection. We predict that the viral barcodes will provide a sensitive method for evaluating the intensity of exposure. Additionally, I will test varying ambient temperature and relative humidity levels to assess the impact of environmental conditions on modes of exposure to IAV and host immune responses. In Aim 1, I will examine the relationship between the intensity of exposure and established barcode diversity given distinct modes of exposure. In Aim 2, I will assess the extent to which environmental conditions impact host immune responses and the efficiency of IAV infection by different modes. The impact of this research is to advance knowledge of external factors defining host susceptibility and IAV infection and transmission potential. Our results will deepen understanding of the roles of different modes of exposure in leading to infection and thereby enable development of effective interventions to limit IAV spread.