The long-term objective of this application is to define the relationship between infant respiratory syncytial virus (RSV) infection and the host response that enables asthma inception, data needed to develop RSV and asthma prevention strategies. There is abundant evidence that children who experience severe RSV during infancy are at greater risk for developing asthma later in childhood; however, the host and viral determinants that lead to asthma development are not known. In our prior funding cycle, we conducted a study considering RSV infection in infancy as a natural quasi-random event and demonstrated that infant RSV infection, not only severe infection, is associated with increased asthma risk, and that missing RSV infection during infancy protects from asthma development. Furthermore, we demonstrated the mediating effect of infant RSV infection on the developing airway microbiome and immune development and risk of wheeze, and in preliminary data, the effect of RSV on long-term airway epithelial cell metabolism. Our overarching hypothesis for this proposal is that age-dependent effects of infant RSV infection contribute to chronic respiratory disease through altering DNA methylation (DNAm) of the airway epithelium, and airway epithelial metabolism and developmental programming, and that identifying gene x RSV interactions will explain extremes of asthma susceptibility following infant RSV infection. This proposal leverages rich data sets and birth cohorts to address these hypotheses, and has the potential to greatly advance understanding of the mechanisms and developmental processes underlying the effect of RSV on recurrent wheeze and asthma. To test these hypotheses we will use a combination of two human natural quasi-randomization studies of infant RSV infection specifically designed to assess effects of infant RSV infection on subsequent respiratory health and the airway epithelium, in vitro models of RSV infection of nasal airway epithelial cells (NAECs), along with cutting edge-laboratory and analytic approaches to integrate the resultant complex data. Project 2 leverages data from the INSPIRE cohort that revealed that increased infant blood levels at birth of L-citrulline (L-CIT) were significantly associated with decreased odds of infant bronchiolitis. These data suggested that higher blood levels of L-CIT at time of birth protected against infant bronchiolitis and may protect against the development of asthma later in childhood. We hypothesize that compared to regular diet (RD), an L-CIT supplemented diet (L-CITsd) fed to parents during gestation and their offspring prevents severe RSV bronchiolitis in the offspring. Our preliminary data strongly support this hypothesis and reveal that the L-CITsd decreased RSV-induced lung IL-13 expression, reduced the frequency of lung IL-13 expressing ILC2, and restrained airways responsiveness. Further, L- CITsd significantly decreased ILC2 expression of IL-13 when the cells were stimulated ex v...