Human norovirus (HuNoV), a NIAID Category B pathogen, is the leading cause of acute gastroenteritis globally, and respiratory syncytial virus (RSV), a Category C pathogen, is the major global respiratory pathogen of children. HuNoV infections result in significant acute morbidity in all age groups, chronic disease in immunocompromised cancer and transplant patients, and death in young children and older adults. RSV is the leading cause of lower respiratory tract morbidity and mortality among children, and contributes significantly to illness and death in the immunocompromised, those with co-morbidities and older adults. The burden of disease and economic impact of these mucosal pathogens have stimulated extensive efforts for vaccine development. With vaccines anticipated in the next 5 years for both pathogens, this pre-licensure window, together with our recent development of a successful cultivation system for HuNoVs using stem cell-derived human intestinal organoids (HIOs) and the recent description of RSV infections in human lung organoids (HLOs) provide an ideal window to apply analytical genomics and functional studies in relevant human culture systems to address key questions about HuNoV and RSV diversity, evolution and virulence. Our proposed studies utilize carefully selected clinical specimens covering the spectrum of HuNoV and RSV illness, including: (i) acute gastroenteritis in immunocompetent children from two populations (USA and Hong Kong), (ii) acute respiratory infections in children in the CDC sponsored New Vaccine Surveillance Network, (iii) well-controlled volunteer HuNoV challenge studies, (iv) transplant and immunocompromised patients with chronic HuNoV infection or acute RSV infection, (v) the first randomized double-blind clinical trial of nitazoxanide (NTZ) for treatment of chronic HuNoV in transplant recipients, and (vi) women and infants in the first phase III trial of an RSV-F vaccine (Novavax) in healthy pregnant women for preventing disease in their infants. In addition to pathogen-targeted sequencing, we will evaluate the role of the ecological niche in viral pathogenesis using high throughput, integrated sequencing approaches to profile the bacteriome and virome of clinical samples. Full-length (FL) genomic analyses of HuNoV and functional studies in HIEs will provide critical information on virus diversity in different populations and over time in diverse hosts, the presence of intragenotypic and intergenotypic recombinants, and the appearance of mutants with treatment resistance to NTZ. FL genomic analyses of RSV and functional studies in HLOs and other cell lines will establish specific viral signatures of RSV disease severity, enable understanding the effects of immune pressure and immunotherapeutics on the viral genome including antigenic site specific motifs, and changes in B and T cell epitopes. The scientific community will benefit immensely from large databases of FL genomes for HuNoV and RSV, and functional studies in novel human cultures that comprehensively address viral genomic signatures, microbial ecology and host responses before vaccine introduction. Identifying biomarkers, viral and host targets will guide treatment and development of therapeutics.