PROJECT SUMMARY Acute gastrointestinal and respiratory infections are leading causes of morbidity and mortality in children worldwide. Among gastrointestinal pathogens, human norovirus (HuNoV, NIAID category B pathogen) is the leading cause of acute and sporadic gastroenteritis and is responsible for over 1 million pediatric health care visits in the US each year. Respiratory syncytial virus (RSV, NIAID category C pathogen) is the major respiratory pathogen in children globally and results in over 1.5 million health care visits annually in the US. In addition to the clinical burden of disease, both viruses cause significant economic burden. There are currently no approved vaccines for children for HuNoV or RSV. However, several vaccines and long-acting monoclonal antibodies are in clinical trials or recently approved and are anticipated to be available to protect children. Immune pressure driven changes in the genomic landscape of both viruses are anticipated with the introduction of interventions. This is in addition to changes in the epidemiology following the COVID-19 pandemic which altered the burden of disease and circulating patterns of these viruses, all posing challenges for the long-term success of interventions. We propose to perform innovative and extensive sequencing of pediatric stool and respiratory samples from racially and ethnically diverse population, together with functional validation in physiologically relevant pediatric ex vivo organoid cultures, to identify viral and host factors affecting strain emergence and disease presentation. Our studies will be focused on pediatric samples and organoids as children have long been considered a key population for emergence of new HuNoV and RSV variants. Study samples will cover four distinct time periods: pre-pandemic, pandemic, post-pandemic, and following the introduction of HuNoV and RSV interventions. The studies are feasible since Project investigators are part of the CDC’s New Vaccine Surveillance Network that evaluates the burden of pediatric gastrointestinal and respiratory illnesses in seven sites across the US, and thus have access to thousands of relevant clinical samples and associated metadata. We will perform full-length HuNoV and RSV genome analysis, microbiome, transcriptome, and virome analysis of clinical samples (Aim 1). Study investigators have also established and characterized pediatric human intestinal and nasal organoids that will serve as physiologically relevant models for functional studies (Aim 2). Our proposed studies will provide a rich repository of genomic data and allow us to (i) track strain emergence and diversity, (ii) discover biomarkers for mucosal gastrointestinal and respiratory disease, (iii) identify viral signatures related to pathogenesis and strain emergence, and (iv) functionally delineate virus–host–microbiome interactions. The timing of these studies is critical in the context of pandemic-induced epidemiological shifts and the potential f...