PROJECT SUMMARY Gastroenteritis (GE) is among the leading causes of mortality globally. Our research focuses on human rotavirus (HRV) and human norovirus (HuNoV), the two leading causes of viral GE worldwide causing over 320,000 deaths annually. No antivirals are available for either virus and there is no vaccine for HuNoVs. While vaccines to HRV are available and are effective in high-income countries (84-90%), the efficacy remains suboptimal (45- 57%) in low-income settings where the burden of disease is greatest. Economically, HuNoV infections result in over $4 billion in direct healthcare costs and over $60 billion in societal costs each year. These data underscore the need for continued investment in studies to overcome mucosal enteric disease. Both these human GI viruses do not infect mice; further, HRV replicates poorly in cultured cells, and HuNoV was noncultivatable for over almost 50 years. Using tissue stem cell-derived human intestinal organoid (HIO) cultures as a replication model for these human GI pathogens, we made some remarkable fundamental discoveries. Key findings include: both human viruses replicate in at least two distinct intestinal cell types (enterocytes and enterendocrine cells) in the small intestine, and HRVs also replicate in the colon. Each virus binds to genetically encoded histo-blood antigens (HBGAs) but these glycans play different roles in infection. HBGA expression does not restrict infection but correlates with severe HRV disease while it is required for infection with HuNoV. Interactions with HBGA are strain-dependent for both viruses. Each virus infects the polarized epithelium by a different route with HRVs infecting basolaterally and HuNoVs infecting apically. Infected HIO cultures produce a new form of HRV released in vesicles that exhibit different properties from standard cell-culture derived virus. Both viruses induce a predominant epithelial innate response of type III interferon (IFN); surprisingly this does not restrict virus replication suggesting type III IFN may have other functions than being antiviral. HIOs allow cultivation of multiple HuNoV strains and bile is a critical factor for replication. Although these previous studies using epithelial-only HIOs provide new knowledge on HRV and HuNoV infections, we still lack a comprehensive understanding of the pathophysiology and host responses that lead to life-threatening disease. Continued development of “human mini-gut” models is required to fully understand human-GI virus interactions linked to pathogenesis and improve mucosal immune responses to viral infections. Using complex biomimetic cultures, we propose to answer two biological questions of fundamental and clinical relevance: What mechanisms mediate severe GI disease during infection with HRV and HuNoV (Aim 1), and what is the role of microbe-microbe interactions in the pathophysiology of viral GE (Aim 2)? Through interactions with Projects 2 and 3 and our two Scientific Cores, we predict...