PROJECT SUMMARY Pediatric diarrheal diseases are a major cause of morbidity in children under 5 years old. Rotavirus is an enteric virus that causes diarrhea and vomiting and can be fatal in children without supportive care. Rotavirus infection results in 128,500 deaths per year. Despite causing severe, potentially life-threatening diarrhea, rotavirus infection is highly localized to the enterocytes in the villus tips of the small intestine. Until recently, it was unclear how these cell-cell signals were propagated from infected cells to surrounding uninfected cells. Using high resolution live imaging, we found that rotavirus infection increases calcium (Ca2+) signaling both within infected enterocytes as well as surrounding uninfected cells, which manifests as intercellular Ca2+ waves. These Ca2+ waves originate from the infected cell and propagate through uninfected cells. We have identified that Ca2+ waves are mediated by extracellular purinergic signaling; primarily through the purine nucleotide ADP and the P2Y1 receptor. However, the functional consequence of these intercellular Ca2+ waves remains unknown. Our central hypothesis is that rotavirus-induced paracrine signaling, via P2Y1 purinergic signaling, functionally dysregulates neighboring uninfected epithelium response and is required for the hallmarks of rotavirus infection. Potential downstream targets of P2Y1 signaling include chloride (Cl-) and mucin secretion. Rotavirus infection is characterized by both Cl- driven secretory diarrhea and loss of mucin-filled goblet cells; hallmarks which are considered to promote infection. We are focusing on rotavirus-epithelial interactions as these events may be able to mediate the major consequence of infection. The objective of this research is to elucidate the role of purinergic signaling in host intestinal epithelial cells in response to rotavirus infection and the mechanistic consequences of rotavirus dysregulation of these pathways. Aim 1 will determine the role of purinergic signaling via P2Y1 during rotavirus infection in vivo. We anticipate deficiency of P2Y1, by genetic loss of P2Y1 (P2Y1-/- mice) or inhibition of P2Y1 with the pharmacological inhibitor MRS2500, will reduce rotavirus symptoms including decreased diarrhea, weight loss, histopathology and cytokine production, and viral shedding. In Aim 2 we will characterize the signaling pathways in vitro that drive epithelial functions (cell susceptibility, Cl- secretion, and mucus expulsion) during rotavirus infection in human intestinal enteroids (HIEs). We predict that pretreatment of HIEs with ADP or P2Y1 agonist increases susceptibility to infection and results in increased infected cells, apical Cl- secretion and fluid secretion. We anticipate ADP activation of P2Y1 stimulations Ca2+ activated chloride channels (CaCC) via PLC and IP3R, which also plays a role in mucin secretion from goblet cells. We will use fluorescent reporters, pharmacological inhibitors and P2Y1 and P2Y2 CRISP...