Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains unclear. We found that rotavirus-infected cells produce paracrine signals that manifested as intercellular calcium waves (ICWs), observed in cell lines and human intestinal enteroids. Rotavirus ICWs were caused by the release of extracellular adenosine 5′-diphosphate (ADP) that activated P2Y1 purinergic receptors on neighboring cells. ICWs were blocked by P2Y1 antagonists or CRISPR-Cas9 knockout of the P2Y1 receptor. Blocking the ADP signal reduced rotavirus replication, inhibited rotavirus-induced serotonin release and fluid secretion, and reduced diarrhea severity in neonatal mice. Thus, rotavirus exploited paracrine purinergic signaling to generate ICWs that amplified the dysregulation of host cells and altered gastrointestinal physiology to cause diarrhea.

26Rotavirus causes severe diarrheal disease in children worldwide. A hallmark of rotavirus 27 infection is an increase in cytosolic calcium in infected small intestine epithelial cells. However, 28 the underlying mechanism(s) of rotavirus-cell signaling remains incompletely characterized. Here 29 we show that rotavirus-infected cells produce paracrine signals that manifest as intercellular 30 calcium waves (ICWs); which are observed in both cell lines and human intestinal enteroids 31 (HIEs). Rotavirus ICWs are caused by the release of extracellular adenosine diphosphate (ADP) 32 that activates P2Y1 purinergic receptors on neighboring cells and are blocked by P2Y1 33 antagonists or CRISPR/Cas9 knockout of P2Y1. This paracrine purinergic signal is critical for 34 rotavirus replication and diarrhea. Blocking the ICW signal reduces rotavirus replication; inhibits 35 rotavirus-induced serotonin release and fluid secretion; and reduces diarrhea severity in neonatal 36 mice. This is the first evidence that viruses exploit intercellular calcium waves to amplify diarrheal 37 signaling; a finding which have broad implications for gastrointestinal physiology. signaling molecules during RV infection, including enterotoxin NSP4, prostaglandins (PGE2), and 57 nitric oxide (NO) 7,15-17 . In this model, enterotoxin NSP4 can bind to neighboring, uninfected 58 enterocytes to activate Ca 2+ -activated chloride channels and cause secretory diarrhea 18,19 , and 59 PGE2 and NO may further activate fluid secretion processes 20,21 . Dysregulation of neighboring 60 enteroendocrine cells triggers the Ca 2+ -dependent release of serotonin, which stimulates the 61 enteric nervous system both to activate vomiting centers in the central nervous system and to 62 activate secretory reflex pathways in the gastrointestinal (GI) tract 5,22 . Thus, this model of RV-63 induced diarrhea addresses how limited infection at the middle-to-upper villi may cause 64 widespread dysregulation of host physiology and life-threatening disease. 65Herein we demonstrate that RV-infected cells signal to uninfected cells via an extracellular 66 purinergic signaling pathway. This newly identified pathway is a dominant driver of observed RV 67 disease processes, including replication, upregulation of PGE2-and NO-producing enzymes, 68 serotonin secretion, fluid secretion, and diarrhea in a neonatal mouse model. Our findings provide 69 new insights into the mechanism(s) of viral diarrhea and gastrointestinal physiology. 70 Results 71 Low multiplicity infection reveals intercellular calcium waves 72Previous studies have shown that RV significantly increases cytosolic Ca 2+ during infection 73 and disrupts host Ca 2+ -dependent processes to cause disease [23][24][25] . We used African Green 74 monkey kidney MA104 cells stably expressing the genetically-encoded calcium indicator (GECI) 75GCaMP5G or GCaMP6s to observe changes in cytosolic Ca 2+ during RV infection using live-cell 76 time-lapse epifluorescence imaging. We did not observe differences in response using either...

Viruses are among the most prevalent enteric pathogens. While virologists historically relied on cell lines and animal models, human intestinal organoids (HIOs) continue to grow in popularity. HIOs are non-transformed, stem cell derived, ex vivo cell cultures that maintain the cell type diversity of the intestinal epithelium. They offer higher throughput than standard animal models while more accurately mimicking the native tissue of infection than transformed cell lines. Here, we review recent literature that highlights virological advances facilitated by HIOs. We discuss the variations and limitations of HIOs, but also how HIOs have allowed for the cultivation of previously uncultivatable viruses and how they have offered insight into tropism, entry, replication kinetics, and host-pathogen interactions. In each case, we discuss exemplary viruses and archetypal studies. We discuss how the speed and flexibility of HIO-based studies contributed to our knowledge of SARS-CoV-2 and anti-viral therapeutics. Finally, we discuss current limitations of HIOs and future directions to overcome these.

Rotavirus is a leading cause of viral gastroenteritis. A hallmark of rotavirus infection is an increase in cytosolic Ca2+ caused by the nonstructural protein 4 (NSP4). NSP4 is a viral ion channel that releases Ca2+ from the endoplasmic reticulum (ER) and the increase in Ca2+ signaling is critical for rotavirus replication. In addition to NSP4 itself, host inositol 1,4,5-trisphosphate receptor (IP3R) ER Ca2+ channels may contribute to rotavirus-induced Ca2+ signaling and by extension, virus replication. Thus, we set out to determine the role of IP3R Ca2+ signaling during rotavirus infection using IP3R-knockout MA104-GCaMP6s cells (MA104-GCaMP6s-IP3R-KO), generated by CRISPR/Cas9 genome editing. Live Ca2+ imaging showed that IP3R-KO did not reduce Ca2+ signaling in infected cells but eliminated rotavirus-induced intercellular Ca2+ waves (ICWs) and therefore the increased Ca2+ signaling in surrounding, uninfected cells. Further, MA104-GCaMP6s-IP3R-TKO cells showed similar rotavirus susceptibility, single-cycle replication, and viral protein expression as parental MA104-GCaMP6s cells. However, MA104-GCaMP6s-IP3R-TKO cells exhibited significantly smaller rotavirus plaques, decreased multi-round replication kinetics, and delayed virus spread, suggesting that rotavirus-induced ICW Ca2+ signaling stimulates virus replication and spread. Inhibition of ICWs by blocking the P2Y1 receptor also resulted in decreased rotavirus plaque size. Conversely, exogenous expression of P2Y1 in LLC-MK2-GCaMP6s cells, which natively lack P2Y1 and rotavirus ICWs, rescued the generation of rotavirus-induced ICWs and enabled plaque formation. In conclusion, this study shows that NSP4 Ca2+ signals fully support rotavirus replication in individual cells; however, IP3R is critical for rotavirus-induced ICWs and virus spread by priming Ca2+-dependent pathways in surrounding cells.

No abstract