Human norovirus infection does not induce serotonin secretion in human intestinal organoids

Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide. The primary symptoms of HuNoV-associated disease are vomiting and diarrhea; yet the mechanisms underlying these symptoms are poorly understood. Neuroactive hormones such as serotonin (5-hydroxytryptamine; 5-HT) secreted by intestinal enteroendocrine cells (EECs) can activate vomiting centers in the brain and play a critical role in regulating emetic signaling. Serotonin release from infected EECs has been implicated in vomiting during other enteric infections. Recent studies have reported the detection of HuNoV RNA and antigen in EECs in jejunal biopsies from immunocompromised patients with chronic HuNoV infection and rhesus macaques. We therefore investigated whether HuNoV infection of human intestinal organoids (HIOs) induces serotonin secretion. We examined infection and serotonin secretion with multiple genogroup II HuNoV strains using adult and infant jejunal HIO models, including a doxycycline-inducible neurogenin-3 HIO line with increased numbers of EECs. HuNoV infection did not induce serotonin secretion in either adult or infant HIOs as measured by ELISA. Consistent with enterocytes being the primary target cell type for HuNoVs, GII.4 HuNoV replication was robust in parental HIOs but reduced in EEC-enriched cultures. Furthermore, supernatants from parental HIOs failed to stimulate serotonin release from EEC-enriched HIOs. Together, these findings demonstrate that HuNoV infection of intestinal epithelial cells is insufficient to trigger serotonin release in HIOs. These results suggest that HuNoV-associated vomiting is unlikely to result from direct epithelial serotonin secretion and may require additional cellular components such as immune or neuronal signaling pathways or may involve other neuroactive hormones.IMPORTANCEVomiting is a hallmark symptom of human norovirus disease. Yet, the biological mechanisms responsible for this response are unknown. Infection of enteroendocrine cells and secretion of serotonin play key roles in the emetic response to other gastrointestinal viruses such as rotavirus. Since recent studies have detected human norovirus antigens and RNA in enteroendocrine cells, we hypothesized that human norovirus infection could stimulate serotonin secretion. We used jejunal human intestinal organoids from adults and infants, including organoids enriched for enteroendocrine cells, and found that infection with several clinically important strains did not induce serotonin secretion, even under conditions supporting robust viral replication. These data indicate that serotonin release from intestinal epithelial cells alone is unlikely to explain norovirus-induced vomiting and highlights the need to investigate alternative mechanisms, including other neuroactive hormones, and neuronal signaling pathways to better understand the pathogenesis of norovirus-associated vomiting.