PROJECT SUMMARY/ABSTRACT Rotavirus (RV) is the leading cause of viral gastroenteritis in children under the age of 5 globally. Despite the introduction of several WHO-approved vaccines, RV infections still cause 114 million diarrhea episodes, 24 million outpatient visits, and approximately 215,000 deaths annually, and therefore necessitates further study. Aside from the typical gastroenteritis, around 75% of infected children have viremia and 90% have antigenemia, and RV detection in the sera is often associated with high fever. Furthermore, various reports indicate the presence of RV RNA or antigen in extraintestinal tissues, including the hepatobiliary system and pancreas, which have been linked to clinical manifestations such as biliary atresia and type 1 diabetes mellitus. The molecular mechanisms by which RV disseminates to systemic organs from the small intestine are not clear. Studies with reovirus, a closely related non-pathogenic human enteric virus, show that the virus can bypass the intact epithelial barrier to establish intestinal infection and spread systemically in mouse models by exploiting the transcytotic nature of microfold (M) cells. M cells are a subset of specialized intestinal epithelial cells that sample and transport content from the lumen to antigen-presenting cells at the basolateral side of the epithelium. Various enteric pathogens exploit M cells to gain access to host systemic sites. RV virions have been visualized in vivo within porcine M cells and human RV was recently shown by our lab to be transcytosed in vitro by human M cells in an ileal enteroid system. However, it is not known whether RV utilizes M cells for intra- and/or extra- intestinal infection in vivo. Additionally, the endocytic pathways responsible for M cell uptake of pathogens like RV have not been defined. The overall hypothesis of this proposed work is that RV is transcytosed by M cells in a caveolin- dependent manner and that this process is key to RV intestinal infection, systemic dissemination, and infection of extraintestinal organs. In Aim 1, the extraintestinal tissues and cell types targeted by RV will be determined following oral inoculation. Next, Villin-Cre Tnfrsf11aflox/flox mice that lack intestinal M cells will be utilized to study the role of M cells in intestinal infection and extraintestinal dissemination. In Aim 2, CRISPR- Cas9 mediated knockout will be used to dissect the endocytic pathway(s) involved in RV transport by M cells in human ileal enteroids. Conceptually, this study will define the mechanisms by which RV, a major childhood enteric pathogen, can disseminate systemically and resolve the longstanding question of how M cells transcytose substrates, paving the path for rational design of M cell-targeted mucosal vaccines for improved immunogenicity and antigen delivery.

PROJECT NARRATIVE Rotavirus infections are a major global health concern. A better understanding of rotavirus pathogenesis is imperative to the development of more effective vaccines and life-saving treatments. The proposed work will identify the mechanism of rotavirus extraintestinal dissemination and tissue tropism, setting the stage for preventing and treating secondary complications after enteric viral infections.