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.
