Efficient Murine Norovirus Transport Across the Intestinal Epithelium Requires Microfold (M) Cells.

Abstract

Microfold (M) cells are known specialized intestinal epithelial cells that internalize particulate antigens and can aid in the establishment of immune responses to enteric pathogens. While certain enteric viruses have been observed within M cells, not much is understood about whether viruses in general use M cells to initiate a productive infection. Noroviruses (NoVs) are single-stranded RNA viruses that infect their host via the fecal-oral route. Murine NoV (MNV) infects intestinal macrophages and dendritic cells of its host and provides a tractable experimental system for understanding how an enteric virus interacts with the intestinal epithelium. This dissertation focuses in the mechanism of how MNV breaches the intestinal epithelial barrier to get in contact with its permissive cells for a productive infection. Using an in vitro transwell model of polarized intestinal epithelial cells, I demonstrated that MNV crosses the cell monolayer in the absence of viral replication or disruption of tight junctions. Additionally, this MNV transport was mediated by a subset of cells in the monolayer with functional M cell properties also termed as M-like cells. In vivo, replication of two divergent MNV strains was reduced in mice depleted of M cells and similar findings were made using reovirus, an enteric double-stranded RNA virus that infects intestinal epithelial cells. Finally, since residual MNV viral titers were still present, perhaps due to an incomplete depletion of M cells, two different genetically M cell deficient mouse models were used for MNV infection studies: Rag2-/-γc-/- and RANKfl/flVillin-Cre mice. Results in both mouse models, show that oral MNV infection is greatly reduced when compared to controls and to our previous conditional M cell depletion model. Taken together, these results demonstrate that M cells are required for MNV pathogenesis. Similar findings were observed for reovirus. Interestingly, preliminary results suggest that, in the large intestine, alternate entry mechanisms (e.g. transepithelial dendritic cells or villous M cells) could take place for MNV infection in a virus-strain dependent way. Further examination into potential alternative MNV-strain dependent entry mechanisms still needs to be performed, but this dissertation has laid a framework for future studies.