To successfully infect their host, pathogens that enter via the gastrointestinal tract must overcome the multi- layered system of intestinal defenses including physical barriers (e.g., mucus, tight junctions) and immune responses (e.g., secretory IgA). Microfold (M) cells, specialized cells in the intestinal epithelium that take up particulate antigens or sIgA-immune complexes, play an important role in the establishment of immune responses to enteric pathogens. Since viruses were visualized in M cells, they may also provide portals for pathogen entry into the gut, but direct experimental evidence that M cells are required for initiating a productive infection is lacking. A better understanding of the interactio of pathogens with the intestine and associated lymphoid tis- sue offers avenues for early intervention, but no information is available for noroviruses (NoVs). NoVs are enteric viruses that infect the intestinal tract in a species-specific manner and are spread by the fecal-oral route. Murine NoVs (MNVs) are the most prevalent viruses in biomedical research colonies, with the potential to alter research outcomes. In contrast to the non-cultivatable human NoVs (HuNoVs), MNV replicates in tissue culture and a small animal host and can be genetically modified. The native murine host also offers genetic tractability and a wide array of available resources. Thus, the MNV system provides a versatile system to study the inter- action of an enteric virus with the epithelial barrier and enables addressing a fundamental question in enteric virus pathogenesis: how do enteric viruses overcome the intestinal barrier of the host to establish a productive infection? Preliminary data demonstrate that in vitro, MNV crosses a differentiated murine intestinal epithelial cell monolayer by intracellular transport in the absenc of viral replication or disruption of the epithelial tight junctions. MNV transport is increased uner M cell-inducing conditions or after virions bind to natural (non- specific) secretory IgA. These data are the foundation of the central hypothesis: MNV exploits gastrointestinal host defenses by using natural secretory IgA and M cells to cross the epithelial barrier and gain access to permissive macrophages and dendritic cells. This hypothesis will be tested by pursuing the fol- lowing specific aims: 1) Determine whether MNV exploits M cells to establish a productive infection in its native murine host, and 2) Determine whether natural sIgA enhances MNV infection of the murine gastrointestinal tract. These objectives will be achieved by exploiting bot in vivo (e.g. M cell deficient, IgA-/- mice) and ex vivo (i.e. ligated ileal loops) systems. Knowledge of how a NoV breaches the intestinal epithelial barrier to establish a productive infection will increase the understanding of fundamental principles during host-pathogen interac tion in the intestine. Principles uncovered herein can later be tested for their applicability to HuNoV and other enteric pathogens. The proposed research is innovative because it experimentally tests for the first time whether intestinal M cells are required during productive infections, and it could provide the first demonstration of viral subversion of the natural sIgA hot defense pathway to enhance viral infections.

Public Health Relevance

In this application we propose to determine how a norovirus crosses the intestinal epithelial barrier, the initial step in infection of the host. Knowledge of these mechanisms is an essential first step toward developing antiviral or oral vaccine strategies to control and/or prevent norovirus infections, which are the most common cause of viral diarrhea worldwide. As such our work is critical to ultimately reduce the social and economic impact of noroviruses, for which there are currently no treatments.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI103961-02
Application #
8604681
Study Section
Virology - B Study Section (VIRB)
Program Officer
Cassels, Frederick J
Project Start
2013-01-15
Project End
2014-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
2
Fiscal Year
2014
Total Cost
$208,003
Indirect Cost
$67,689
Name
University of Michigan Ann Arbor
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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Hwang, Seungmin; Alhatlani, Bader; Arias, Armando et al. (2014) Murine norovirus: propagation, quantification, and genetic manipulation. Curr Protoc Microbiol 33:15K.2.1-15K.2.61
Jones, Melissa K; Watanabe, Makiko; Zhu, Shu et al. (2014) Enteric bacteria promote human and mouse norovirus infection of B cells. Science 346:755-9
Gonzalez-Hernandez, Mariam B; Liu, Thomas; Payne, Hilary C et al. (2014) Efficient norovirus and reovirus replication in the mouse intestine requires microfold (M) cells. J Virol 88:6934-43