This is an A1 (year -06 149=13.5%) competing renewal for grant R01 AI054883 entitled Immunity and Pathogenesis of a Novel Norovirus. Noroviruses (NoV, Category B Biodefense agents) are the major etiologic agent of nonbacterial, epidemic gastroenteritis worldwide, causing significant suffering, morbidity, and economic loss. Eighty to 96% of all cases of non-bacterial epidemic gastroenteritis in the US are attributed to NoV. Human NoV are not readily cultured and do not infect small animals. There is no NoV vaccine, although significant insight has come from studies of the immune response to virus-like-particles generated when NoV capsid proteins are over expressed. We identified murine NoV (MNV) in 20031, and during the first grant period made significant strides towards developing MNV infection into a robust infection-only experimental model for understanding aspects of NoV replication, immunity, and pathogenesis. We developed the first NoV culture system in 20042, and the first NoV reverse genetics system coupled to a culture system in 20073. MNV was found to infect a significant proportion of laboratory mice, requiring a significant investment in developing a closed mouse breeding colony to generate non-immune mice with which to perform immunology and pathogenesis experiments. Further work has led to isolation and full sequencing of 21 new strains, and the solution of the cryo-EM structure of both the T=3 virion and a fascinating smaller T=1 particle assembled from the MNV capsid protein. Of fundamental importance, we show here that it is possible to effectively vaccinate against MNV, and that clearance of MNV from intestine involves B cells, antibody, CD4 T cells, CD8 T cells, and perforin. Interestingly, certain MNV strains persistently replicate in intestine of WT mice and are shed in feces for weeks despite a significant immune response. The combination of reverse genetics, a vaccination model, mouse genetics, and persistent and non-persistent MNV strains will allow us to define molecular and immunologic mechanisms of MNV immunity through the following Specific Aims: Aim 1. Determine the mechanism(s) responsible for vaccination against MNV. Aim 2. Determine the mechanism(s) responsible for persistent shedding of MNV in feces. Aim 3. Test the hypothesis that production of the T=1 form of MNV is an immune evasion strategy. 1. Karst, et al. STAT1-dependent innate immunity to a Norwalk-like virus. 2003. Science. 299:1575. 2. Wobus et al., Replication of a Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. 2004. PLOS Biology. E432. 3. Ward, et al., Recovery of infectious murine norovirus using polII-driven expression of full-length cDNA. 2007. PNAS. June 20 [Epub ahead of print]. PROJECT NARRATIVE: Noroviruses are responsible for more than 90% of the epidemic gastroenteritis in the world, causing untold harm. We have developed a mouse model for norovirus infection, and have shown for the first time that we can vaccinate against oral norovirus infection. In addition we have set up molecular methods that will allow us to map viral genes responsible for important biological phenotypes. In this grant we propose to define the mechanisms responsible for norovirus immunity and vaccination, determine the mechanisms that allow the virus to persist despite an active immune response, and define the role in infection of a novel viral particle made in norovirus infected cells that we hypothesize may be a viral tool to evade immunity.
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