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The goal of the current proposal is to advance the use of 3-D intestinal cell cultures to use them as an analytical tool of broad applicability to food safety laboratories. We recently reported the use of 3D intestinal cultures in the first successful propagation of human noroviruses in vitro. We now propose to extend these studies, with an emphasis on using these cultures for developing a robust method for assessing norovirus infectivity that can be utilized in routine assays (such as in food safety diagnostic programs). Development of a practical assay for human norovirus will potentially facilitate both detection and intervention strategies for human norovirus by reducing the minimum sensitivity needed for a positive RT PCR test and permitting determination of the infectious/noninfectious nature of virus samples. Currently, inactivation and mitigation strategies for human norovirus are severely hampered by the inability to determine if the virus has been efficiently inactivated. <P> The hypothesis to be tested in the proposed research is: Can the 3D intestinal cell culture model be adapted to create a robust bioassay system that can be used in food safety laboratories to unambiguously establish the present of infectious noroviruses in food materials. <P>
The Specific Objectives of the research are: <OL> <LI> To characterize the time course and amplitude of virus replication in the 3D intestinal cell culture model. <LI>To evaluate the potential to enhance virus propagation. <LI> To establish a beta test site for 3D cell cultures for norovirus replication in a USDA food safety research facility to establish that routine and robust analyses of norovirus infectivity can be readily achieved.
NON-TECHNICAL SUMMARY: Human noroviruses are a leading cause of gastrointestinal illness, and account for approximately 700 million infections per annum worldwide. In the United States, it is estimated that approximately 1 person in 10 to 15 persons contracts norovirus annually. Forty percent of norovirus infections are thought to be foodborne, making norovirus the number one cause of food-borne illness in the United States. Norovirus is a significant problem for the molluscan shellfish industry since wild-caught and aquaculture products are often consumed raw and these bivalves can readily bioconcentrate virus particles contaminating their production areas. For the produce industry, norovirus are a significant issue since this virus and other enteric viruses can be inadvertently sprayed on crops by irrigation with virus contaminated water or spread by workers with unclean hands who tend and harvest the crops. This latter produce issue has become an important one since increasingly produce consumed in the US is imported from developing regions and countries (i.e., Latin America), and virus outbreaks have been associated with these imports. There are several important reasons for developing a practical in vitro replication system for human noroviruses. First, current testing technologies for food safety can only detect the presence of virus and cannot determine if the food sample actually contains infectious virus. The second reason for developing a practical in vitro replication system is that while many current detection technologies are quite sensitive, most do not have sensitivity at the low level of an infectious viral dose (10 to 100 virions). To overcome these limitations, we propose to use our 3D intestinal cultures to develop a robust practical method that can be routinely used by food safety laboratories to a) determine the infectious/noninfectious nature of virus samples, and b) reduce the minimum sensitivity needed for positive virus detection.
<P>APPROACH: The detailed Specific Objectives of the research are: <BR><BR>1. To characterize the time course and amplitude of virus replication in the current 3D intestinal cell culture model. We will evaluate the time course and amplitude of replication for Genotype I and Genotype II noroviruses in the current monotypic 3D cell culture system (comprised of intestinal epithelial cells) using multiple criteria, including quantitative real-time RT PCR methods. Radiolabeling and immunoprecipitation of replicated capsid with 35S methionine and/or radiolabling of RNA with 3H Uracil will be used to validate replication for both GI and GII viruses. RT PCR directed against the negative sense (replicating template) RNA strand during infection is also proposed as an additional tool for unambiguous evidence of viral replication. <BR><BR>2. To evaluate the potential to enhance virus propagation. We will develop a second 3D intestinal model that will expand upon the model used in Aim 1; this cell culture system will include the coculture of macrophage cells in combination with the epithelial cells. This multicellular coculture model more closely approximates the parental tissue in vivo, and may allow further cellular differentiation that mimics in vivo cellular organization; the studies will also reveal if addition of macrophages enhances viral replication. We will identify the pattern of intestinal cell differentiation needed to support norovirus replication in 3D culture using confocal imaging with fluorescent antibodies against the viral capsid and against cell specific markers. <BR><BR>3. Establish a beta test site for 3D cell cultures for norovirus replication in a USDA food safety research facility to establish that routine and robust analyses of norovirus infectivity can be readily achieved. Transfer of technology under this grant will accompany the development of new tools for validation of the norovirus replication assays. An important goal of this objective is to define variables that have made it difficult for other laboratories to establish their own 3D cell culture systems for norovirus analysis, and identify protocols to overcome these constraints.