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The overall goal is to validate and expand a newly developed human norovirus replication system using the 3-D tissue culture model developed in the NASA bioreactor at Arizona State University (Straub et al., 2007). We intend to adapt it for use as a pragmatic assay for human norovirus infectivity. This work will be performed by Dr. David H. Kingsley (USDA), Dr. Cheryl Nickerson (ASU) and Dr. Charles Arntzen (ASU). <P>
The grant objectives are three fold. First we will characterize norovirus replication in the bioreactor by performing single step growth curves using, radiolabeling of the virion, identify the differentiated cell type in which norovirus is replicating, and identify replicating RNA. <P>Second, we will seek to improve norovirus replication in the bioreactor. Central to the second aim, a co-culture model 3D model will be established by addition of macrophages. <P>Third we will establish a Beta testing site at the USDA Dover DE laboratory in preparation for adapting and disseminating this technology for applied food safety research.
APPROACH: The primary portions of the research that ARS will be responsible for are: 1) generation of single-step growth curves and time course of viral RNA produced during human norovirus infection by quantitiative Real-time RT-PCR; 2) identifcation of replicating RNA in theses cells; and 3) the ARS laboratory will be responsible for objective 3 using the Dover DE ARS laboratory as a 'beta test site' for adapting methodologies developed for virologic food safety testing.