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The purpose of this work is to develop simple, broadly reactive methods to detect the epidemiologically relevant foodborne viruses (NoV and HAV) in candidate foods. The underlying hypothesis is that specific ligands can be designed and applied for the efficient capture (concentration) of viruses from foods, after which the contaminant can be rapidly detected and enumerated using real-time nucleic acid amplification methods. The specific aims are as follows: <ol>
<li> identify broadly reactive ligands that can be used to rapidly and efficiently concentrate HAV and NoV from candidate artificially contaminated food matrices; <li> develop broadly reactive molecular amplification methods that can be applied for the detection of HAV and the NoV derived from food matrices; <li> characterize the sensitivity, specificity, and limit of detection of the combined virus concentration-detection scheme using food products artificially contaminated with representative virus strains; and <li> apply the methods developed to the detection of HAV and NoV in naturally contaminated foods and clinical isolates associated with foodborne outbreaks.
NON-TECHNICAL SUMMARY: Human enteric viruses, i.e., hepatitis A virus and particularly the noroviruses, are the leading cause of foodborne disease. For a number of unique reasons, detection of these viruses in foods is very difficult. There is a need to develop better virus detection methods to facilitate our understanding and control of these important foodborne disease agents. The purpose of this work is to develop broadly reactive and highly sensitive, yet simple and inexpensive, methods to detect the epidemiologically relevant enteric viruses in candidate foods.
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APPROACH: Ligands will be identified using two approaches, i.e., based on previous characterization of virus binding receptors (carbohydrate binding assays) and the selective identification of specific nucleic acid binding molecules (aptamers). Quantitative real-time RT-PCR methods will be further developed with specific consideration of the unique issues associated with the detection of viruses in contaminated foods. The combined virus concentration and detection approach will be evaluated using artifically contaminated food matrices, and also using foods implicated in disease outbreaks. Successful completion this study will result in a simple and inexpensive method to detect the epidemiologically relevant foodborne virueses. Such methods can be used for epidemiological investigation of foodborne viral disease outbreaks; for the tracking of virus transmission during food production, processing and handling; and to evaluate the efficacy of proposed mitigation strategies for foods at-risk of viral contamination.