Persistence, Transfer and Detection of Human Enteric Viruses in Foods.

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Title: Persistence, Transfer and Detection of Human Enteric Viruses in Foods.
Author: Papafragkou, Efstathia Miss
Advisors: Dr. Lee-Ann Jaykus, Committee Chair
Abstract: The Human Noroviruses ( NoVs) and hepatitis A virus (HAV) are currently recognized as the two most epidemiologically important foodborne virus pathogens. They are both highly infectious and can lead to widespread outbreaks. Complicating factors that enhance their wide and easy spread include the fact that theoretically any product could be contaminated with these viral agents, their infectious dose is usually low, and they demonstrate considerable resistance to a number of inactivation agents and processes. Their detection in food, clinical, and environmental samples usually rely on molecular-based methods, as there are no susceptible cell lines yet available that support the proliferation of neither the wild-type HAV, nor the NoVs. The most crucial, and difficult step in virus detection is the extraction of virus particles from the food matrix, as it is of great necessity that during the isolation all the matrix-associated inhibitors are removed. In an effort to address these concerns, four individual studies were designed and conducted. We first considered the Pathatrix system as an alternative sample processing technique to concentrate HAV from representative foods. The virus capture is believed to happen when the negatively charged proteins in the virus capisd bind to the positively charged magnetic particles used in the Pathatrix scheme. We were able to use this machine and consistently concentrate HAV from a variety of at-risk food items including lettuce, green onions, strawberries, deli turkey, oysters, and cake. We further amplified the virus RNA with RT-PCR and confirmed HAV detection with dot-blot hybridization. We were able to detected HAV in all food items at a seeding level of 100PFU per 25g sample, which is consistent with the virus load to be anticipated in naturally contaminated products. We were also able to develop a rapid and quantitative approach to detect HAV in food matrices; when we used a Real-time RT-PCR protocol that included a synthetic HAV RNA standard that allowed for simultaneous quantification of the virus load. Another objective was to investigate the persistence and transfer of NoV within food preparation, by conducting preliminary studies on the virus transfer from stainless steel surfaces to a model RTE food (lettuce). This study verified the ease of NoVs transmission in the environment that is likely to contribute to its spread by foodborne routes. In this study we used a surrogate for performing the mammalian cell infectivity assays, as the NoVs are not cultivable. This approach lead us to our next study which was to evaluate and compare alternative surrogates for NoV that may be used in environmental persistence and transfer studies. In this regard, we assessed virus stability to pH, heat and various organic solvents as well as their persistence on stainless steel surfaces by comparing the behavior of a feline calicivirus (FCV F9) and the recently discovered first mouse norovirus (MNV-1). The major finding was that as MNV-1 was consistently more stable in a wide variety of pHs in comparison to FCV, it may be a more suitable surrogate in studies involving the environmental stability and transmission of the NoVs. Our last objective was to monitor the environmental persistence of HAV through the anticipated shelf-life of representative food items, as well as on surfaces commonly found in food preparation venues. Our studies confirmed that HAV is highly stable in contaminated foods and can survive storage at either room or refrigerator temperature. In addition, our findings indicated that HAV can remain infectious on food preparation surfaces for extended periods of time. Taken together, this research contributes new information about the survival and transfer characteristics of enteric viruses that can assist in the design of effective intervention strategies for lowering the risk of viral foodborne illnesses. In addition, it provides useful methodology for the isolation and study of those agents in a research setting, either on a routine basis or during an outbreak situation.
Date: 2007-12-18
Degree: PhD
Discipline: Food Science
URI: http://www.lib.ncsu.edu/resolver/1840.16/4890


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