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The overall goal in this project is to obtain a better understanding of the transmission of human noroviruses (NoV) from the environment to food and subsequently to the public. <P>Specific objectives: 1. Characterize novel surrogates for the study of human noroviruses. 2. Investigate environmental reservoirs for the transmission human norovirus surrogates via fresh produce. From this project, it is expected that 1) better NoV surrogates will be identified and utilized for the development of mitigation strategies to control NoV in our food systems and 2) the interaction of NoV with fresh produce via association with produce "microflora" will be established.
Non-Technical Summary:<br/>
An estimated 9.4 million episodes of foodborne illnesses due to known etiologies occur annually in the United States of which 5.45 million (58%) are caused by human norovirus (NoV). Food types most commonly associated with NoV outbreaks include ready to eat foods and in general, foods that are subject to minimal processing. Between 1990-2005, 64, 67, and 47% of outbreaks in the U.S. involving greens-based salads, fruits, and lettuce, respectively, were attributed to NoV, by far exceeding the contribution of all bacterial and protozoan foodborne pathogens. One of the major challenges the food industry faces today with respect to controlling NoV is the lack of 1) sensitive and specific detection methodologies for screening foods and water for virus contamination; and 2) a comprehensive understanding of environmental transmission. This research explores the characterization and use of novel NoV surrogates in research to understand transmission of NoV in fresh produce packinghouse environments.
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Approach:<br/>
Virus [Tulane virus (TV), porcine sapovirus (PoSV)] survival at 37, 56, and 72 degrees C will be evaluated over various time points up to 5 days. Virus stability at pH 2, 3, 7, 9, and 10 will be evaluated at room temperature (RT) and 37 degrees C over various time points up to 3 hrs. Virus stability when exposed to 3 different concentrations each of peroxide, bleach, and ethanol will be evaluated at room temperature over various time points up to 1 hour. Virus survival in soil and water matrices will be evaluated at various time periods, temperatures, and levels of relative humidity. Virus persistence in surface water and groundwater will be determined at 4 degrees C and RT by inoculating the water samples at 105 PFU/ml. The samples will be incubated in the dark with continuous mixing for up to 5 weeks. Periodically, 1.5 ml subsamples will be removed and analyzed for virus infectivity by plaque assay using the corresponding host cell line. For virus survival in soil matrices, various types of soil will be collected, characterized (e.g., nutrients, metal oxides, conductivity, saturation pH, etc.), and inoculated with prepared virus surrogates at 105 PFU/g in a total of 5 g. The soil samples will be incubated for up to 8 weeks at optimal temperatures for growing leafy greens (i.e. 15 to 18 degrees C) as well as at 4, 10 and 25 degrees C. At each predetermined time point, subsamples will be removed and organic solvent extraction of viruses from the soil matrix will be applied. For all experiments the samples will be analyzed for virus infectivity by plaque assay.
<br/>Obj 2: Free-living amoebae (FLA) and virus interactions will be evaluated. For co-culture of amoebae with NoV surrogates, experiments will be performed in suspension with a multiplicity of infection [MOI] of 0.33. The amoebae - virus suspensions will be incubated for one hour at 30 degrees C followed by centrifugation to pellet amoebic cells. The pellet will then be washed with PBS in order to remove unabsorbed viruses and maintained at 30 degrees C for up to 6 days. During this 6-day incubation, samples will be taken at 0, 6, 24, 48, 72, 96, 120, and 144 hours post-infection to determine virus titer within amoebae as well as cell-free supernatant. A plaque assay technique will be used to detect infectious virus particles. The transfer efficiency of FLA (with and without viruses) in water and on various surfaces to fresh produce will be determined. For transfer of FLA in water to fresh produce (i.e., leafy greens), experiments will be conducted with both ATCC and a subsample of wild-type Acanthamoebae spp. High and low level contamination experiments for each FLA and produce type will be repeated in triplicate. For transfer of FLA from contaminated surfaces to fresh produce, experiments will be conducted with both ATCC and a subsample of wild-type type FLA. For each FLA, 5 by 5 cm coupons of stainless steel or rubber conveyor belt material will be inoculated with high (106) or low (103) levels of FLA. High and low level contamination experiments for each FLA and produce type will be repeated 3 times.