Enteric viral interactions on fresh produce

Foodborne viruses are an important cause of gastroenteritis in the US and worldwide and have been the cause of major outbreaks associated with produce commodities. Among enteric viruses, human noroviruses and hepatitis A virus are the leading cause of foodborne illness with human adenoviruses also responsible for a large portion of disease. All three viruses are transmitted through the fecal-oral route and are a concern for produce contamination. Spinach and green onions have been implicated in major outbreaks in the last decade which has brought them to the forefront as commodities that are high-risk for pathogen contamination. In efforts to reduce contamination events Good Agricultural Practices are commonly used and the Food and Drug Administration produce regulations are under development; however, despite large efforts by researchers to understand these interactions of human pathogens with plants, the variety of growing conditions, commodities and cultivars, and practices employed for plant health reasons makes developing best practices for safe produce production a challenge. In efforts to better prevent outbreaks from foodborne enteric viruses, an understanding of the potential interactions of pathogens with produce commodities at risk is important. Many researchers have focused on bacterial pathogens, but little scientific data exists on the viral interactions with fresh produce. In addition, assessing norovirus infectivity still poses a challenge and impacts the ability to assess virus stability and processing technologies. The objectives of this project were to 1) understand the interaction of viral pathogens on spinach and green onions, through assessing the potential for root uptake in soil and hydroponic systems; 2) address how these internalized viruses are affected by post-harvest processing intervention strategies including traditional chlorine washes as compared to novel non-thermal processing technologies of UV, ozone and high pressure; 3) develop an assay to determine norovirus infectivity through the use of norovirus attachment properties and 4) understand what factors affect norovirus survival on spinach leaves. If contamination were to occur in the soil in the field either through contaminated irrigation water, flooding, or manure/biosolid application, viruses are unlikely to be internalized into spinach or green onion tissues. However; if hydroponic growing systems, either nutrient film technique or floating systems, become contaminated through inputs or foodhandlers, internalization of enteric viruses to titers comparable to an infectious dose is likely. Once viruses are internalized into green onions, they were shown to be protected from inactivation by traditional chlorine sprays; however, overall, no significant difference in nonthermal processing technologies inactivation of internalized MNV, HAV and Ad41 as compared to externally inoculated viruses by UV, ozone, and pressure were observed. These results indicate that these novel nonthermal technologies are more effective at reducing human enteric viruses present both externally and internalized into green onions. Factors affecting norovirus surrogate survival on spinach leaves was assessed if contamination were to occur through irrigation or rain splash. In addition to the commonly used murine norovirus surrogate, a newly discovered Calicivirus, Tulane virus, was used and viruses persisted on spinach plants for up to 14 days. No significant difference was observed between virus type, spinach cultivar (smooth leaf or semi-savoy leaf); however, norovirus surrogate survival was significantly lower on adaxial leaf surfaces as compared to abaxial leaf surfaces. Scanning electron microscopy images showed differences between these leaf surfaces that might explain the differences observed in virus survival. Exposure to UVA/UVB significantly impacted virus survival on smooth spinach leaves, but not on semi-savoy spinach leaves. Lastly, one of the major problems assessing norovirus persistence in the environment and inactivation by food processing technologies is due to the fact that human noroviruses are non-cultivable. Using the attachment properties of human noroviruses, an Enzyme-linked Immunosorbant Assay (ELISA) was developed to determine if infectivity correlated with attachment of the virus capsid to carbohydrate cellular receptors. Results show that processes, such as heat, whereby the capsid was destroyed is necessary to correlate attachment with infectivity. However, processes that mildly affect viral capsid proteins such as UV, ozone and pressure, were not correlated with infectivity. Given the low infectious dose of these enteric viruses, even a low amount of contamination, survival or internalization has the potential to cause illness.