Effects of Abiotic and Biotic Stresses on the Internalization and Dissemination of Human Norovirus Surrogates in Growing Romaine Lettuce

Abstract

Human norovirus (NoV) is the major causative agent of fresh-produce-related outbreaks of gastroenteritis; however, the ecology and persistence of human NoV in produce systems are poorly understood. In this study, the effects of abiotic and biotic stresses on the internalization and dissemination of two human NoV surrogates (murine norovirus 1 [MNV-1] and Tulane virus [TV]) in romaine lettuce were determined. To induce abiotic stress, romaine lettuce was grown under drought and flood conditions that mimic extreme weather events, followed by inoculation of soil with MNV-1 or TV. Independently, lettuce plants were infected with lettuce mosaic virus (LMV) to induce biotic stress, followed by inoculation with TV. Plants were grown for 14 days, and viral titers in harvested tissues were determined by plaque assays. It was found that drought stress significantly decreased the rates of both MNV-1 and TV internalization and dissemination. In contrast, neither flood stress nor biotic stress significantly impacted viral internalization or dissemination. Additionally, the rates of TV internalization and dissemination in soil-grown lettuce were significantly higher than those for MNV-1. Collectively, these results demonstrated that (i) human NoV surrogates can be internalized via roots and disseminated to shoots and leaves of romaine lettuce grown in soil, (ii) abiotic stress (drought) but not biotic stress (LMV infection) affects the rates of viral internalization and dissemination, and (iii) the type of virus affects the efficiency of internalization and dissemination. This study also highlights the need to develop effective measures to eliminate internalized viruses in fresh produce. Human norovirus (NoV) is the major cause of nonbacterial gastroenteritis, contributing to Ͼ95% of nonbacterial acute gastroenteritis worldwide and Ͼ60% of all food-borne illnesses reported annually in the United States (1-4). High-risk foods for human NoV contamination include fresh produce, shellfish, and ready-to-eat foods. As individuals are increasingly striving to achieve healthier diets, the consumption of fresh produce has increased in recent years, and fresh produce is now recognized as a leading cause of food-borne illness in the United States (5, 6). Human NoV alone accounts for more than 40% of the freshproduce-related illnesses reported each year in the United States (1, 2, 4, 6-9). Outbreaks in many diverse types of produce, including fresh cut fruit, lettuce, tomatoes, melons, salads, green onions, strawberries, blueberries, raspberries, and salsa, have been attributed to human NoV (6, 7, 10-15). Human NoV is highly infectious, is resistant to common disinfectants, has a low infectious dose, and is highly stable in the environment, all features that contribute to the high prevalence of food-borne outbreaks associated with the virus and its presence and persistence in food commodities (3,5,7,(16)(17)(18)(19).Fresh produce can become contaminated with human NoV at any step from production to processing. In a survey of the point of...

Abiotic Stress and Phyllosphere Bacteria Influence the Survival of Human Norovirus and Its Surrogates on Preharvest Leafy Greens

“…The 14-day study period was chosen because by 7 and 14 days plants can be harvested and because of the importance of the period between a contamination event and harvest time. In addition, the 14-day study period used in our study is similar to or longer than the study periods reported for studies dealing with the survival of HuNoV (7 days) or TV and MNV (14 days) on leaf surfaces (45,46). Collectively, because HuNoV RNA showed a higher degree of stability than MNV, SaV, and TV RNAs and RNA titers correlated significantly with infectivity titers for all surrogates, our results suggest that infectious HuNoVs contaminating leafy greens close to the harvest time may persist until the vegetables are harvested.…”

“…A previous study, however, implemented an extreme weather situation where drought or flooding was applied for 14 days (45). That study showed a negative effect of drought on virus internalization.…”

“…conditions (45). However, only one study investigated the preharvest survival of HuNoV on leaf surfaces, reporting that the virus can persist on the leaves of mature spinach plants and showing no significant differences in viral RNA titers during the 7-day study period (46).…”

“…The first study investigated the role of sunlight (UVA/UVB) on the survival of the surrogate viruses MNV and TV on spinach for 7 days and reported that it had a negative effect only when viruses were inoculated on the adaxial surfaces (46). The other study reported that extreme drought decreased the rate of MNV and TV internalization in lettuce plants (45). However, both studies relied on surrogates and did not assess the survival of HuNoV on plants subjected to abiotic stress.…”

Effect of Leaf Surface Chemical Properties on Efficacy of Sanitizer for Rotavirus Inactivation

“…Viruses, including norovirus and rotavirus, cause foodborne outbreaks, especially due to their persistence in the environment and their low infectious doses (10 to 100 particles) (7,8). Murine norovirus, Tulane virus, and sapovirus have been found to attach to the surface of or within the tissues of romaine lettuce and strawberries (9)(10)(11)(12)(13), which suggests that preharvest viral contamination is a public health concern. To reduce the incidence of viral foodborne illnesses caused by contaminated produce, it is essential to understand the environmental and agricultural factors that control the stability and, therefore, the infectivity of foodborne viruses.…”