Internalization and Dissemination of Human Norovirus and Animal Caliciviruses in Hydroponically Grown Romaine Lettuce

bHuman norovirus (HuNoV) is the leading cause of foodborne illnesses, with an increasing number of outbreaks associated with leafy greens. Because HuNoV cannot be routinely cultured, culturable feline calicivirus (FCV), murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV) have been used as surrogates. These viruses are generated in different cell lines as infected cell lysates, which may differentially affect their stability. Our objective was to uniformly compare the survival of these viruses on postharvest lettuce while evaluating the effects of cell lysates on their survival. Viruses were semipurified from cell lysates by ultrafiltration or ultracentrifugation followed by resuspension in sterile water. Virus survival was examined before and after semipurification: in suspension at room temperature (RT) until day 28 and on lettuce leaves stored at RT for 3 days or at 4°C for 7 and 14 days. In suspension, both methods significantly enhanced the survival of all viruses. On lettuce, the survival of MNV in cell lysates was similar to that in water, under all storage conditions. In contrast, the survival of FCV, SaV, and TV was differentially enhanced, under different storage conditions, by removing cell lysates. Following semipurification, viruses showed similar persistence to each other on lettuce stored under all conditions, with the exception of ultracentrifugation-purified FCV, which showed a higher inactivation rate than MNV at 4°C for 14 days. In conclusion, the presence of cell lysates in viral suspensions underestimated the survivability of these surrogate viruses, while viral semipurification revealed similar survivabilities on postharvest lettuce leaves. Human noroviruses (HuNoVs) are the leading cause of acute viral gastroenteritis in the United States (1). These viruses are 28 to 35 nm in diameter, nonenveloped, single-stranded RNA viruses that are members of the Caliciviridae family. An increasing number of HuNoV outbreaks have been associated with leafy greens (2). For example, food was the primary vehicle of transmission in 1,008 HuNoV outbreaks reported between 2009 and 2012 in the United States, and leafy greens were the primary food associated with these outbreaks (1). Therefore, there is a need to understand the occurrence and survival/persistence of HuNoV on leafy greens. However, assessing the survival of HuNoVs on leafy greens is hampered by the fact that these viruses are still refractory to routine growth in cell culture; hence, their infectivity cannot be readily quantified (3, 4). Therefore, surrogate culturable viruses are often used as proxies to investigate HuNoV transmission routes, disinfection, and survival in the environment.Among members of the Caliciviridae family, feline calicivirus (FCV) has been the most widely used HuNoV surrogate since the 1990s (5). In 2004, murine norovirus (MNV), a virus genetically more closely related to HuNoV, was propagated in cell culture (6) and, since then, it has been widely used as a HuNoV surrogate (7). In 2004, bile acids in i...

Section: Discussionmentioning

confidence: 99%

Feline Calicivirus, Murine Norovirus, Porcine Sapovirus, and Tulane Virus Survival on Postharvest Lettuce

Esseili

Saif

Farkas

et al. 2015

“…Because of the speed with which it acts, this technology offers several advantages over continuous UV treatment. It could be used to deal with contamination issues, including contamination of shellfish by municipal sewage released into marine product growing areas (47) and contamination of vegetables by irrigation (48,49).…”

Section: CMmentioning

confidence: 99%

Efficacy and Mechanisms of Murine Norovirus Inhibition by Pulsed-Light Technology

Vimont

Fliss

Jean

2015

Pulsed light is a nonthermal processing technology recognized by the FDA for killing microorganisms on food surfaces, with cumulative fluences up to 12 J cm ؊2 . In this study, we investigated its efficacy for inactivating murine norovirus 1 (MNV-1) as a human norovirus surrogate in phosphate-buffered saline, hard water, mineral water, turbid water, and sewage treatment effluent and on food contact surfaces, including high-density polyethylene, polyvinyl chloride, and stainless steel, free or in an alginate matrix. The pulsed-light device emitted a broadband spectrum (200 to 1,000 nm) at a fluence of 0.67 J cm ؊2 per pulse, with 2% UV at 8 cm beneath the lamp. Reductions in viral infectivity exceeded 3 log 10 in less than 3 s (5 pulses; 3.45 J cm ؊2 ) in clear suspensions and on clean surfaces, even in the presence of alginate, and in 6 s (11 pulses; 7.60 J cm ؊2 ) on fouled surfaces except for stainless steel (2.6 log 10 ). The presence of protein or bentonite interfered with viral inactivation. Analysis of the morphology, the viral proteins, and the RNA integrity of treated MNV-1 allowed us to elucidate the mechanisms involved in the antiviral activity of pulsed light. Pulsed light appeared to disrupt MNV-1 structure and degrade viral protein and RNA. The results suggest that pulsed-light technology could provide an effective alternative means of inactivating noroviruses in wastewaters, in clear beverages, in drinking water, or on food-handling surfaces in the presence or absence of biofilms.H uman noroviruses are responsible for one-fifth of all cases of acute gastroenteritis worldwide (1). They spread directly via person-to-person contact (fecal-oral and vomitus-oral) or indirectly through food, water, and the environment (2). Natural biofilms in sewage treatment effluent could be responsible for many persistent waterborne outbreaks, since such biofilms have been found to harbor noroviruses (3, 4). Infection spreads quickly and widely, affecting individuals of all ages, especially where many people gather or live in close proximity, such as in long-term care residences, retirement homes, cruise ships, and the like. Infectious doses as low as 2,800 particles, the high viral loads in feces and vomit (up to 10 9 genomic copies per gram), persistence in the environment, prolonged duration of viral shedding even after symptoms have resolved, and inadequate long-term immunity all contribute to the high incidence of norovirus illness (5). Although the illness is usually mild and self-limiting, the large number of cases per year represents a considerable loss in productivity and constitutes a substantial burden on society (2, 6).Among the physical disinfection processes, pulsed-light treatment appears well suited for the disinfection of food contact surfaces in industrial and health care settings and for decontaminating water and beverages. It has been proposed as a means of nonthermal pasteurization for food preservation and for decontaminating air and packaging materials (7). It has proven effective for inactivati...

“…A number of studies reported that HuNoV and its surrogates can internalize inside lettuce and spinach plants under normal conditions (28,43,44) and in lettuce under certain biotic and extreme abiotic stress a Numbers in bold indicate a significant effect (P Ͻ 0.05) for that factor. Statistical analyses were performed by two-way ANOVA.…”

Section: Discussionmentioning

confidence: 99%

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

Esseili

Gao

Tegtmeier

et al. 2016

Foodborne outbreaks of human noroviruses (HuNoVs) are frequently associated with leafy greens. Because there is no effective method to eliminate HuNoV from postharvest leafy greens, understanding virus survival under preharvest conditions is crucial. The objective of this study was to evaluate the survival of HuNoV and its surrogate viruses, murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV), on preharvest lettuce and spinach that were subjected to abiotic stress (physical damage, heat, or flood). We also examined the bacteria culturable from the phyllosphere in response to abiotic stress and in relation to viral persistence. Mature plants were subjected to stressors 2 days prior to inoculation of the viruses on leaves. We quantified the viral RNA, determined the infectivity of the surrogates, and performed bacterial counts on postinoculation days (PIDs) 0, 1, 7, and 14. For both plant types, time exerted significant effects on HuNoV, MNV, SaV, and TV RNA titers, with greater effects being seen for the surrogates. Infectious surrogate viruses were undetectable on PID 14. Only physical damage on PID 14 significantly enhanced HuNoV RNA persistence on lettuce, while the three stressors differentially enhanced the persistence of MNV and TV RNA. Bacterial counts were significantly affected by time and plant type but not by the stressors. However, bacterial counts correlated significantly with HuNoV RNA titers on spinach and with the presence of surrogate viruses on both plant types under various conditions. In conclusion, abiotic stressors and phyllosphere bacterial density may differentially influence the survival of HuNoV and its surrogates on lettuce and spinach, emphasizing the need for the use of preventive measures at the preharvest stage.H uman noroviruses (HuNoVs) are the leading cause of acute gastroenteritis and foodborne illnesses in the United States, causing 19 million to 21 million cases, 570 to 800 deaths, and $777 million in health care-associated costs annually (1). These singlestranded RNA viruses belong to the Caliciviridae family and are 28 to 35 nm in diameter and nonenveloped. Although these viruses cause self-limiting gastroenteritis, more serious infections can develop in high-risk groups, such as the elderly and immunocompromised populations (2). The virus is mainly transmitted to humans via the fecal-oral route either by contact with an infected person or fomites or by ingestion of contaminated food and water (2). Leafy greens are frequently associated with HuNoV outbreaks (1) and are globally recognized to be a high priority in terms of the microbial safety of fresh produce (3). Contamination of leafy greens with HuNoVs can occur at any stage along the farm-to-fork chain (3) through a number of sources, including fecally contaminated water used for irrigation, improperly treated sewage sludge used for fertilization, and food harvesters or food handlers who use improper hygiene practices (4, 5). Because HuNoVs have a low infectious dose (ϳ18 to 1,000 viral particles) (6) ...