Search

Foodborne viral contamination of fresh produce has been associated with numerous outbreaks. Detection of such contaminated foods is important in protecting public health. Here, we demonstrate for the first time the capability of the U.S. Food and Drug Administration Enteric Viruses tiling microarray (FDA-EVIR) to perform rapid molecular identification of hepatitis A virus (HAV) and human norovirus extracted from artificially inoculated fresh produce. Two published viral extraction strategies, total RNA extraction or virus particle isolation, were used to prepare the viral targets. The total RNA extraction method was used on material eluted from tomatoes, using an alkaline Tris-glycine-beef extract (TGBE) buffer. Optimization procedures including DNase treatment and poly(A)-RNA enrichment were adopted to improve microarray sensitivity. For green onions or celery, material was eluted using either glycine buffer or TGBE buffer supplemented with pectinase, respectively, and then virus particles were concentrated by ultracentrifugation. We also assessed the amount of viral RNA extracted from celery using three commercially available kits and how well that RNA performed on FDA-EVIR. Our results confirm that FDA-EVIR can identify common enteric viruses isolated from fresh produce when present as either a single or mixed species of viruses. Using total RNA extraction from tomatoes yielded a limit of detection of 1.0 × 105 genome equivalents (ge) of HAV per array input. The limit of detection for viral RNA obtained using ultracentrifugation was 1.2 × 105 ge of HAV from green onions and 1.0 × 103 ge of norovirus from celery per array input. Extending microarray methods to other food matrices should provide important support to surveillance and outbreak investigations. HIGHLIGHTS

Reduction of foodborne illness caused by norovirus (NoV) continues to be a focus for the food safety community. Using a previously published quantitative risk assessment model, we evaluated more than 60 scenarios examining the impact of implementation of and compliance with risk management strategies identified in the U.S. Food and Drug Administration Food Code for (a) surface cleaning and sanitizing, (b) hand hygiene, (c) exclusion, or (d) restriction of ill employees. Implementation of and compliance with hand hygiene and ill food employee exclusion strategies had the largest impact on the predicted number of highly contaminated food servings and associated consumer illnesses. In scenarios in which gloves were always worn and hand washing compliance was 90%, the model estimated reductions in the number of highly contaminated food servings and ill consumers to 39 and 43% of baseline estimates (i.e., typical practice), respectively. Reductions were smaller when gloves were never worn. Hand washing compliance after using the restroom strongly impacted predicted numbers of highly contaminated servings and consumer illnesses. Ten percent compliance with removing or excluding ill food employees was predicted to increase the number of highly contaminated food servings and ill consumers to 221 and 213% of baseline estimates, respectively. Ninety-four percent compliance with exclusion of ill food employees was predicted to decrease these numbers to 69 and 71% of baseline estimates, respectively. Surface cleaning in food establishments had a relatively small impact on these measures. Restriction of food employees (removed from contact with food and food contact equipment and utensils) was not effective for reducing NoV illness unless this restriction included additional provisions. The results from this study can help risk managers prioritize mitigation strategies and their implementation for controlling the transmission of NoV and subsequent consumer foodborne illness.

We developed a quantitative risk assessment model using a discrete event framework to quantify and study the risk associated with norovirus transmission to consumers through food contaminated by infected food employees in a retail food setting. This study focused on the impact of ill food workers experiencing symptoms of diarrhea and vomiting and potential control measures for the transmission of norovirus to foods. The model examined the behavior of food employees regarding exclusion from work while ill and after symptom resolution and preventive measures limiting food contamination during preparation. The mean numbers of infected customers estimated for 21 scenarios were compared to the estimate for a baseline scenario representing current practices. Results show that prevention strategies examined could not prevent norovirus transmission to food when a symptomatic employee was present in the food establishment. Compliance with exclusion from work of symptomatic food employees is thus critical, with an estimated range of 75–226% of the baseline mean for full to no compliance, respectively. Results also suggest that efficient handwashing, handwashing frequency associated with gloving compliance, and elimination of contact between hands, faucets, and door handles in restrooms reduced the mean number of infected customers to 58%, 62%, and 75% of the baseline, respectively. This study provides quantitative data to evaluate the relative efficacy of policy and practices at retail to reduce norovirus illnesses and provides new insights into the interactions and interplay of prevention strategies and compliance in reducing transmission of foodborne norovirus.

Although the incidence rate of hepatitis A virus (HAV) infection has been on the decline in developed countries, in part due to immunization availability, high profile outbreaks continue to be reported. Hepatitis E virus has been recognized as an emerging pathogen in industrialized countries. While associated with waterborne illnesses, particularly in undeveloped countries, several animal species have been identified as reservoirs for the virus. Consequently, the potential of zoonotic transmission exists as a function of the consumption of infected animals. In this review we provide a comparative overview of these two virus species with regard to their known virus properties, discuss extraction methodologies, and describe some basic principles and methodology applied toward the isolation of these viruses (as particles or their isolated genomes) from food commodities. We also discuss the challenges that remain as experimental hurdles to extraction of such viruses from food. As HAV has been the most extensively studied with regard to virus detection in foods, it often serves as a model virus for current and future development of sample preparation methodology for foodborne virus detection. Lastly, we discuss the application and role of current and developing technologies in the post-extraction detection and identification of these viruses from foods.

The development of rapid and sensitive detection methods for human noroviruses (HuNoV) in produce items is critical, especially with the recent rise in outbreaks associated with this food commodity. In this study, 50-g portions of various produce items linked to a norovirus outbreak (celery, cucumber, lettuce, grapes, and radish) were artificially inoculated with murine norovirus (MNV-1) and concentrated either by ultracentrifugation or polyethylene glycol (PEG) precipitation after elution with an alkaline Tris-glycine-beef extract buffer supplemented with pectinase. As a viral concentration step following virus elution and clarification, ultracentrifugation yielded a faster method (

Next generation sequencing (NGS) holds promise as a single application for both detection and sequence identification of foodborne viruses; however, technical challenges remain due to anticipated low quantities of virus in contaminated food. In this study, with a focus on data analysis using several bioinformatics tools, we applied NGS toward amplification-independent detection and identification of norovirus at low copy (

Analysis of enterovirus infection is difficult in animals because they express different virus receptors than humans, and static cell culture systems do not reproduce the physical complexity of the human intestinal epithelium. Here, using coxsackievirus B1 (CVB1) as a prototype enterovirus strain, we demonstrate that human enterovirus infection, replication and infectious virus production can be analyzed in vitro in a human Gut-on-a-Chip microfluidic device that supports culture of highly differentiated human villus intestinal epithelium under conditions of fluid flow and peristalsis-like motions. When CVB1 was introduced into the epithelium-lined intestinal lumen of the device, virions entered the epithelium, replicated inside the cells producing detectable cytopathic effects (CPEs), and both infectious virions and inflammatory cytokines were released in a polarized manner from the cell apex, as they could be detected in the effluent from the epithelial microchannel. When the virus was introduced via a basal route of infection (by inoculating virus into fluid flowing through a parallel lower 'vascular' channel separated from the epithelial channel by a porous membrane), significantly lower viral titers, decreased CPEs, and delayed caspase-3 activation were observed; however, cytokines continued to be secreted apically. The presence of continuous fluid flow through the epithelial lumen also resulted in production of a gradient of CPEs consistent with the flow direction. Thus, the human Gut-on-a-Chip may provide a suitable in vitro model for enteric virus infection and for investigating mechanisms of enterovirus pathogenesis.

The human noroviruses (NoV) are the major cause of acute non-bacterial gastroenteritis and are commonly transmitted by foodborne routes. Epidemiological evidence from propagated outbreaks, as well as environmental sampling, suggest that these viruses are environmentally stable. The purpose of this study was to examine the persistence of representative human NoV on the fingertips of volunteers and on commonly used food preparation surfaces. Human fingerpads and surfaces (stainless steel, Formica®, and ceramic) were inoculated with 20% fecal suspensions of Norwalk virus (NV) or Snow Mountain virus (SMV). The virus inocula were recovered by elution at serial time points ranging from 0 to 120 min post-inoculation (for fingerpads) and after up to 42 days (for surfaces). The quantity of detectable viral RNA, expressed as genome equivalent particles (GEP) was evaluated using quantitative real-time RT-PCR (RT-qPCR). The amount of NV RNA on the surface decreased gradually over time, with an average reduction ranging from 1.5 to 2.9 log10 GEP after 21–28 days storage under ambient conditions. SMV showed greater environmental persistence, with a 0.4–1.2 log10 GEP reduction on all three surfaces after 42 days of ambient storage. On fingerpads, the amount of human NoV RNA declined slightly (

The number of foodborne outbreaks linked to the consumption of fresh produce has been on the rise (Gould et al. 2013). Over 45 % of outbreaks of known etiology reported to the Centers for Disease Control and Prevention (CDC) between 1998 and 2008 were caused by foodborne viruses, especially human norovirus (HuNoV) and to a lesser extent hepatitis A virus (HAV) (Gould et al. 2013). In one recent study, a majority of viral outbreaks were attributed to the consumption of leafy vegetables (35 %) or fruits and nuts (15 %) (Painter et al. 2013). Contamination of fresh produce may occur prior to distribution in the wholesale or retail market through contaminated irrigation water, poor hygiene of harvesters, and/or during the processing and packaging steps due to improperly sanitized equipment or wash water. Contamination during production and processing has recently led to large scale outbreaks of HuNoV associated with strawberries in 2012 (Bernard et al. 2014; Made et al. 2013) and of HAV in green onions in 2003 (Wheeler et al. 2005).

The noroviruses (NoV) are a common cause of human gastroenteritis whose transmission by foodborne routes is well documented. Fecally contaminated surfaces are likely to contribute to this foodborne transmission and to the propagation of viral disease outbreaks. The purpose of this study was to (i) investigate the stability of NoV on various food preparation surfaces; and (ii) evaluate the degree of virus transfer from these surfaces to a model-ready-to-eat (RTE) food. For the virus persistence experiments, stainless steel, formica and ceramic coupons were artificially contaminated with Norwalk virus (NV), the prototype genogroup I NoV; NV RNA; or feline calicivirus (FCV) F9 (a NoV surrogate), stored at ambient temperature for up to 7 d, and periodically assayed for detection. In the transfer experiments, stainless steel coupons were inoculated with NV or FCV F9 and allowed to dry for 10, 30 and 60 min, after which lettuce leaves were exposed to the surface of the coupons at various contact pressures (10, 100, and 1000 g/9 cm2). Virus recovery was evaluated by RT-PCR (for NV and NV RNA) or by plaque assay (for FCV F9) using Crandell Reese Feline Kidney (CRFK) cells. NV and FCV were detected on all three surfaces for up to 7 d post-inoculation; for FCV, there was an approximate 6 to 7-log10 drop in virus titer over the 7 d evaluation period. By contrast, when stainless steel was inoculated with purified NV RNA, RT-PCR detection was not possible beyond 24 h. Transfer of both NV and FCV from stainless steel surfaces to lettuce occurred with relative ease. This study confirms lengthy NoV persistence on common food preparation surfaces and their ease of transfer, confirming a potential role for environmental contamination in the propagation of viral gastroenteritis.

Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407) or human epithelial colorectal adenocarcinoma cells (Caco-2) growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D) cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin). Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8). At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus.

Recently, there have been increasing reports of foodborne illnesses associated with the consumption of fresh produce. Among these, hepatitis A virus (HAV) remains epidemiologically important and has been continually implicated in several outbreaks. We describe a rapid method (

Hepatitis A virus (HAV) is an important cause of foodborne disease worldwide. The detection of this virus in naturally contaminated food products is complicated by the absence of a reliable culture method, low levels of contamination, and the presence of matrix-associated compounds which inhibit molecular detection. In this study, we report a novel method to concentrate HAV from foods prior to the application of reverse transcription-PCR (RT-PCR) for detection. Specifically, we used cationically charged magnetic particles with an automated capture system (Pathatrix™) to concentrate the virus from 25 g samples of artificially contaminated lettuce, strawberries, green onions, deli-turkey, oysters, and cake with frosting. Detection limits varied according to the product but in most cases, the virus could be consistently detected at input levels corresponding to 10 2 PFU/25 g food sample. For some products, detection was possible at levels as low as 10 −1 PFU/25 g. The assay was applied by a second independent laboratory and was also used to confirm viral contamination of produce items associated with a recent HAV outbreak. Parallel infectivity assays demonstrated that the cationically charged particles bound approximately 50% of the input virus. This is the first application of the automated magnetic capture technology to the concentration of viruses from foods, and it offers promise for facilitating the rapid detection of HAV from naturally contaminated products.

Human noroviruses (NoVs) are the leading cause of food- and waterborne outbreaks of acute nonbacterial gastroenteritis worldwide. As a result of the lack of a mammalian cell culture model for these viruses, studies on persistence, inactivation, and transmission have been limited to cultivable viruses, including feline calicivirus (FCV). Recently, reports of the successful cell culture of murine norovirus 1 (MNV-1) have provided investigators with an alternative surrogate for human NoVs. In this study, we compared the inactivation profiles of MNV-1 to FCV in an effort to establish the relevance of MNV-1 as a surrogate virus. Specifically, we evaluated (i) stability upon exposure to pH extremes; (ii) stability upon exposure to organic solvents; (iii) thermal inactivation; and (iv) surface persistence under wet and dry conditions. MNV-1 was stable across the entire pH range tested (pH 2 to 10) with less than 1 log reduction in infectivity at pH 2, whereas FCV was inactivated rapidly at pH values3 and9. FCV was more stable than MNV-1 at 56 degrees C, but both viruses exhibited similar inactivation at 63 and 72 degrees C. Long-term persistence of both viruses suspended in a fecal matrix and inoculated onto stainless steel coupons were similar at 4 degrees C, but at room temperature in solution, MNV-1 was more stable than FCV. The genetic relatedness of MNV-1 to human NoVs combined with its ability to survive under gastric pH levels makes this virus a promising and relevant surrogate for studying environmental survival of human NoVs.

Spores of Bacillus subtilis lacking all germinant receptors germinate >500-fold slower than wild-type spores in nutrients and were not induced to germinate by a pressure of 100 MPa. However, a pressure of 550 MPa induced germination of spores lacking all germinant receptors as well as of receptorless spores lacking either of the two lytic enzymes essential for cortex hydrolysis during germination. Complete germination of spores either lacking both cortex-lytic enzymes or with a cortex not attacked by these enzymes was not induced by a pressure of 550 MPa, but treatment of these mutant spores with this pressure caused the release of dipicolinic acid. These data suggest the following conclusions: (i) a pressure of 100 MPa induces spore germination by activating the germinant receptors; and (ii) a pressure of 550 MPa opens channels for release of dipicolinic acid from the spore core, which leads to the later steps in spore germination.