Quantitative Risk Assessment of Norovirus Transmission in Food Establishments: Evaluating the Impact of Intervention Strategies and Food Employee Behavior on the Risk Associated with Norovirus in Foods.

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.

Whole-Genome Sequencing-Based Confirmatory Methods on RT-qPCR Results for the Detection of Foodborne Viruses in Frozen Berries.

Accurate detection, identification, and subsequent confirmation of pathogens causing foodborne illness are essential for the prevention and investigation of foodborne outbreaks. This is particularly true when the causative agent is an enteric virus that has a very low infectious dose and is likely to be present at or near the limit of detection. In this study, whole-genome sequencing (WGS) was combined with either of two non-targeted pre-amplification methods (SPIA and SISPA) to investigate their utility as a confirmatory method for RT-qPCR positive results of foods contaminated with enteric viruses. Frozen berries (raspberries, strawberries, and blackberries) were chosen as the food matrix of interest due to their association with numerous outbreaks of foodborne illness. The hepatitis A virus (HAV) and human norovirus (HuNoV) were used as the contaminating agents. The non-targeted WGS strategy employed in this study could detect and confirm HuNoV and HAV at genomic copy numbers in the single digit range, and in a few cases, identified viruses present in samples that had been found negative by RT-qPCR analyses. However, some RT-qPCR-positive samples could not be confirmed using the WGS method, and in cases with very high Ct values, only a few viral reads and short sequences were recovered from the samples. WGS techniques show great potential for confirmation and identification of virally contaminated food items. The approaches described here should be further optimized for routine application to confirm the viral contamination in berries.

Use of Human Intestinal Enteroids for Recovery of Infectious Human Norovirus from Berries and Lettuce.

Norovirus (NoV) is the leading cause of viral foodborne gastroenteritis globally. Currently, the gold standard for detecting NoV in clinical, food, and environmental samples is via molecular-based methods, primarily RT-PCR. Nevertheless, there is a great need for confirmatory assays that can determine the infectivity of viral particles recovered from contaminated matrices. The use of the human intestinal enteroids system (HIEs) has allowed for the expansion of norovirus replication, although it still suffers from limitations of strain preferences and the requirement of high titer stocks for infection. In this study, we wanted to explore the feasibility of using the HIEs to support the replication of NoV that had been recovered from representative food matrices that have been associated with foodborne illness. We first confirmed that HIEs can support the replication of several strains of NoV as measured by RT-qPCR. We subsequently chose two of those strains that reproducibly replicated, GII.4 and GII.6, to evaluate in a TCID50 assay and for future experiments. Infectious NoV could be recovered and quantified in the HIEs from lettuce, frozen raspberries, or frozen strawberries seeded with high titers of either of these strains. While many experimental challenges still remain to be overcome, the results of this study represent an important step toward the detection of infectious norovirus from representative produce items.

Evaluation of the Impact of Compliance with Mitigation Strategies and Frequency of Restaurant Surface Cleaning and Sanitizing on Control of Norovirus Transmission from Ill Food Employees Using an Existing Quantitative Risk Assessment Model.

ABSTRACT: 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.

Evaluation of U.S. Food and Drug Administration Enteric Viruses Microarray for Detection of Hepatitis A Virus and Norovirus in Inoculated Tomatoes, Green Onions, and Celery.

ABSTRACT: 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.

Rapid and sensitive detection of hepatitis A virus in representative food matrices.

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.

Testing for Human Norovirus and Recovery of Process Control in Outbreak-Associated Produce Items.

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 (<8 h, including reverse transcription quantitative PCR), with MNV-1 recoveries similar to or better, than those obtained with PEG precipitation. The addition of polyvinylpyrrolidone to the elution buffer, to remove polyphenolic inhibitors, improved MNV-1 recoveries by over two- and fivefold for cucumber and grapes, respectively. However, despite MNV-1 recoveries ranging from 10 to 38% as calculated with 10-fold diluted RNA, contaminating HuNoV was not detected in any of the outbreak-associated samples tested. For store-bought produce samples, the limit of detection for artificially seeded HuNoV GII.4 was determined to be 103 copies per 50 g, with reproducible detection achieved in grapes, radish, and celery. The results support the use of ultracentrifugation as an alternative approach to PEG precipitation to concentrate norovirus from a variety of produce items.

Application of next generation sequencing toward sensitive detection of enteric viruses isolated from celery samples as an example of produce.

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 (<103 copies) or within multiple strains from produce. Celery samples were inoculated with human norovirus (stool suspension) either as a single norovirus strain, a mixture of strains (GII.4 and GII.6), or a mixture of different species (hepatitis A virus and norovirus). Viral RNA isolation and recovery was confirmed by RT-qPCR, and optimized for library generation and sequencing without amplification using the Illumina MiSeq platform. Extracts containing either a single virus or a two-virus mixture were analyzed using two different analytic approaches to achieve virus detection and identification. First an overall assessment of viral genome coverage for samples varying in copy numbers (1.1×103 to 1.7×107) and genomic content (single or multiple strains in various ratios) was completed by reference-guided mapping. Not unexpectedly, this targeted approach to identification was successful in correctly mapping reads, thus identifying each virus contained in the inoculums even at low copy (estimated at 12 copies). For the second (metagenomic) approach, samples were treated as "unknowns" for data analyses using (i) a sequence-based alignment with a local database, (ii) an "in-house" k-mer tool, (iii) a commercially available metagenomics bioinformatic analysis platform cosmosID, and (iv) an open-source program Kraken. Of the four metagenomics tools applied in this study, only the local database alignment and in-house k-mer tool were successful in detecting norovirus (as well as HAV) at low copy (down to <103 copies) and within a mixture of virus strains or species. The results of this investigation provide support for continued investigation into the development and integration of these analytical tools for identification and detection of foodborne viruses.

Human Gut-On-A-Chip Supports Polarized Infection of Coxsackie B1 Virus In Vitro.

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.

Persistence of caliciviruses on environmental surfaces and their transfer to food.

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.

Surrogates for the study of norovirus stability and inactivation in the environment: aA comparison of murine norovirus and feline calicivirus.

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 values < 3 and > 9. 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.

Review: Approaches to the Viral Extraction, Detection, and Identification of Hepatitis Viruses, HAV and HEV, in Foods.

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.

Challenges of culturing human norovirus in three-dimensional organoid intestinal cell culture models.

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 ß-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.

Development of a rapid total nucleic acid extraction method for the isolation of hepatitis A virus from fresh produce.

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 (<8h) for the isolation and subsequent detection with real-time quantitative PCR (RT-qPCR) of the HAV HM-175 cytopathic strain seeded onto baby spinach and sliced tomatoes using a total RNA extraction method, utilizing a high concentration (4M) guanidine thiocyanate buffer. Consistent detection of HAV genome from both produce items was achieved at an inoculation level of 3×10³ PFU/25 g of food, with less consistent detection achieved at 3×10² PFU/25g. Initial studies revealed that a final precipitation of recovered RNA with potassium acetate to reduce carryover of polysaccharides and the addition of polyvinylpyrrolidone to remove polyphenolics in spinach were essential. For tomatoes, virus isolation was achieved with the incorporation of either an elution step with a high pH Tris-glycine-beef extract (TGBE) buffer or with an enzymatic digestion with pectinase. We also describe the development of a protocol for the detection of HAV from tomatoes utilizing a Luminex® microbead-based suspension array. The results correlated well with the RT-qPCR assay suggesting the feasibility of the Bioplex® as a detection platform for viruses isolated from foods.

Surface plasmon resonance biosensor for detection of feline calicivirus, a surrogate for norovirus.

The human noroviruses are the most common non-bacterial cause of gastroenteritis and are responsible for as much as 50% of all gastroenteritis outbreaks worldwide. Norovirus (NoV), a single stranded RNA virus, is highly contagious with an infectious dose of less than 100 viral particles. While techniques exist for the identification of NoV, the lack of a reliable cell culture system, NoV genetic variability, and time-consuming sample preparation steps required to isolate the virus (or its genome) prior to molecular based methods has hindered rapid virus detection. To better protect the public from virus-contaminated food and enable better detection in clinical and environmental samples, sensitive and selective methods with simple sample preparation are needed. Surface plasmon resonance (SPR) biosensors represent an emerging detection platform, and this approach has been applied to the rapid detection of foodborne small molecule toxins, protein toxins, and bacteria. This analytical technique, however, has yet to be fully investigated for rapid virus detection, especially for intact viral particles extracted from food matrices. For this study, the culturable, non-human pathogen feline calicivirus (FCV), which has similar morphology and is genetically related to NoV, was chosen as a surrogate virus for designing and evaluating an SPR assay. An antibody-based assay was performed by first immobilizing anti-FCV to an SPR chip surface and then directly measuring virus binding and subsequent secondary antibody binding. The resulting biosensor directly detected intact FCV particles with limits of detection of approximately 10(4)TCID50FCV/mL from purified cell culture lysates. In addition, intact virus detection in FCV-spiked oyster matrix was possible when using a simple extraction procedure and employing a secondary antibody to FCV for quantitation. The results from these preliminary studies show promise for the development of a rapid assay for detecting intact viruses, such as NoV, using an SPR biosensor. While the current level of sensitivity achieved with this SPR biosensor may be more applicable to virus detection in clinical specimens, broader application and increased sensitivity of this method for foodborne viruses may be achieved when performed in conjunction with efficient virus extraction and concentration methods.

Mechanisms of induction of germination of Bacillus subtilis spores by high pressure.

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.