Reduction of Enteric Viruses by Blueberry Juice and Blueberry Proanthocyanidins

Blueberry and blueberry extracts are known for their health benefits and antimicrobial properties. Natural therapeutic or preventive options to decrease the incidences of foodborne viral illnesses are becoming popular and being researched. This study aimed to determine the antiviral effects of blueberry juice (BJ) and blueberry proanthocyanidins (BB-PAC, B-type PAC structurally different from A-type PAC found in cranberries) against the infectivity of hepatitis A virus (HAV) and human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) at 37 °C over 24 h using standard plaque assays. Viruses at ~5 log PFU/ml were mixed with equal volumes of BJ (pH 2.8), neutralized BJ (pH 7.0), BB-PAC (1, 2, 4, and 10 mg/ml), malic acid (pH 3.0), or phosphate-buffered saline (pH 7.2) and incubated over 24 h at 37 °C. Each experiment was carried out in duplicate and replicated thrice. FCV-F9 titers were found to be reduced to undetectable levels with 1 and 2 mg/ml BB-PAC after 5 min, with 0.5 mg/ml BB-PAC after 1-h, and with BJ after 3-h. MNV-1 titers were reduced to undetectable levels after 3 h with 1, 2, and 5 mg/ml BB-PAC and after 6 h with BJ. HAV titers were reduced to undetectable levels after 30 min with 2 and 5 mg/ml BB-PAC, after 3 h with 1 mg/ml BB-PAC, and by ~2 log PFU/ml with BJ after 24-h. BB-PAC shows preventive potential against infection by the tested enteric viruses in a dose- and time-dependent manner, although further in vitro studies in model food systems and in vivo studies using animal models are warranted.     

Detection of Potential Infectious Enteric Viruses in Fresh Produce by (RT)-qPCR Preceded by Nuclease Treatment

Foodborne illnesses associated with contaminated fresh produce are a common public health problem and there is an upward trend of outbreaks caused by enteric viruses, especially human noroviruses (HNoVs) and hepatitis A virus (HAV). This study aimed to assess the use of DNase and RNase coupled to qPCR and RT-qPCR, respectively, to detect intact particles of human adenoviruses (HAdVs), HNoV GI and GII and HAV in fresh produce. Different concentrations of DNase and RNase were tested to optimize the degradation of free DNA and RNA from inactivated HAdV and murine norovirus (MNV), respectively. Results indicated that 10 µg/ml of RNase was able to degrade more than 4 log₁₀ (99.99%) of free RNA, and 1 U of DNase degraded the range of 0.84–2.5 log₁₀ of free DNA depending on the fresh produce analysed. The treatment with nucleases coupled to (RT)-qPCR was applied to detect potential infectious virus in organic lettuce, green onions and strawberries collected in different seasons. As a result, no intact particles of HNoV GI and GII were detected in the 36 samples analysed, HAdV was found in one sample and HAV was present in 33.3% of the samples, without any reasonable distribution pattern among seasons. In conclusion, RT-qPCR preceded by RNase treatment of eluted samples from fresh produce is a good alternative to detect undamaged RNA viruses and therefore, potential infectious viruses. Moreover, this study provides data about the prevalence of enteric viruses in organic fresh produce from Brazil.     

Comparative Uptake of Enteric Viruses into Spinach and Green Onions

Root uptake of enteric pathogens and subsequent internalization has been a produce safety concern and is being investigated as a potential route of pre-harvest contamination. The objective of this study was to determine the ability of hepatitis A virus (HAV) and the human norovirus surrogate, murine norovirus (MNV), to internalize in spinach and green onions through root uptake in both soil and hydroponic systems. HAV or MNV was inoculated into soil matrices or into two hydroponic systems, floating and nutrient film technique systems. Viruses present within spinach and green onions were detected by RT-qPCR or infectivity assays after inactivating externally present viruses with Virkon^®. HAV and MNV were not detected in green onion plants grown up to 20 days and HAV was detected in only 1 of 64 spinach plants grown in contaminated soil substrate systems up to 20 days. Compared to soil systems, a drastic difference in virus internalization was observed in hydroponic systems; HAV or pressure-treated HAV and MNV were internalized up to 4 log RT-qPCR units and internalized MNV was shown to remain infectious. Understanding the interactions of human enteric viruses on produce can aid in the elucidation of the mechanisms of attachment and internalization, and aid in understanding risks associated with contamination events.     

Potential Risk of Norovirus Infection Due to the Consumption of “Ready to Eat” Food

In this study, we investigated the presence of enteric viruses such as norovirus (NoV), hepatitis A virus (HAV), hepatitis E virus (HEV), and adenovirus (HAdV), in vegetables available on the Italian markets. For this aim, 110 national and international ??ready to eat?? samples were collected and analyzed by biomolecular tests and positive samples were confirmed by sequencing. All samples (100?%) were negative for HAV, HEV, and HAdV, while 13.6?% (15/110) were positive for NoV. Actually there is not a formal surveillance system for NoV infections in Italy but we clearly demonstrated a potential risk associated with the consumption of ??ready to eat?? vegetables. This study confirmed for the first time in Italy the presence of norovirus in semi-dried tomatoes by PCR technique.     

Naturally Occurring Flavonoids Against Human Norovirus Surrogates

Naturally occurring plant-derived flavonoids are reported to have antibacterial, antiviral, and pharmacological activities. The objectives of this study were to determine the antiviral effects of four flavonoids (myricetin, l-epicatechin, tangeretin, and naringenin) on the infectivity of food borne norovirus surrogates after 2 h at 37 °C. The lab-culturable surrogates, feline calicivirus (FCV-F9) at titers of ~7 log₁₀ PFU/ml (high titer) or ~5 log₁₀ PFU/ml (low titer) and murine norovirus (MNV-1) at ~5 log₁₀ PFU/ml, were mixed with equal volumes of myricetin, l-epicatechin, tangeretin, or naringenin at concentrations of 0.5 or 1 mM, and incubated for 2 h at 37 °C. Treatments of viruses were neutralized in cell culture medium containing 10 % heat-inactivated fetal bovine serum, serially diluted, and plaque assayed. Each treatment was replicated thrice and assayed in duplicate. FCV-F9 (low titer) was not found to be reduced by tangeretin or naringenin, but was reduced to undetectable levels by myricetin at both concentrations. Low titer FCV-F9 was also decreased by 1.40 log₁₀ PFU/ml with l-epicatechin at 0.5 mM. FCV-F9 at high titers was decreased by 3.17 and 0.72 log₁₀ PFU/ml with myricetin and l-epicatechin at 0.5 mM, and 1.73 log₁₀ PFU/ml with myricetin at 0.25 mM, respectively. However, MNV-1 showed no significant inactivation by the four tested treatments. The antiviral effects of the tested flavonoids are dependent on the virus type, titer, and dose. Further research will focus on understanding the antiviral mechanism of myricetin and l-epicatechin.     

Detection and Typing of Norovirus from Frozen Strawberries Involved in a Large-Scale Gastroenteritis Outbreak in Germany

During September/October 2012, a norovirus gastroenteritis outbreak affecting about 11,000 people occurred in Germany. Epidemiological studies suggested that frozen strawberries represented the vehicle of infection. We describe here the analysis of frozen strawberries for the presence of norovirus. Samples were taken by applying a stratified subsampling scheme. Two different methods for virus extraction from strawberries were compared. First, viruses were eluted from strawberries under alkaline conditions and concentrated using a polyethylene glycol precipitation. Second, ultrafiltration was applied for concentration of viruses rinsed off of the berries. In both cases, RNA was extracted and analyzed by real-time RT-PCR. Application of the ultrafiltration method generally resulted in a lower detection rate. Noroviruses were detected in 7/11 samples derived from the lot of strawberries implicated in the outbreak using the precipitation method. Typing of norovirus revealed three different genotypes including a combination of norovirus genotype II.16 (viral polymerase) and II.13 (viral capsid). This genotype combination was also found in some of the patients that were involved in the outbreak, but that had not been reported in Germany so far. In conclusion, heterogeneously distributed noroviruses in frozen strawberries can be detected by applying an optimized combination of sampling procedures, virus extraction methods, and real-time RT-PCR protocols. The detection of several different genotypes in the strawberries may suggest contamination from sewage rather than from a single infected food handler.     

An Optimised Direct Lysis Method for Viral RNA Extraction and Detection of Foodborne Viruses on Fruits and Vegetables

Detection of norovirus (NoV) and hepatitis A virus (HAV) on fruits and vegetables using current standard methodologies can be inefficient. Method optimisat     

High Pressure Processing and its Application to the Challenge of Virus-Contaminated Foods

High pressure processing (HPP) is an increasingly popular non-thermal food processing technology. Study of HPP’s potential to inactivate foodborne viruses has defined general pressure levels required to inactivate hepatitis A virus, norovirus surrogates, and human norovirus itself within foods such as shellfish and produce. The sensitivity of a number of different picornaviruses to HPP is variable. Experiments suggest that HPP inactivates viruses via denaturation of capsid proteins which render the virus incapable of binding to its receptor on the surface of its host cell. Beyond the primary consideration of treatment pressure level, the effects of extending treatment times, temperature of initial pressure application, and matrix composition have been identified as critical parameters for designing HPP inactivation strategies. Research described here can serve as a preliminary guide to whether a current commercial process could be effective against HuNoV or HAV.     

Heat Inactivation of Hepatitis A Virus in Shellfish Using Steam

Shellfish are an important cause of foodborne viral illness. Consumer-friendly cooking recommendations for shellfish could improve food safety and decrease the risk for infection from contaminated products. Thermal inactivation parameters were established for hepatitis A virus (HAV) in mussels and validated with cooking experiments. Steaming for only 2–5 min was not sufficient to inactivate HAV in mussels in all layers of a steamer. Steaming mussels for 6 min was sufficient to inactivate HAV in all layers. These cooking guidelines produce shellfish with a reduced risk for foodborne virus transmission.     

Occurrence of Norovirus,Rotavirus, Hepatitis a Virus,and Enterovirus in Berries in Argentina

Food and Environmental Virology - Berries have been implicated as the probable vehicle of infection in multiple outbreaks of norovirus and hepatitis A virus (HAV). These foods often receive minimal...     

High Pressure Inactivation of HAV Within Mussels

The potential of high pressure processing to inactivate hepatitis A virus (HAV) within Mediterranean mussels (Mytilus galloprovincialis) and blue mussels (Mytilus edulis) was evaluated. HAV was bioaccumulated within mussels to approximately 6-log₁₀ PFU by exposure of mussels to HAV-contaminated seawater. After shucking, 5 min pressure treatments of 300, 325, 350, 375, and 400 MegaPascals (MPa) were performed at room temperature (18–22°C). For blue mussels, log₁₀ PFU reductions of HAV averaged 2.1 and 3.6 for treatments of 350 and 400 MPa, while for Mediterranean mussels reductions of 1.7 and 2.9 log₁₀ PFU MPa were observed for equivalent treatments. These results demonstrate that high pressure processing is capable of inactivating HAV within mussels.     

Detection of Hepatitis A Virus and Norovirus in Different Food Categories: A 6-Year Survey in Italy

Food and Environmental Virology - To observe the prevalence of contamination by hepatitis A virus (HAV) and norovirus (NoV) in different food types, 9242 samples were analyzed over a 6-year period...     

Simple and Rapid Detection of Human Norovirus from Produce Using SYBR Green I-based Real-time RT-PCR

Several foodborne norovirus gastroenteritis outbreaks have been linked to fresh produce. Rapid and sensitive detection can help prevent the release of contaminated produce items in the market. The objectives of this study were to apply a relatively inexpensive SYBR Green I-based real-time RT-PCR assay for the rapid detection of human norovirus (NoV) GI and GII on the surfaces of lettuce, cherry tomatoes, and green onions. Each washed produce commodity (25 g) was spiked with serial dilutions of NoV GI and GII stool samples. RNA was eluted from the produce surface and extracted using the TRIzol^? method. This was followed by detection using SYBR Green I real-time RT-PCR with primers specific for NoV GI (COG1F-COG1R) and GII (COG2F-COG2R) along with an internal RNA amplification control. End-point detection limits from lettuce and tomatoes were found to be 10 RT-PCR units/25 g for GI and GII and 1 RT-PCR unit/25 g for GI and 10 RT-PCR units/25 g for GII from green onions. These results were confirmed by Tm analysis (showing peaks at 81.5 and 84°C for GI and GII, respectively; and 83°C for the IAC) as well as agarose gel electrophoresis that confirmed products of ~95 bp for GI and GII and ~155 bp for the RNA IAC. Results could be obtained within one working day, showing potential for routine use in diagnostics and monitoring of NoV contamination by the produce industry.     

Persistence of Hepatitis A Virus in Fresh Produce and Production Environments,and the Effect of Disinfection Procedures: A Review

Although information is limited, it is evident that prolonged persistence of infectious Hepatitis A virus (HAV) is a factor in the transmission of the virus via fresh produce. Consequently, data on persistence of the virus on produce, and in environments relevant to production, such as soils, water and surfaces, are required to fully understand the dynamics of transmission of HAV via foods. Furthermore, information on effective disinfection procedures is necessary to implement effective post-harvest control measures. This review summarises current information on HAV persistence in fresh produce and on relevant disinfection procedures. On vegetables, HAV can remain infectious for several days; on frozen berries, it can persist for several months. HAV can remain infectious on surfaces for months, depending on temperature and relative humidity, and can survive desiccation. It can survive for several hours on hands. Washing hands can remove the virus, but further data are required on the appropriate procedure. Chlorination is effective in water, but not when HAV is associated with foodstuffs. Bleach and other sodium hypochlorite disinfectants at high concentrations can reduce HAV on surfaces, but are not suitable for use on fresh produce. There is only limited information on the effects of heating regimes used in the food industry on HAV. HAV is resistant to mild pasteurisation. Some food components, e.g. fats and sugars, can increase the virus’ resistance to higher temperatures. HAV is completely eliminated by boiling. Quantitative prevalence data are needed to allow the setting of appropriate disinfection log reduction targets for fresh produce.     

Synergistic Effects of Combined Chlorine and Vitamin B1 on the Reduction of Murine Norovirus-1 on the Oyster (Crassostrea gigas) Surface

This study investigated the synergistic effects of combined chlorine (200, 500, 700, and 1000 ppm) and vitamin B₁ (1000, 2000, and 3000 ppm) on the murine norovirus-1 (MNV-1), a human norovirus (NoV) surrogate, on oyster surface. Vitamin B₁ slightly reduced MNV-1 (0.04–0.3 log-reduction), whereas chlorine significantly reduced MNV-1 (0.4–1.0 log-reduction). The combined chlorine and vitamin B₁ resulted in a 0.52–1.97 log-reduction of MNV-1. The synergistic reduction in the MNV titer was not dependent on the concentrations of chlorine and vitamin B₁, and it ranged between 0.08 and 1.03 log₁₀ PFU/mL. The largest synergistic reduction observed was for the combined 700 ppm chlorine and 1000 ppm vitamin B₁. The pH and mechanical texture of the oysters were not significantly changed by the combined 0–1000 ppm chlorine and 3000 ppm vitamin B₁. The overall sensory acceptability were significantly (P < 0.05) reduced in oysters treated with 1000 ppm chlorine and 3000 ppm vitamin B₁ than in those treated with 0–700 ppm chlorine and 3000 ppm vitamin B₁. This study suggests that the combined 700 ppm chlorine and 3000 ppm vitamin B₁ could potentially be used to reduce NoV on oyster surface without causing concomitant changes in the mechanical texture, pH, or sensory qualities of the oysters.

     

Survival of Respiratory Viruses on Fresh Produce

In addition to enteric viruses of fecal origin, emerging zoonotic viruses such as respiratory coronaviruses and influenza viruses may potentially be transmitted via contaminated foods. The goal of this study was to determine the recovery efficiencies and the survival of two respiratory viruses, namely, adenovirus 2 (Ad2) and coronavirus 229E (CoV229E), on fresh produce in comparison to the enteric poliovirus 1 (PV1). Adenovirus was recovered with efficiencies of 56.5, 31.8, and 34.8 % from lettuce, strawberries, and raspberries, respectively. Coronavirus was recovered from lettuce with an efficiency of 19.6 % yet could not be recovered from strawberries. Poliovirus was recovered with efficiencies of 76.7 % from lettuce, but only 0.06 % from strawberries. For comparison purposes, the survival of Ad2, CoV229E, and PV1 was determined for periods up to 10 days on produce. The enteric PV1 survived better than both respiratory viruses on lettuce and strawberries, with only ≤1.03 log₁₀ reductions after 10 days of storage at 4 °C compared to CoV229E not being recovered after 4 days on lettuce and reductions of 1.97 log₁₀ and 2.38 log₁₀ of Ad2 on lettuce and strawberries, respectively, after 10 days. Nevertheless, these respiratory viruses were able to survive for at least several days on produce. There is therefore the potential for transfer to the hands and subsequently to the mucosa via rubbing the eyes or nose. In addition, some respiratory coronaviruses (e.g., severe acute respiratory syndrome coronavirus) and adenoviruses are also capable of replication in the gut and there is thus some potential for acquisition through the consumption of contaminated produce.     

Comparative Analysis of Viral Concentration Methods for Detecting the HAV Genome Using Real-Time RT-PCR Amplification

Hepatitis A is a major infectious disease epidemiologically associated with foodborne and waterborne outbreaks. Molecular detection using real-time RT-PCR to detect the hepatitis A virus (HAV) in contaminated vegetables can be hindered by low-virus recoveries during the concentration process and by natural PCR inhibitors in vegetables. This study evaluated three virus concentration methods from vegetables: polyethylene glycol (PEG) precipitation, ultrafiltration (UF), and immunomagnetic separation (IMS). UF was the most efficient concentration method, while PEG and IMS were very low for the recovery rate of HAV. These results demonstrate that UF is the most appropriate method for recovering HAV from contaminated vegetables and that this method combined with the real-time RT-PCR assay may be suitable for routine laboratory use.     

Somatic Coliphage Profiles of Produce and Environmental Samples from Farms in Northern México

Somatic coliphages were quantified in 459 produce and environmental samples from 11 farms in Northern Mexico to compare amounts of somatic coliphages among different types of fresh produce and environmental samples across the production steps on farms. Rinsates from cantaloupe melons, jalapeño peppers, tomatoes, and the hands of workers, soil, and water were collected during 2011–2012 at four successive steps on each farm, from the field before harvest through the packing facility, and assayed by FastPhage MPN Quanti-tray method. Cantaloupe farm samples contained more coliphages than jalapeño or tomato (p range <0.01–0.03). Across production steps, jalapeños had higher coliphage percentages before harvest than during packing (p = 0.03), while tomatoes had higher coliphage concentrations at packing than all preceding production steps (p range <0.01–0.02). These findings support the use of targeted produce-specific interventions at multiple points in the process of growing and packing produce to reduce the risk of enteric virus contamination and improve food safety during fruit and vegetable production.