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Among the enteric viruses implicated in foodborne outbreaks, the human norovirus and hepatitis viruses A and E (HAV and HEV) represent a serious public health concern. International standard ISO 15216 proposes methods for detecting HAV and norovirus (genogroups I and II) RNA from soft fruit, leaf, stem and bulb vegetables, bottled water or food surfaces. These methods had not previously been validated for detecting the targeted viruses in other foodstuffs such as multicomponent foods, nor for detecting other viruses in foodstuffs. The aim of this study was to characterise a method derived from the vegetable method described in ISO 15216 to detect HAV, HEV and norovirus in artificially-contaminated multicomponent foodstuffs according to the recent international standard ISO 16140-4. Results showed that the mean recovery rates for all settings did not differ according to the operator. The mean extraction yields ranged from 0.35% to 40.44% for HAV, 5.19% to 100% for HEV, 0.10% to 40.61% for norovirus GI and 0.88% to 69.16% for norovirus GII. The LOD 95 was 10 2 genome copies/g for HAV, HEV and norovirus GII and 10 3 genome copies/g for norovirus GI. The LOQ was 2.90 × 10 4 , 1.40 × 10 3 , 1.60 × 10 4 and 1.30 × 10 4 genome copies/g for HAV, HEV, norovirus GI and norovirus GII respectively. The MNV-1 process control was detected in 120 out of 128 RNA extracts analysed and was recovered with an efficiency of between 3.83% and 50.22%. The mean inhibition rates of quantitative real-time RT-PCR reaction ranged from 3.25% to 28.70% and varied significantly with the type of food matrix. The described method could be used to detect viruses in composite food products for routine diagnosis needs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Strawberries are often consumed fresh or only receive minimal processing, inducing a significant health risk to the consumer if contamination occurs anywhere from farm to fork. Outbreaks of foodborne illness associated with strawberries often involve a broad range of microbiological agents, from viruses (human norovirus) to bacteria (Salmonella spp. and Listeria monocytogenes). The addition of sanitizers to water washes is one of the most commonly studied strategies to remove or inactivate pathogens on berries as well as avoid cross contamination due to reuse of process wash water. The risk posed with the safety issues of by-products from chlorine disinfection in the fruit industry has led to a search for alternative sanitizers. We evaluated the applicability of different chemical sanitizers (peracetic acid (PA), hydrogen peroxide (H 2 O 2 ), citric acid (CA), lactic acid (LA) and acetic acid (AA)) for the inactivation of S. enterica, L. monocytogenes and murine norovirus (MNV-1) on strawberries. A control treatment with chlorine (NaClO) (100 ppm) was included. For each sanitizer, different doses (40, 80 and 120 ppm for PA and 1, 2.5 and 5% for H 2 O 2 , LA, AA and CA) and time (2 and 5 min) were studied in order to optimize the decontamination washing step. The best concentrations were 80 ppm for PA, 5% for H 2 O 2 and 2.5% for organic acids (LA, AA and CA) after 2 min treatment. Results indicate that the sanitizers selected may be a feasible alternative to chlorine (100 ppm) for removing selected pathogenic microorganisms (P > 0.05), with reductions about ≥2 log for bacterial strains and ≥ 1.7 log for MNV-1. As the washing water may also increase the microbial counts by cross-contamination, we observed that no pathogenic bacteria were found in wash water after 5% H 2 O 2 and 80 ppm PA after 2 min treatment. On the other hand, we also reported reductions about total aerobic mesophyll (TAM) (0.0-1.4 log CFU/g) and molds and yeasts (M&Y) (0.3-1.8 log CFU/g) with all alternative sanitizers tested. Strawberries treated did not shown significant differences about physio-chemical parameters compared to the untreated samples (initial). For this study, the optimal sanitizer selected was PA, due to the low concentration and cost needed and its microbiocidal effect in wash water and fruit. Notwithstanding the results obtained, the effect of PA in combination with other non-thermal technologies such as water-assisted ultraviolet (UV-C) light should be studied in future research to improve the disinfection of strawberries., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Norovirus (NoV) is the leading cause of epidemic and sporadic gastroenteritis worldwide; a high number of those cases are attributed to the consumption of contaminated food. Crop producers have used several strategies to inactivate the virus present in these products and thus stop the NoV transmission chain. Physical methods such as gamma radiation show excellent results in the inactivation of bacteria, but its effect on NoV has been little studied. This study aimed to evaluate the effect of gamma radiation for NoV inactivation, and over the surface topographic characteristics of strawberry cells, as a prototype of soft fruit. A 10% suspension of GII norovirus-positive stool samples were treated with either 200 mg/L of sodium hypochlorite (NaClO) or gamma-irradiated at doses of 5, 10, 15 and 20 kilograys (kGy). Viral inactivation was determined by measuring the integrity of viral capsid using RNase A alone or in combination with proteinase K followed by RT-qPCR. The effect over cellular surface topology characteristics of the fruit was measured by atomic force microscopy (AFM) and confocal microscopy. High doses of radiation (20 kGy) were necessary to detect a significant (p < 0.05) decrease of up to 1.26 log 10 viral copy number. This dose significantly (p < 0.05) raises the root means square roughness (Rq), which affects directly the quality and texture of the product. The gamma irradiation doses tested in this study were not enough to inactivate NoV. The allowed gamma irradiation doses for fresh produce does not alter the surface topology of the fruit, but they affect the content of fluorescent compounds, responsible for the antioxidant activity of the fruit., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Virus detection in food requires appropriate elution and concentration techniques which need to be adapted for different food matrices. ISO/TS-15216-1:2017 and ISO/TS-15216-2:2019 describe standard methods for hepatitis A virus (HAV) research in some food only. Milk-based products containing one or more types of fruit are not covered by ISO procedures, even though they can be contaminated by fruit added to these products or by the food handlers. The aim of this work was to identify an efficient method for the detection of HAV in milk-based products. Four methods were tested to recover HAV from artificially contaminated milk, yoghurt and ice cream containing soft fruits. Results showed that the efficiency of the tested methods depends on the analyzed matrix. In milk we obtained a mean recovery from 13.4% to 1.9%; method based on high speed centrifuge gave the best values. The average recovery in yoghurt was between 3.3% and 114.4%, the latter value achieved by method with beef extract at 3% as eluent. Finally, two methods gave the best results in ice cream with similar recoveries: 29.1% and 27.7% respectively. The first method used glycine as eluent while the other one was based on high speed centrifugation. The ISO method has never proved to be the most efficient in the matrices studied. Therefore, based on the results obtained, a complete rethinking of the ISO method may be necessary to improve its recovery for some products such as milk, while only small changes would be sufficient for other products, such as yoghurt and ice cream., Competing Interests: Declaration of Competing Interest The authors declare there are no conflicts of interest regarding the study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Osmotically dehydrated and air dried berry fruits are used as ingredients for the production of yoghurts, chocolates, cereal bars and mixes, ice creams and cakes and these fruits are often subjected to mild thermal treatments only, posing questions around their microbiological safety. As osmotic dehydration methods and parameters vary considerably within the industry and minimally processed high quality fruits are increasingly sought, the scope of this study was to determine which temperatures are required for the inactivation of relevant bacteria and viruses during osmotic dehydration of berries, using blueberries as a model berry in a thawed state to mimic common industrial practices. Additionally, we studied the inactivation of osmotic dehydration at 23 °C, sometimes referred to "cold infusion" followed by air drying at 100 °C to determine the microbiological safety achieved by this combined treatment. Four pathogens (Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes and hepatitis A virus (HAV)) and five surrogates (Enterococcus faecium, Escherichia coli P1, Listeria innocua, murine Norovirus (MNV) and bacteriophage MS2) were inoculated on blueberries and reductions were measured after different treatment combinations. After osmotic dehydration of bacterial strains at 40 °C no survivors were detected on blueberries, with the exception of E. faecium. Inactivation of the viruses at 45 °C showed no survivors for MS2 and mean reductions of 1.5 and 3.4 log 10 median tissue culture infectious dose (TCID 50 )/g for HAV and MNV, respectively. Similarly, in the sugar solution at 40 °C, no survivors were observed, with the exception of E. faecium and the three viruses. The combined process (osmotic dehydration at 23 °C followed by air-drying at 100 °C) achieved an >6 log reduction of all tested bacterial strains and MS2. For HAV and MNV, 2.6 and >3.4 log 10 TCID 50 /g were measured. In summary, the present study shows that osmotic dehydration appears an efficient control measure for the control of L. monocytogenes, S. enterica and E. coli O157:H7 if carried out at 40 °C or at 23 °C and followed by air-drying at 100 °C. Based on the results generated with MNV, the combined treatment is also expected to reduce human Norovirus (NoV) but does not appear to be sufficient to fully control HAV. The results contribute to a better management of the microbial safety of osmotically dehydrated and dried berries and especially the results generated for the viruses emphasize that within a robust food safety management system, safety must be assured through the entire food supply chain and therefore must start at primary production with the implementation of Good Agricultural Practices (GAP)., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Strawberry production and exports have been increasing in Spain in recent decades. However, little information is available about their microbiological quality. Due to the growing concern about the microbial safety of these fruits, the objective of this investigation was to study the microbiological quality and the prevalence of the main foodborne pathogens on strawberries sold in Spain. Fresh (n = 152) and frozen (n = 31) samples were obtained from marketplaces and fields in 2017 and 2018. The samples were assayed for total aerobic mesophilic microorganisms (TAM), moulds and yeasts (M&Y), total coliforms (TC), Escherichia coli, Salmonella spp., Listeria monocytogenes as well as Norovirus (NoV) GI and GII. The microbiological counts ranged from <1.70 (detection limit, dl) - 5.89 log 10 CFU/g (mean 3.78 log 10 CFU/g) for TAM; 2.10-5.86 log 10 CFU/g (mean 3.80 log 10 CFU/g) for M&Y; and <0.70 (dl) - 4.91 log 10 CFU/g (mean 2.15 log 10 CFU/g) for TC in fresh strawberries. In frozen strawberries, the counts were <1.70 (dl) - 3.66 log 10 CFU/g (mean 2.30 log 10 CFU/g) for TAM; <1.70 (dl) - 2.76 log 10 CFU/g (mean 1.82 log 10 CFU/g) for M&Y; and <0.70(dl) - 1.74 log 10 CFU/g (mean 0.77 log 10 CFU/g) for TC. All the samples in this study tested negative for Salmonella spp., L. monocytogenes. E. coli and NoV GI and GII genome. A global overview of all the data was executed using Principal Component Analysis (PCA), and the results showed that the scores and loadings according to principal components 1 (PC1) and 2 (PC2) accounted for 75.9% of the total variance, allowing a distinction between fresh and frozen samples. The presence of moulds was significantly higher in the supermarket samples whereas the presence of total coliforms was significantly higher in the field samples (p < 0.05). Although pathogenic microorganisms were not found, preventative measures and prerequisites in the strawberry production chain must be considered in order to avoid possible foodborne diseases related to the microbiological quality of the fruit., (Copyright © 2019. Published by Elsevier B.V.)

The apparent international rise in foodborne virus outbreaks attributed to fresh produce and the increasing importance of fresh produce in the Australian diet has led to the requirement to gather information to inform the development of risk management strategies. A prevalence survey for norovirus (NoV) and hepatitis A virus (HAV) in fresh Australian produce (leafy greens, strawberries and blueberries) at retail was undertaken during 2013-2014 and data used to develop a risk profile. The prevalence of HAV in berries and leafy greens was estimated to be <2%, with no virus detected in produce during the yearlong survey. The prevalence of NoV in fresh strawberries and blueberries was also estimated to be <2% with no virus detected in berries, whilst for leafy greens the NoV prevalence was 2.2%. Prevalence of a bacterial hygiene indicator, Escherichia coli, was also investigated and found to range from <1% in berries to 10.7% in leafy greens. None of the NoV positive leafy green samples tested positive for E. coli, indicating it is a poor indicator for viral risk. The risk was evaluated using standard codex procedures and the Risk Ranger tool. Taking all data into account, including the hazard dose and severity, probability of exposure, probability of infective dose and available epidemiological data, the risk of HAV and NoV foodborne illness associated with fresh Australian berries (strawberries and blueberries) sold as packaged product was deemed to be low. The risk of foodborne illness from HAV associated with leafy greens was also deemed to be low, but higher than that for fresh berries, due mainly to the potential for recontamination post-processing if sold loose. The risk of foodborne illness from NoV associated with leafy greens was deemed to be low/moderate. Despite the prevalence of NoV in leafy greens being low and the inability to discriminate between infective and non-infective virus using PCR based methodologies, the fact that NoV was detected resulted in a higher risk associated with this pathogen-product pairing; compounded by the higher prevalence of NoV within the community compared to HAV, and the potential for leafy greens to become contaminated following processing if sold loose., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

The environmental stability of enteric viruses and resistance to conventional treatments and common disinfectants, leads to their persistence in waters and food, causing serious implications on public health. Among non-thermal treatment methods, ionizing radiation is recognized as a useful and effective mean of disinfection. The objective of this study was to estimate the inactivation of enteric virus by gamma radiation in raw berry fruits, in order to evaluate the potential of this technology to be applied as a disinfection treatment. Fresh strawberries and raspberries were inoculated either individually with murine norovirus type 1 (MuNoV; as a human norovirus surrogate) and human adenovirus type 5 (HAdV) or with a viral pool of both viruses, and irradiated in a Co-60 equipment at doses of 1 kGy up to 11 kGy. The infectivity of viral particles of MuNoV and HAdV was assessed by plaque assay using Raw 264.7 and A549 cells, respectively. A 2 log PFU/g reduction on MuNoV and HAdV titers was obtained after treatment with a dose of 4 kGy for both fruits. However, non-linear inactivation survival curves were obtained for MuNoV and HAdV in fresh fruits, leading to the detection of infective viral particles at a dose of 11 kGy. The irradiation process indicated virucidal potential, although the estimated gamma radiation dose to attain food safety (> 7 kGy) would compromise the preservation of food quality. Nevertheless, the irradiation technology could be an effective virus mitigation tool to treat polluted waters, which are a major vehicle of contamination for fresh produce., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human noroviruses (NoV) are recognized worldwide as important pathogens and the primary cause of foodborne disease outbreaks from contaminated food in the U.S. They are often transmitted by infected food handlers manipulating foods during preparation, such as fresh fruits and vegetables. This paper provides a study to model the transfer of NoV between food handlers and vegetables during salad preparation in school food services based on direct observation data. Three transfer pathways were modeled by considering different initial contamination sources (environment, handlers and contaminated produce). The probability of infection by NoV was also estimated based on the NoV levels at consumption obtained from each simulated transfer pathway. A scenario analysis ranging a wide concentration from 10 2 to 10 7 NoV infective particles was performed to represent different levels of NoV in the initial contamination sources. In addition, a sensitivity analysis was applied to identify the most important model inputs and determine the safest handling practices to be implemented in school food service operations. The pathway describing transfer from contaminated surfaces or handlers to foods indicated that initial levels of ≤10 4 NoV particles/fomite resulted in <0.5% cases per serving of NoV infection. When initial levels were higher, % cases of NoV infection was estimated to be ca. 3%. This rise in % cases of infection was linked to higher doses (5% serving with ≥15 NoV particles/serving) and prevalence levels at consumption (>0.2). In the pathway modeling cross contamination from contaminated vegetables to non-contaminated vegetables, all scenarios could lead to infected individuals, although number of cases of infection were lower (<1.3%), despite concentration levels were higher. On the contrary, for this pathway, prevalence was 2-fold lower than that observed in the pathways describing transfer from contaminated surfaces and hands. Based on the sensitivity analysis, NoV transfers to fresh produce may be minimized by improving hand washing, and therefore effective training programs need to be carried out specifically addressing hand washing. Moreover, the produce's washing step showed to be an effective control measure, depending on the desinfectant efficacy, by reducing % cases of NoV infection from 6 to 1%. The model in this study might be used, in the future, to evaluate the impact on the risk associated with NoV transmission of specific and effective training programs, aimed at food chain operators., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness, with common vehicles including bivalve molluscan shellfish, soft fruit and various vegetables. Outbreaks of viral illness due to contamination of the surfaces of foods, or food preparation surfaces by for example infected food handlers are also common. Virus analysis of food matrices can contribute towards risk management for these hazards and a two-part technical specification for determination of Hepatitis A virus and norovirus in food matrices (ISO/TS 15216:2013) was published jointly by the European Committee for Standardisation and the International Organization for Standardization in 2013. As part of the European Mandate No. M381 to validate 15 standards in the field of food microbiology, an international validation study involving 18 laboratories from 11 countries in Europe was conducted between 2012 and 2014. This study aimed to generate method characteristics including limit of detection, limit of quantification, repeatability and reproducibility for ISO 15216 - Part 1, the method for quantification, in seven food matrices. The organization and results of this study, including observations that led to improvements in the standard method are presented here. After its conclusion, the method characteristics generated were added to the revised international standard, ISO 15216-1:2017, published in March 2017., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hepatitis A virus (HAV) is well known worldwide as a causative virus of acute hepatitis. In recent years, numerous cases of HAV infection caused by HAV-contaminated berries have occurred around the world. Because berries are often consumed without prior heating, reliable disinfection of the raw fruit is important in order to prevent HAV outbreaks. Previous studies have found that murine norovirus strain 1 (MNV-1) and human norovirus GII.4 were inactivated in heat-denatured lysozyme solution. In this study, we investigated whether or not heat-denatured lysozyme is effective in inactivating HAV and whether it could be an effective disinfectant for berries contaminated with HAV or MNV-1. We examined the inactivating effect of heat-denatured lysozyme on three strains of HAV and found that it reduced the infectivity of all three strains. We then immersed blueberries and mixed berries into solutions of HAV or MNV-1, and disinfected them by soaking them in 1% heat-denatured lysozyme for 1min. Consequently, the infectious HAV and MNV-1 contaminating the berries were decreased by >3.1 log units in all samples. Our results demonstrate that heat-denatured lysozyme effectively inactivates HAV and suggest that heat-denatured lysozyme may be an effective disinfectant for berry fruit, which is a potential source of HAV food poisoning., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Raw fruits may harbour many pathogens of public health concern including enteric viruses, which are the leading cause of foodborne outbreaks. Recently, consumption of soft berries has been associated with increasing reports of norovirus and hepatitis A virus outbreaks in Europe. Due to their low infectious doses and low concentrations in food samples, an efficient and sensitive analytical method is required for virus detection. In this study we explored two different ways to improve the reference method for the detection of enteric viruses in soft fruits (ISO/TS 15216-1; 15216-2): an additional purification step after RNA extraction; and the detection of enteric viral genome by an absolute quantification method (microfluidic digital RT-PCR). Both of these approaches led to an improvement of enteric virus detection in soft berries by greatly lowering PCR inhibition, raising viral extraction efficiencies and enabling validation of controls using pure RNA extracts. The PCR inhibitor removal step can be easily included in the routine method. Absolute quantification by digital RT-PCR may be a relevant alternative method to standardize quantification of enteric viruses in foodstuffs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A quantitative human norovirus (NoV) exposure model describing transmission of NoV during pre-harvest, harvest and further processing of soft red fruits exemplified by raspberries is presented. The outcomes of the model demonstrate the presence of NoV in raspberry puree or individual quick frozen (IQF) raspberry fruits and were generated by Monte Carlo simulations by combining GoldSim® and @Risk® software. Input data were collected from scientific literature, observational studies and assumptions. NoV contamination of soft red fruits is assumed to take place at farms by application of contaminated water for pesticides dilution or by berries' pickers shedding NoV. The model was built simulating that a collection center received berries from ten farms with a total of 245 food handlers picking soft red fruits during a 10-hour day shift. Given 0, 5 and 20 out of 245 berries' pickers were shedding NoV, these conditions were calculated to result in a mean NoV contamination of respectively 0.47, 14.1 and 36.2 NoV particles per kg raspberries in case all raspberries are mixed to one day-batch of 11tons. The NoV contamination of the fruits was mainly driven by the route of NoV shedding food pickers (95.8%) rather than by spraying contaminated pesticide water (4.2%) (baseline scenario with 5 shedding pickers and contaminated pesticide water). Inclusion of appropriate hand washing procedures or hand washing followed by hand disinfection resulted in estimated reductions of the mean NoV levels from 14.1 to 0.16 and 0.17 NoV particles per kg raspberries, respectively, for the baseline scenario with 5 out of 245 food pickers shedding NoV. The use of a mild heat treatment (30s at 75°C) during further processing of berries to purees was noted to reduce mean NoV levels substantially from 14.1 to 0.2 NoV particles per kg raspberry puree. For IQF raspberries, the NoV contamination is heterogeneously distributed and resulted in a mean contamination of 3.1 NoV particles per 250g package containing approximately 115 berries. This farm-to-fork model is a useful tool for evaluating NoV mitigation strategies in the soft red fruit supply chain., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human norovirus (HuNoV) is the primary cause of viral gastroenteritis worldwide. Fresh blueberries are among high risk foods associated with norovirus related outbreaks. Therefore, it is important to assess intervention strategies to reduce the risk of foodborne illness. The disinfection efficiency of decontamination methods is difficult to evaluate for fruits and vegetables due to an inconsistent degree of contamination and irregular surface characteristics. The inactivation efficiency and mechanism of murine norovirus 1 (MNV-1, a surrogate for HuNoV) was studied on an experimentally prepared solidified agar matrix (SAM) to simulate blueberries using different wavelengths (A, B, C) of UV light both with and without TiO 2 photocatalysis (TP). MNV-1 was inoculated on exterior and interior of SAM and inactivation efficiencies of different treatments were investigated using a number of assays. Initial inoculum levels of MNV-1 on the SAM surface and interior were 5.2logPFU/mL. UVC with TiO 2 (UVC-TP) achieved the highest level of viral reduction for both externally inoculated and internalized MNV-1. Externally inoculated MNV-1 was reduced to non-detectable levels after UVC-TP treatment for 5min while there was still a 0.9 log viral titer after UVC alone. For internalized MNV-1, 3.2 log and 2.7 log reductions were obtained with UVC-TP and UVC alone treatments for 10min, respectively. The Weibull model was applied to describe the inactivation behavior of MNV-1, and the model showed a good fit to the data. An excellent correlation between the steady-state concentration of OH radicals ([OH] ss ) and viral inactivation was quantified using a para-chlorobenzoic acid (pCBA) probe compound, suggesting that OH radicals produced in the UV-TP reaction were the major species for MNV-1 inactivation. Transmission electron microscopy images showed that the structure of viral particles was completely disrupted with UVC-TP and UVC alone. SDS-PAGE analysis showed that the major capsid protein VP1 was degraded after UVC-TP and UVC alone. Real-time RT-qPCR analysis showed that UVC-TP and UVC alone caused a reduction in the level of viral genomic RNA. Propidium monoazide (PMA) pretreatment RT-qPCR analysis showed that UVC-TP caused damage to the viral capsid protein in addition to viral genomic RNA. UVC both with and without TiO 2 was more effective for MNV-1 inactivation than UVB and UVA. Thus, UVC-TP disinfection aimed to reduce levels of food-borne viruses can inactivate viruses present on the surface and internalized in the interior of blueberries., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Human norovirus (HuNoV) has been an increasing concern of foodborne illness related to fresh and frozen berries. In this study, high hydrostatic pressure (HHP) inactivation of HuNoV on fresh strawberries, blueberries, and raspberries and in their purees was investigated. Porcine gastric mucin (PGM)-conjugated magnetic beads (PGM-MBs) and real-time reverse transcriptional polymerase chain reaction (RT-qPCR) were utilized for infectious HuNoV discrimination and quantification. Strawberry puree inoculated with HuNoV genogroup I.1 (GI.1) strain was HHP-treated at 450, 500 and 550 MPa for 2 min each at initial sample temperatures of 0, 4 and 20 °C. HuNoV GI.1 strain became more sensitive to HHP treatment as the temperature decreased from 20 to 0 °C. HuNoV GI.1 or genogroup II.4 (GII.4) strains were inoculated into three types of berries and their purees and treated at pressure levels from 250 to 650 MPa for 2 min at initial sample temperature of 0 °C. For the purees, the HHP condition needed to achieve >2.9 log reduction of HuNoV GI.1 strain and >4.0 log reduction of HuNoV GII.4 strain was found to be ≥ 550 MPa for 2 min at 0 °C. HHP treatment showed better inactivation effect of HuNoV on blueberries than on strawberry quarters and raspberries. HuNoV GI.1 strain was more resistant to HHP treatment than HuNoV GII.4 strain under different temperatures and environment. The physical properties and sensory qualities of HHP-treated and untreated blueberries and the three types of berry purees were evaluated. Color, pH and viscosity of blueberries and three berry purees showed no or slight changes after HHP treatment. Sensory evaluation demonstrated that HHP treatment of 550 MPa for 2 min at 0 °C did not significantly reduced the sensory qualities of three berry purees. The results demonstrated that the HHP treatment of 550 MPa for 2 min at 0 °C could be a potential nonthermal intervention for HuNoV in berry purees without adversely affecting their sensory qualities and physical properties., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Noroviruses are the leading cause of foodborne illness, and ready-to-eat foods are frequent vehicles of their transmission. Studies of the disinfection of fruits and vegetables are becoming numerous. It has been shown that strong oxidizing agents are more effective than other chemical disinfectants for inactivating enteric viruses. The aim of this study was to evaluate the efficacy of oxidizing disinfectants (sodium hypochlorite, chloride dioxide and peracetic acid) at inactivating noroviruses on fruits and vegetables, using a norovirus surrogate, namely murine norovirus 3, which replicates in cell culture. Based on plaque assay, solutions of peracetic acid (85 ppm) and chlorine dioxide (20 ppm) reduced the infectivity of the virus in suspension by at least 3 log10 units after 1 min, while sodium hypochlorite at 50 ppm produced a 2-log reduction. On the surface of blueberries, strawberries and lettuce, chlorine dioxide was less effective than peracetic acid and sodium hypochlorite, which reduced viral titers by approximately 4 logs. A surprising increase in the efficacy of sodium hypochlorite on surfaces fouled with artificial feces was noted., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this study, a novel set-up using water-assisted UV processing was developed and evaluated for its decontamination efficacy against murine norovirus (MNV-1) inoculated on fresh blueberries for both small and large-scale experimental setups. Blueberries were skin-inoculated with MNV-1 and treated for 1-5 min with UV directly (dry UV) or immersed in agitated water during UV treatment (water-assisted UV). The effect of the presence of 2% (v/v) blueberry juice or 5% crushed blueberries (w/w) in wash water was also evaluated. Results showed that water-assisted UV treatment generally showed higher efficacies than dry UV treatment. With 12,000 J/m(2) UV treatment in small-scale setup, MNV reductions of >4.32- and 2.48-log were achieved by water-assisted UV and dry UV treatments, respectively. Water-assisted UV showed similar inactivating efficacy as 10-ppm chlorine wash. No virus was detected in wash water after UV treatment or chlorine wash. MNV-1 was more easily killed on skin-inoculated blueberries compared with calyx-inoculated berries. When clear water was used as wash water in the large-scale setup, water-assisted UV treatment (UV dose of 12,000 J/m(2)) resulted in >3.20 log and 1.81 log MNV-1 reductions for skin- and calyx-inoculated berries, respectively. The presence of 2% blueberry juice in wash water decreased the decontamination efficacy of water-assisted UV and chlorine washing treatments. To improve the inactivation efficacy, the effect of combining water-assisted UV treatment with chlorine washing was also evaluated. The combined treatment had better or similar inactivation efficacy compared to water-assisted UV treatment and chlorine washing alone. Findings of this study suggest that water-assisted UV treatment could be used as an alternative to chlorine washing for blueberries and potentially for other fresh produce., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Fresh produce that is contaminated with viruses may lead to infection and viral gastroenteritis or hepatitis when consumed raw. It is thus important to reduce virus numbers on these foods. Prevention of virus contamination in fresh produce production and processing may be more effective than treatment, as sufficient virus removal or inactivation by post-harvest treatment requires high doses that may adversely affect food quality. To date knowledge of the contribution of various potential contamination routes is lacking. A risk assessment model was developed for human norovirus, hepatitis A virus and human adenovirus in raspberry and salad vegetable supply chains to quantify contributions of potential contamination sources to the contamination of produce at retail. These models were used to estimate public health risks. Model parameterization was based on monitoring data from European supply chains and literature data. No human pathogenic viruses were found in the soft fruit supply chains; human adenovirus (hAdV) was detected, which was additionally monitored as an indicator of fecal pollution to assess the contribution of potential contamination points. Estimated risks per serving of lettuce based on the models were 3×10(-4) (6×10(-6)-5×10(-3)) for NoV infection and 3×10(-8) (7×10(-10)-3×10(-6)) for hepatitis A jaundice. The contribution to virus contamination of hand-contact was larger as compared with the contribution of irrigation, the conveyor belt or the water used for produce rinsing. In conclusion, viral contamination in the lettuce and soft fruit supply chains occurred and estimated health risks were generally low. Nevertheless, the 97.5% upper limit for the estimated NoV contamination of lettuce suggested that infection risks up to 50% per serving might occur. Our study suggests that attention to full compliance for hand hygiene will improve fresh produce safety related to virus risks most as compared to the other examined sources, given the monitoring results. This effect will be further aided by compliance with other hygiene and water quality regulations in production and processing facilities., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Berries, in particular raspberries, have been associated with multiple recalls due to norovirus contamination and were linked to a number of norovirus (NoV) outbreaks. In the present study a total of 130 samples of frozen raspberries were collected from 26 batches in four different raspberry processing companies. In two companies the samples consisted of bulk frozen raspberries serving as raw material for the production of raspberry puree (an intermediate food product in a business to business setting). In two other companies, the samples consisted of bulk individually quick frozen (IQF) raspberries serving as raw material for the production of frozen fruit mixes (as a final food product for consumer). Enumeration of Escherichia coli and coliforms was performed as well as real-time reverse transcription PCR (RT-qPCR) detection of GI and GII NoV (in 2 × 10 g). In addition, in cases where positive NoV GI or GII RT-qPCR signals were obtained, an attempt to sequence the amplicons was undertaken. Six out of 70 samples taken from the 14 batches of frozen raspberries serving raspberry puree production provided a NoV RT-qPCR signal confirmed by sequencing. Four of these six positive samples clustered in one batch whereas the other two positive samples clustered in another batch from the same company. All six positive samples showed NoV RT-qPCR signals above the limit of quantification of the RT-qPCR assay. These two positive batches of frozen raspberries can be classified as being of insufficient sanitary quality. The mean NoV level in 20 g of these raspberry samples was 4.3 log genomic copies NoV GI/20 g. The concern for public health is uncertain as NoV RT-qPCR detection is unable to discriminate between infectious and non-infectious virus particles. For the IQF raspberries, one batch out of 12 tested NoV positive, but only 1 out of the 5 samples analyzed in this batch showed a positive RT-qPCR GI NoV signal confirmed by sequencing. The RT-qPCR signal was below the limit of quantification of the assay used (<3.7 log genomic copies/20g). It was shown that the applied protocol for sequencing of the amplicon to confirm the specificity of the RT-qPCR signal was successful for GI NoV amplicons but often failed and provided an inconclusive result for GII NoV amplicons., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In recent years, numerous foodborne outbreaks due to consumption of berry fruit contaminated by human enteric viruses have been reported. This European multinational study investigated possible contamination routes by monitoring the entire food chain for a panel of human and animal enteric viruses. A total of 785 samples were collected throughout the food production chain of four European countries (Czech Republic, Finland, Poland and Serbia) during two growing seasons. Samples were taken during the production phase, the processing phase, and at point-of-sale. Samples included irrigation water, animal faeces, food handlers' hand swabs, swabs from toilets on farms, from conveyor belts at processing plants, and of raspberries or strawberries at points-of-sale; all were subjected to virus analysis. The samples were analysed by real-time (reverse transcription, RT)-PCR, primarily for human adenoviruses (hAdV) to demonstrate that a route of contamination existed from infected persons to the food supply chain. The analyses also included testing for the presence of selected human (norovirus, NoV GI, NoV GII and hepatitis A virus, HAV), animal (porcine adenovirus, pAdV and bovine polyomavirus, bPyV) and zoonotic (hepatitis E virus, HEV) viruses. At berry production, hAdV was found in 9.5%, 5.8% and 9.1% of samples of irrigation water, food handlers' hands and toilets, respectively. At the processing plants, hAdV was detected in one (2.0%) swab from a food handler's hand. At point-of-sale, the prevalence of hAdV in fresh raspberries, frozen raspberries and fresh strawberries, was 0.7%, 3.2% and 2.0%, respectively. Of the human pathogenic viruses, NoV GII was detected in two (3.6%) water samples at berry production, but no HAV was detected in any of the samples. HEV-contaminated frozen raspberries were found once (2.6%). Animal faecal contamination was evidenced by positive pAdV and bPyV assay results. At berry production, one water sample contained both viruses, and at point-of-sale 5.7% and 1.3% of fresh and frozen berries tested positive for pAdV. At berry production hAdV was found both in irrigation water and on food handler's hands, which indicated that these may be important vehicles by which human pathogenic viruses enter the berry fruit chain. Moreover, both zoonotic and animal enteric viruses could be detected on the end products. This study gives insight into viral sources and transmission routes and emphasizes the necessity for thorough compliance with good agricultural and hygienic practice at the farms to help protect the public from viral infections., (© 2013.)

Multiple outbreaks of human norovirus (hNoV) have been associated with fresh produce, such as soft berries and lettuce. Even though food handlers are considered an important source for the introduction of hNoV into food chains, their contribution to public health risks associated with hNoV remains unknown. To assess to which extent food handlers contribute to the introduction and spread of hNoV in fresh produce chains quantitative virus transfer data are needed. We estimated transfer proportions of hNoV GI.4, GII.4, murine norovirus (MNV-1), a culturable surrogate of hNoV, and human adenovirus (hAdV-2), a human pathogen proposed as an indicator for human faecal pollution, between gloved fingertips and raspberries, strawberries, and lettuce, by quantitative RT-PCR and cell culture if applicable. Virus transfer proportions were corrected for virus-matrix specific recoveries, and variability and uncertainty of the parameters were estimated. Virus transfer from gloves to soft berries was generally lower as compared to lettuce, with mean transfer proportions ranging between 0.1 to 2.3% and 9 to 10% for infectious MNV-1 and hAdV-2, respectively. Transfer from produce to glove was mostly greater than transfer from glove to produce, adding to the likelihood of virus transfer due to cross contamination from contaminated produce via food handlers. HNoV GI.4 and hNoV GII.4 showed no significant difference between their mean transfer proportions. Using the estimated transfer proportions, we studied the impact of low and high transfer proportions on the public health risk, based on a scenario in which a food handler picked raspberries with contaminated fingertips. Given the made assumptions, we could show that for a pathogen as infectious as hNoV, low transfer proportions may pose a greater public health risk than high transfer proportions, due to a greater viral spread. We demonstrated the potential of food handlers in spreading hNoV in food chains, showing that prevention of virus contamination on food handlers' hands is crucial for food safety. Nevertheless, complete prevention of virus contamination on fresh produce cannot be achieved in reality, and reliable and effective intervention measures are consequently required. We estimated that, especially for low transfer proportions, a robust one log10-unit reduction of infectious hNoV on contaminated produce, and on food handlers' hands, could lower the public health risk substantially. Using the obtained data in quantitative risk assessment will aid in elucidating the contribution of food handlers in hNoV transmission., (© 2013 Elsevier B.V. All rights reserved.)

Determining the stability, or persistence in an infectious state, of foodborne viral pathogens attached to surfaces of soft fruits and salad vegetables is essential to underpin risk assessment studies in food safety. Here, we evaluate the effect of temperature and sunlight on the stability of infectious human adenoviruses type 2 and MS2 bacteriophages on lettuce and strawberry surfaces as representative fresh products. Human adenoviruses have been selected because of their double role as viral pathogens and viral indicators of human fecal contamination. Stability assays were performed with artificially contaminated fresh samples kept in the dark or under sunlight exposure at 4 and 30°C over 24h. The results indicate that temperature is the major factor affecting HAdV stability in fresh produce surfaces, effecting decay between 3 and 4 log after 24h at 30°C. The inactivation times to achieve a reduction between 1 and 4-log are calculated for each experimental condition. This work provides useful information to be considered for improving food safety regarding the transmission of foodborne viruses through supply chains., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Human noroviruses (hNoV) have been detected on soft fruits. Especially raspberries have been found to be associated with outbreaks of gastroenteritis suggesting persistence of hNoV on these fruits. Therefore, the persistence of hNoV GII.4 and GI.4, murine norovirus (MNV-1, a culturable surrogate for hNoV), and human adenovirus (hAdV, an indicator for human fecal contamination), on raspberries, strawberries and in phosphate buffered saline (PBS) at 4°C, 10°C and 21°C, mimicking commonly applied storage conditions was studied by molecular and cell culture techniques. Monophasic, biphasic and Weibull models were fitted to virus counts with maximum likelihood estimation. The tested viruses were persistent (≤0.5 log(10)-unit reduction in viral titer) under all studied conditions in PBS, at 4°C and 10°C on raspberries, and at 4°C on strawberries. The difference in viral persistence on raspberries and strawberries was most pronounced at 21°C. Here, infectious MNV-1 and hAdV particles decayed rapidly on strawberries with TFL-values (time for the first log(10)-unit reduction) of only 1day (95% CI of 0.6-1 and 0.8-1days, respectively). On raspberries, however, the TFL-value of infectious MNV-1 was found to likely exceed the shelf life of the berries with 3days (95% CI of 2.8-3.1days); hAdV remained infectious with only 0.3 log(10)-unit reduction (95% CI of 0.2-0.4) in viral titer. For hNoV GI, a TFL-value of 2days (95% CI 1-4days) was determined based on the targeted genome fragment, whereas the TFL-value of hNoV GII exceeded the shelf life of strawberries at 21°C. The greater viral persistence on raspberries as compared to strawberries, especially at 21°C, may at least in part explain why raspberries are more frequently associated with hNoV outbreaks than strawberries. Moreover, our results show that due to the high persistence of the virus already low contamination levels of the highly infectious hNoV may be associated with an infection risk of humans after consumption of raspberries. The estimated decay parameters and uncertainties of this study serve as important input requirements in the quantitative assessment of public health risks from the consumption of soft fruits., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Foodborne viruses, especially noroviruses (NoV), are increasingly reported as the cause of foodborne outbreaks. NoV outbreaks have been reported linked to fresh soft red fruits and leafy greens. Belgium, Canada and France were the first countries to provide data about the prevalence of NoV on fresh produce. In total, 867 samples of leafy greens, 180 samples of fresh soft red fruits and 57 samples of other types of fresh produce (tomatoes, cucumber and fruit salads) were analyzed. Firstly, the NoV detection methodology, including virus and RNA extraction, real-time RT-PCR and quality controls were compared among the three countries. In addition, confirmation and genotyping of the NoV strains was attempted for a subset of NoV positive samples using conventional RT-PCR targeting an alternative region followed by sequencing. Analysis of the process control showed that 653, 179 and 18 samples of the leafy greens, soft red fruits and other fresh produce types were valid for analysis based on the recovery of the process control. NoV was detected by real-time RT-PCR in 28.2% (N=641), 33.3% (N=6) and 50% (N=6) of leafy greens tested in Canada, Belgium and France, respectively. Soft red fruits were found positive by real-time RT-PCR in 34.5% (N=29) and 6.7% (N=150) of the samples tested in Belgium and France, respectively. 55.5% (N=18) of the other fresh produce types, analyzed in Belgium, were found NoV positive by real-time RT-PCR. Conventional RT-PCR resulted in an amplicon of the expected size in 19.5% (52/266) of the NoV positive samples where this assay was attempted. Subsequent sequencing was only successful in 34.6% (18/52) of the suspected amplicons obtained by conventional RT-PCR. From this study, using the described methodology, NoV genomes were frequently detected in fresh produce however sequence confirmation was not successful for the majority of the samples tested. Infection or outbreaks were rarely or not known to be related to the NoV positive samples. With the increase in sensitivity of the detection methodology, there is an increasing concern about the interpretation of positive NoV results by real-time amplification. Strategies to confirm the results by real-time RT-PCR should be developed in analogy with the detection of microbial pathogens in foods. Detection might indicate contact with NoV in the fresh produce chain. Consequently, a potential risk for infection cannot be excluded but the actual risk from RT-PCR NoV positive produce is still unknown. Studies should be designed determining the probability of infection related to the presence or levels of NoV genomic copies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Norovirus (NV) and hepatitis A virus (HAV) are foodborne enteric viruses associated with outbreaks of disease following consumption of fresh or frozen produce. Model experiments were performed to determine the effectiveness of certain commercial processes for the removal of enteric viruses that might be present in berries and herbs. The survival and persistence of HAV, NV, rotavirus (RV) and feline calicivirus (FCV), a surrogate for NV, in frozen produce over time were determined. Survival and inactivation of HAV, RV and FCV were assessed by viral culture and quantitative reverse transcription-PCR (RT-PCR), whereas NV persistence was determined by quantitative RT-PCR only. Freezing did not significantly reduce the viability of any of the viruses except the infectivity of FCV in strawberries. Frozen storage for 3 months had limited effects on HAV and RV survival in all tested food products, whereas in frozen raspberries and strawberries FCV infectivity showed the highest decay rate due to acid pH. To simulate postharvesting conditions, fresh berries and herbs were rinsed with tap, warm or chlorinated water or with a chlorine dioxide (ClO(2)) solution. Available chlorine at a concentration of 200 ppm and ClO(2) at 10 ppm reduced measurable enteric viruses in raspberry and parsley samples by less than 2 log(10) units.

Disinfection of produce is principally used to inactivate spoilage microbes and may also reduce the risk of consumer exposure to enteric pathogens. However, the rate and extent of enteric virus inactivation by free chlorine on produce has not been adequately characterized. Experiments were performed to determine the kinetics of free chlorine inactivation of hepatitis A virus (HAV) and the indicator virus coliphage MS2 on strawberries (SBs), cherry tomatoes (CTs), and head lettuce (HL). The oxidant demand of these produce items also was determined. When produce items were exposed to approximately 20 parts per million (ppm) solution of free chlorine for 5-10 min, HAV and MS2 were inactivated by 90-99% and in some cases virus inactivation was > or =99%. Exposure of strawberries to approximately 200 ppm free chlorine resulted in more rapid and extensive inactivation of both viruses. The produce items tested in this study exhibited a demand for chlorine which varied by produce type, and chlorine residuals declined over time. These results demonstrate the potential for chlorine to reduce the levels of infectious viruses on different produce types, but adequate contact time and chlorine residual are required to achieve maximum virus inactivation. The difference in chlorine demand between SBs, CTs, and HL suggests that varying disinfection practices are needed for the wide variety of processed fruits and vegetables. The inactivation kinetics of MS2 and HAV were similar, suggesting that MS2 and perhaps other similar bacterial viruses may be used as process indicators and surrogates for determining the disinfection efficacy of produce in the laboratory or in actual practice.

Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform.

A method was developed for detection of hepatitis A virus (HAV) in soft fruits (raspberries and strawberries). After washing the sample in 1 M sodium bicarbonate with added soya protein, fruits were removed by slow speed centrifugation, then particulate material and residual pectin were removed from the supernatant by flocculation and pectinase treatment during another slow speed centrifugation. Virus particles were then sedimented by ultracentrifugation. RNA was extracted from the virus particles, and nested RTPCR was performed on the nucleic acid extract. Nested RTPCR comprised an RTPCR, followed by PCR to amplify sequences within the amplicon. Internal amplification controls (IACs) were constructed for both the RTPCR and the PCR. The sensitivity of the nested RTPCR was approximately 10 RTPCRU. The overall method was shown to be able to detect 10(4) RTPCRU HAV in 90 g fresh strawberries, and 10(3) RTPCRU HAV in 60 g fresh raspberries. It is estimated that the lowest possible limit of detection of the method should be between 40 and 400 RTPCRU HAV per fruit sample. The method can be performed within one day, in suitably equipped microbiological laboratories, and is suitable for routine screening of food samples, and for analysis of suspected samples, e.g. during outbreak investigations.

Following an acute foodborne gastroenteritis outbreak in southern Sweden, stool specimens from five of nine ill patients were found positive for norovirus using reverse transcriptase polymerase chain reaction. Epidemiological data pointed to raspberry cakes as the source of the outbreak. Using a combination of generic and patient-specific primers and novel food analysis methodology (with extraction efficiency control and inhibitor removal), norovirus strains from two different genogroups were directly identified in the contaminated raspberries.