Adhesion of human pathogenic enteric viruses and surrogate viruses to inert and vegetal food surfaces

Enteric viruses, particularly human Noroviruses (NoV) and hepatitis A virus (HAV), are key food-borne pathogens. The attachment of these pathogens to foodstuff and food-contact surfaces is an important mechanism in the human contamination process. Studies were done to investigate the nature of the physicochemical forces, such as hydrophobic and electrostatic ones, involved in the interaction virus/matrix but, at this day, only few data are available concerning surface properties of viruses and prediction of the adhesion capacity of one specific virus onto matrices is still very difficult. The purpose of this study was to propose a reference system, including a representative virus surrogate, able to predict as close as possible behaviour of pathogenic viruses in term of adhesion on inert (stainless steel and polypropylene) and food surfaces (lettuce leaves, strawberries and raspberries). The adhesion of human pathogenic enteric viruses, cultivable strain of HAV and non-cultivable strains of human NoV (genogroups I and II), have been quantified and compared to these of human enteric viruses surrogates, included the MNV-1 and three F-specific RNA bacteriophages (MS2, GA and Qβ). A standardized approach was developed to assess and quantify viral adhesion on tested matrices after a contact time with each virus using real-time RT-PCR. Methods used for virus recovery were in accordance with the CEN recommendations, including a bovine Enterovirus type 1 as control to monitor the efficiency of the extraction process and amplification procedure from directly extracted or eluted samples. The adhesion of human pathogenic viruses, ranging from 0.1 to 2%, could be comparable for all matrices studied, except for NoV GII on soft fruits. Adhesion percentages obtained for the studied surrogate virus and phages were shown to be comparable to those of HAV and NoV on inert and lettuce surfaces. The MNV-1 appeared as the best candidate to simulate adhesion phenomena of all human pathogenic enteric viruses on all studied surfaces, while MS2 and GA bacteriophages could be a good alternative as model of viral adhesion on inert and lettuce surfaces. These results will be usable to design relevant experimental systems integrating adhesion behaviour of enteric viruses in the assessment of the efficiency of a technological or hygienic industrial process.     

Foodborne viruses: an emerging problem

Several groups of viruses may infect persons after ingestion and then are shed via stool. Of these, the norovirus (NoV) and hepatitis A virus (HAV) are currently recognised as the most important human foodborne pathogens with regard to the number of outbreaks and people affected in the Western world. NoV and HAV are highly infectious and may lead to widespread outbreaks. The clinical manifestation of NoV infection, however, is relatively mild. Asymptomatic infections are common and may contribute to the spread of the infection. Introduction of NoV in a community or population (a seeding event) may be followed by additional spread because of the highly infectious nature of NoV, resulting in a great number of secondary infections (50% of contacts). Hepatitis A is an increasing problem because of the decrease in immunity of populations in countries with high standards of hygiene. Molecular-based methods can detect viruses in shellfish but are not yet available for other foods. The applicability of the methods currently available for monitoring foods for viral contamination is unknown. No consistent correlation has been found between the presence of indicator microorganisms (i.e. bacteriophages, E. coli) and viruses. NoV and HAV are highly infectious and exhibit variable levels of resistance to heat and disinfection agents. However, they are both inactivated at 100 degrees C. No validated model virus or model system is available for studies of inactivation of NoV, although investigations could make use of structurally similar viruses (i.e. canine and feline caliciviruses). In the absence of a model virus or model system, food safety guidelines need to be based on studies that have been performed with the most resistant enteric RNA viruses (i.e. HAV, for which a model system does exist) and also with bacteriophages (for water). Most documented foodborne viral outbreaks can be traced to food that has been manually handled by an infected foodhandler, rather than to industrially processed foods. The viral contamination of food can occur anywhere in the process from farm to fork, but most foodborne viral infections can be traced back to infected persons who handle food that is not heated or otherwise treated afterwards. Therefore, emphasis should be on stringent personal hygiene during preparation. If viruses are present in food preprocessing, residual viral infectivity may be present after some industrial processes. Therefore, it is key that sufficient attention be given to good agriculture practice (GAP) and good manufacturing practice (GMP) to avoid introduction of viruses onto the raw material and into the food-manufacturing environment, and to HACCP to assure adequate management of (control over) viruses present during the manufacturing process. If viruses are present in foods after processing, they remain infectious in most circumstances and in most foods for several days or weeks, especially if kept cooled (at 4 degrees C). Therefore, emphasis should be on stringent personal hygiene during preparation. For the control of foodborne viral infections, it is necessary to: Heighten awareness about the presence and spread of these viruses by foodhandlers; Optimise and standardise methods for the detection of foodborne viruses; Develop laboratory-based surveillance to detect large, common-source outbreaks at an early stage; and Emphasise consideration of viruses in setting up food safety quality control and management systems (GHP, GMP, HACCP).     

Fate of Foodborne Viruses in the “Farm to Fork” Chain of Fresh Produce

Norovirus (NoV) and hepatitis A virus (HAV) are the most important foodborne viruses. Fresh produce has been identified as an important vehicle for their transmission. In order to supply a basis to identify possible prevention and control strategies, this review intends to demonstrate the fate of foodborne viruses in the farm to fork chain of fresh produce, which include the introduction routes (contamination sources), the viral survival abilities at different stages, and the reactions of foodborne viruses towards the treatments used in food processing of fresh produce. In general, the preharvest contamination comes mainly from soli fertilizer or irrigation water, while the harvest and postharvest contaminations come mainly from food handlers, which can be both symptomatic and asymptomatic. Foodborne viruses show high stabilities in all the stages of fresh produce production and processing. Low‐temperature storage and other currently used preservation techniques, as well as washing by water have shown limited added value for reducing the virus load on fresh produce. Chemical sanitizers, although with limitations, are strongly recommended to be applied in the wash water in order to minimize cross‐contamination. Alternatively, radiation strategies have shown promising inactivating effects on foodborne viruses. For high‐pressure processing and thermal treatment, efforts have to be made on setting up treatment parameters to induce sufficient viral inactivation within a food matrix and to protect the sensory and nutritional qualities of fresh produce to the largest extent.     

Prevalence of human pathogenic enteric viruses in bivalve molluscan shellfish and cultured shrimp in south west coast of India

The prevalence of human enteric viruses in bivalve molluscan shellfish and shrimp collected off the south west coast of India was studied to assess the extent of fecal pollution of coastal environment. Out of 194 samples analyzed, 37% of oyster, 46% of clam and 15% of shrimp samples were positive for enteroviruses (EV). Adenoviruses (ADV) were detected in 17% of oyster and 27% of clam samples. However, other enteric viruses such as noroviruses (NoV) and hepatitis A virus (HAV) were not detected in any of the samples. High prevalence of EV and ADV was noticed between May to December. Thirty four percent of oyster and 49% of clam samples showed fecal coliform values higher than the limit. MS-2 phage was detected in 57% of oyster and 73% of clam samples. The presence of MS-2 phage and human enteric viruses showed association while fecal coliforms and enteric viruses showed no association. However, 17 samples, which were positive for enteric viruses (EV and ADV), were negative for MS-2 phage.     

Polymers and Biopolymers with Antiviral Activity: Potential Applications for Improving Food Safety

Gastroenteritis and hepatitis, caused by human noroviruses (HuNoVs) and hepatitis A virus (HAV), respectively, are the most common illnesses resulting from the consumption of food contaminated with human enteric viruses. Food‐grade polymers can be tailor designed to improve food safety, either as novel food‐packaging materials imparting active antimicrobial properties, applied in food contact surfaces to avoid cross‐contamination, or as edible coatings to increase fresh produce's shelf life. The incorporation of antimicrobial agents into food‐grade polymers can be used to control the food microbiota and even target specific foodborne pathogens to improve microbiological food safety and to enhance food quality. Enteric viruses are responsible for one fifth of acute gastroenteritis cases worldwide and the development of food‐grade polymers and biopolymers with antiviral activity for food applications is a topic of increased interest, both for academia and the food industry, even though developments are still limited. This review compiles existing studies in this widely unexplored area and highlights the potential of these developments to improve viral food safety.     

Comparison of chlorine and peroxyacetic-based disinfectant to inactivate Feline calicivirus, Murine norovirus and Hepatitis A virus on lettuce

In recent years, raw fruits and vegetables have frequently been involved in foodborne transmission to humans of enteric viruses, particularly noroviruses and hepatitis A virus (HAV). Although viral contamination can occur during all steps of food processing, primary production is a critical stage on which prevention measures must be focused to minimize the risk of infection to consumers. Postharvest sanitation may be a valid technological solution for decreasing the bacterial load on fresh raw material, but there is a lack of data concerning the effectiveness of this process on enteric viruses. In this study, we compared the survival of two human norovirus surrogates, the feline calicivirus (FCV), and the murine norovirus (MNV-1), and of HAV on lettuce after water washing with bubbles and with or without ultrasound, and washing with bubbles in the presence of active chlorine (15 ppm) or peroxyacetic acid-based disinfectant (100 ppm). Cell culture and quantitative RT-PCR assays were used to detect and quantify the viruses on the surface of the lettuce after the sanitizing treatments. Levels of viral inactivation on the lettuce leaves were not significantly different between washing with bubbles and washing with bubbles plus ultrasound and were not dependant on the quantification method. A simple washing without disinfectant resulted in a decrease of approximately 0.7 log units in the quantity of virus detected for HAV and FCV and of 1.0 log unit for MNV-1.In the experimental set-up including a washing step (with or without ultrasound) followed by washing for 2 min in the presence of disinfectants, 15 ppm of active chlorine was found more effective for inactivating FCV (2.9 log units) than HAV and MNV-1 (1.9 log units and 1.4 log units, respectively) whereas 100 ppm of peroxyacetic-based biocide was found effective for inactivating FCV (3.2 log units) and MNV-1 (2.3 log units), but not HAV (0.7 log units). Quantitative RT-PCR results indicated that the presence of viral RNA did not correlate with the presence of infectious viruses on disinfected lettuce, except for MNV-1 processed with chlorine (15 ppm). In comparison with water washing, a substantial additional decrease of genomic FCV titer (1.1 log units) but no significant reduction of the genomic titers of HAV and MNV-1 were found on lettuce treated with chlorine (15 ppm). No significant effect of the disinfection step of lettuce with peroxyacetic-based biocide (100 ppm peracetic acid) was found by qRT-PCR on all genomic viral titers tested. This study illustrates the necessity of determining the effectiveness of technological processes against enteric viruses, using a relevant reference such as HAV, in order to reduce the risk of hepatitis and gastroenteritis by exposure to vegetables.     

Norovirus, hepatitis A virus and enterovirus presence in shellfish from high quality harvesting areas in Portugal

This is the first report on the screening of shellfish from Portugal for the presence of human enteropathogenic viruses. Approximately 2000 shellfish (Curbicula fluminea, Ruditapes decussatus, Tellina crassa, Spisula solida, Dosinia exoleta, Ensis spp., Mytilus spp., Ostrea edulis and Cerastoderma edule), organized in 49 batches, were collected between March 2008 and February 2009. They were tested for norovirus (NoV), hepatitis A virus (HAV) and enterovirus (EV) by RT-PCR followed by nucleotide sequencing. Bacterial contamination was also evaluated by Escherichia coli counts. Viral contamination was detected throughout the year in all shellfish species and in all collection areas, independently of their harvesting areas classification. Overall, 67% of all analyzed batches were contaminated by at least one of the studied viruses while the simultaneous presence of two and three viruses was detected in 22% and 6% batches, respectively. Of the three viruses, NoV was detected in 37% of the batches, followed by EV in 35%, and HAV in 33%. Nucleotide sequencing of the NoV and HAV RT-PCR products demonstrated that all strains belonged to NoV genotype GII.4 and HAV subgenotype 1B. The presence of NoV and HAV in shellfish from “A class” harvesting areas of Portugal can represent a potential health risk.     

含4 种食源性病毒检测靶标多联装甲RNA的制备、纯化与定值

基于Qβ噬菌体装甲RNA制备平台构建同时含有诺如病毒（norovirus,NoV）、甲肝病毒（hepatitis A virus,HAV）、轮状病毒（rotavirus,RV）、星状病毒（astrovirus,AstV）检测靶标RNA的多联装甲RNA（multiplex armored RNA,AR-MulV）,并进行纯化与初步定值。结果表明,重组质粒在大肠杆菌中成功表达,表达产物大小约为14.1 kDa;制备的AR-MulV经纯化后无杂蛋白与残留重组质粒,电镜下可见大量结构形态完整的病毒样颗粒,大小约为25 nm。初步定值结果显示,AR-MulV中GI型NoV、GII型NoV、HAV、RV和AstV检测靶标RNA的含量分别为（1.24±0.2）×107、（2.54±0.6）×107、（2.24±0.3）×107、（2.96±0.5）×107 copies/μL和（3.19±0.4）×107 copies/μL。本研究基于Qβ噬菌体装甲RNA制备平台成功制备同时包含4 种食源性病毒标准方法检测靶标的多联装甲RNA,为食源性病毒的分子检测以及多重实时荧光定量逆转录-聚合酶链式反应阳性质控样品的研发提供新思路。     

Chlorine disinfection of produce to inactivate hepatitis A virus and coliphage MS2

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 >/=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.     

Detection of noroviruses in foods: a study on virus extraction procedures in foods implicated in outbreaks of human gastroenteritis

Disease outbreaks in which foods are epidemiologically implicated as the common source are frequently reported. Noroviruses and enteric hepatitis A viruses are among the most prevalent causative agents of foodborne diseases. However, the detection of these viruses in foods other than shellfish is often time-consuming and unsuccessful. In this study, three virus concentration methods were compared: polyethylene glycol (PEG) plus NaCl, ultracentrifugation, and ultrafiltration. Two RNA extraction methods, TRIzol and RNeasy Mini Kit (Qiagen), were compared for detection of viruses in whipped cream and lettuce (as representatives of the dairy and vegetable-fruit food groups, respectively). A seeding experiment with canine calicivirus was conducted to determine the efficiency of each virus extraction procedure. The PEG-NaCl-TRIzol method was most efficient for the detection of viruses in whipped cream and the ultracentrifugation-RNeasy-Mini Kit procedure was best for detection on lettuce. Based on the seeding experiments, food items implicated in norovirus-associated gastroenteritis outbreaks were subjected to the optimal procedure for a specific composition and matrix. No noroviruses were detected in the implicated food items, possibly because the concentration of virus on the food item was too low or because of the presence of inhibitory factors. For each food group, a specific procedure is optimal. Inhibitory factors should be controlled in these procedures because they influence virus detection in food.     

Closed-circuit system for the depuration of mussels experimentally contaminated with hepatitis A virus

In Italy, the consumption of raw or slightly cooked mussels represents the most important risk factor for the transmission of hepatitis A virus (HAV). Although there exist effective methods for the bacterial depuration of contaminated mussels, these methods are poorly effective on enteric viruses. The objective of the present study was to evaluate the effectiveness of a closed-circuit depuration system that uses both ozone and UV light for disinfecting water and that allows salinity and temperature, important parameters for the metabolism of mussels (Mytilus galloprovincialis), to be maintained at constant levels. The results showed that this depuration method decreased the viral load (from 1.72 log 50% tissue culture infective dose [TCID50] ml(-1) to <1 log TCID50 ml(-1) within 24 h and from 3.82 log TCID50 ml(-1) to <1 log TCID50 ml(-1) within 48 h). However, in both cases, after 120 h of depuration, a residual amount of virus capable of replicating in cells was detected. These results show that depuration, even if performed with advanced systems, may not guarantee the absence of virus.     

Simultaneous recovery of bacteria and viruses from contaminated water and spinach by a filtration method

Water and leafy vegetables eaten fresh are increasingly reported as being involved in food-borne illness cases. The pathogenic agents responsible for these infections are mainly bacteria and viruses and are present in very small quantities on the contaminated food matrices. Laboratory techniques used to isolate or detect the contaminating agent differ enormously according to the type of microorganisms, generating time and economical losses. The purpose of this study was to optimize a single method which allows at the same time the recovery and concentration of these two main types of pathogenic organisms. Water and spinach samples were artificially contaminated with the feline calicivirus (FCV), rotavirus, hepatitis A virus (HAV), Escherichia coli, Listeria monocytogenes, Campylobacter jejuni and Salmonella Typhimurium. The principle behind the recovery technique is based on the use of a positively charged membrane which adsorbs both viruses and bacteria present in the water or in the rinse from the vegetables. Using conventional microbiology, PCR and RT-PCR, this filtration technique allowed a detection level superior to 10² CFU/g for S. Typhimurium, E. coli, L. monocytogenes and C. jejuni and to 10¹ PFU/g for FCV, HAV and rotavirus. This combined method can also be applied to other bacterial and viral species for the identification of the responsible agent for food-borne illnesses.