Quantitative farm-to-fork risk assessment model for norovirus and hepatitis A virus in European leafy green vegetable and berry fruit supply chains.

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.

Hepatitis E virus in young pigs in Finland and characterization of the isolated partial genomic sequences of genotype 3 HEV.

Hepatitis E virus (HEV) infections occur in swine worldwide. The porcine infection is usually subclinical, but HEV genotypes 3 and 4 are zoonotic agents that cause sporadic, indigenous human cases of hepatitis E. The aims of this study were to investigate the occurrence and dynamics of HEV infections in young pigs by analyzing a total of 273 fecal samples collected from six farrowing farms, to genetically characterize the HEV isolates obtained, and to examine the phylogenetic relationships of HEV isolates occurring at different swine farms in Finland. Fecal shedding of HEV of individual piglets was followed at two farms that were selected from five farms identified as HEV RNA positive. Excretion of HEV was detected in 87.5% of the piglets during the survey. Piglets contracted primary HEV infection 3-8 weeks after weaning, and at the time they were transferred to fattening farms, practically all (96.6%) of the pigs with a sample available at this occasion still excreted the virus. According to phylogenetic analysis, all HEV isolates obtained belonged to HEV genotype 3, subtype e, and a separate, farm-specific isolate originated from 10 of 11 farms examined. The results of our study show that HEV infections are highly common in young pigs, and HEV RNA-positive pigs enable HEV transmission from farrowing to fattening farms, creating a possible risk of infection for pig handlers, and that genetic variations in HEVs originating from different farms occur.

Tracing enteric viruses in the European berry fruit supply chain.

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.

Evaluation of four virus recovery methods for detecting noroviruses on fresh lettuce, sliced ham, and frozen raspberries.

Although noroviruses play a significant role in causing foodborne illness in developed countries, no standardised method for detecting noroviruses in foodstuffs is currently available. This study compared four virus recovery methods based on ultrafiltration, immunomagnetic separation, ultracentrifugation and PEG precipitation techniques using identical real-time RT-PCR protocols for detection of RNA in eluates from lettuce, sliced ham and raspberries inoculated artificially with genogroup II norovirus. Noroviruses in all the food source matrices were successfully detected by all four methods. Ultracentrifugation yielded the highest recovery efficiencies in lettuce and ham, whereas PEG precipitation recovered the highest yield of noroviruses from raspberries. The repeatability of the results and the applicability of the methods to all food matrices were best with PEG precipitation, which had average virus recoveries of 19%, 47% and 28% for lettuce, ham and raspberries (viral RNA in dilution 1:10), respectively. In each case, a tenfold dilution of the extracted RNA clearly reduced the level of PCR inhibitors, which were released from raspberries in particular. The results of this study show that the detection of noroviruses in food is challenging, and more efforts to develop sensitive methods are still needed to detect noroviruses in food containing viruses in low numbers.

Presence of human noro- and adenoviruses in river and treated wastewater, a longitudinal study and method comparison.

Norovirus (NoV) is one of the most common causative agents of waterborne gastroenteritis outbreaks. The main objective of the study was to determine the presence of human NoVs in river water and in treated wastewater (TW) released into the river. During a one-year survey in 2007/2008, NoVs were detected in 30.8% of river samples (20/65), and 40.5% of TW samples (17/45) with a real-time reverse transcription-PCR assay. NoVs were present in the river water in the winter and spring, coinciding with the NoV epidemiological peak in the community and the presence of NoVs in TW. Later in 2009, the concentration method used, pre-filtration with a Waterra filter combined with filtration through a negatively charged membrane, was evaluated against glass wool filtration and freeze-drying for the detection of adenoviruses in river water. The virus amounts measured varied greatly depending on the virus concentration method. The continued monitoring in the spring of 2009 also revealed that the average concentration of noro- and adenoviruses in TW was 2.64 × 10(3) and 1.29 × 10(4) pcr units per mL, respectively. No correlation between the presence of viruses and Escherichia coli was found. These results may be useful for risk assessment studies.

Pet dogs--a transmission route for human noroviruses?

BACKGROUND: Human noroviruses (HuNoVs) are one of the leading causes of diarrhoeal diseases worldwide in all age groups. Virus transmission can occur via the faecal-oral route from person to person or via contaminated food, water, or surfaces. The most common NoV strains circulating among humans belong to genogroup GII. Thus far, to our knowledge, no HuNoVs have been detected in pets. OBJECTIVES: We investigated whether pet dogs could serve as carriers for HuNoVs and thereby transmit the infection to humans. STUDY DESIGN: Ninety-two faecal samples of indoor pet dogs were obtained. The main criteria for sample collection were that the dog or humans in the household had suffered from diarrhoea or vomiting. All samples were screened for HuNoV genogroups GI, GII, and GIV by real-time one-step RT-PCR. RESULTS: We detected HuNoV in four faecal samples from pet dogs that had been in direct contact with symptomatic persons. Three of the positive samples contained genotype GII.4 variant 2006b or 2008 and one GII.12. All NoV-positive dogs lived in households with small children and two dogs showed mild symptoms. CONCLUSIONS: Our results suggest that HuNoVs can survive in the canine gastrointestinal tract. Whether these viruses can replicate in dogs remains unresolved, but an association of pet dogs playing a role in transmission of NoVs that infect humans is obvious.

Virus hazards from food, water and other contaminated environments.

Numerous viruses of human or animal origin can spread in the environment and infect people via water and food, mostly through ingestion and occasionally through skin contact. These viruses are released into the environment by various routes including water run-offs and aerosols. Furthermore, zoonotic viruses may infect humans exposed to contaminated surface waters. Foodstuffs of animal origin can be contaminated, and their consumption may cause human infection if the viruses are not inactivated during food processing. Molecular epidemiology and surveillance of environmental samples are necessary to elucidate the public health hazards associated with exposure to environmental viruses. Whereas monitoring of viral nucleic acids by PCR methods is relatively straightforward and well documented, detection of infectious virus particles is technically more demanding and not always possible (e.g. human norovirus or hepatitis E virus). The human pathogenic viruses that are most relevant in this context are nonenveloped and belong to the families of the Caliciviridae, Adenoviridae, Hepeviridae, Picornaviridae and Reoviridae. Sampling methods and strategies, first-choice detection methods and evaluation criteria are reviewed.

Detection method for avian influenza viruses in water.

Recent events have shown that humans may become infected with some pathogenic avian influenza A viruses (AIV). Since soil and water, including lakes, rivers, and seashores, may be contaminated by AIV excreted by birds, effective methods are needed for monitoring water for emerging viruses. Combining water filtration with molecular methods such as PCR is a fast and effective way for detecting viruses. The objective of this study was to apply a convenient method for the detection of AIV in natural water samples. Distilled water and lake, river, and seawater were artificially contaminated with AIV (H5N3) and passed through a filter system. AIV was detected from filter membrane by real-time RT-PCR. The performance of Zetapor, SMWP, and Sartobind D5F membranes in recovering influenza viruses was first evaluated using contaminated distilled water. SWMP, which gave the highest virus recoveries, was then compared with a pre-filter combined GF/F filter membrane in a trial using natural water samples. In this study, the cellulose membrane SMWP was found to be practical for recovery of AIVs in water. Viral yields varied between 62.1 and 65.9% in distilled water and between 1 and 16.7% in natural water samples. The borosilicate glass membrane GF/F combined with pre-filter was also feasible in filtering natural water samples with viral yields from 1.98 to 7.33%. The methods described can be used for monitoring fresh and seawater samples for the presence of AIV and to determine the source of AIV transmission in an outbreak situation.

Occurrence of human enteric viruses in commercial mussels at retail level in three European countries.

In this study, the prevalence of different enteric viruses in commercial mussels was evaluated at the retail level in three European countries (Finland, Greece and Spain). A total of 153 mussel samples from different origins were analysed for human norovirus (NoV) genogroups I and II, hepatitis A virus (HAV) and hepatitis E virus (HEV). Human adenovirus (HAdV) was also tested as an indicator of human faecal contamination. A full set of controls (such as sample process control, internal amplification controls, and positive and negative controls) were implemented during the process. The use of a sample process control allowed us to calculate the efficiencies of extraction, which ranged from 79 to 0.5 %, with an average value of 10 %. Samples were positive in 41 % of cases, with HAdV being the most prevalent virus detected (36 %), but no significant correlation was found between the presence of HAdV and human NoV, HAV and HEV. The prevalences of human norovirus genogroup II, HEV and human NoV genogroup I were 16, 3 and 0.7 %, respectively, and HAV was not detected. The estimated number of PCR detectable units varied between 24 and 1.4 × 10(3) g(-1) of digestive tract. Interestingly, there appeared to be a significant association between the type of mussel species (M. galloprovincialis) and the positive result of samples, although a complete overlap between country and species examined required this finding to be confirmed including samples of both species from all possible countries of origin.

Hepatitis E virus in patients with unexplained hepatitis in Finland.

BACKGROUND: Hepatitis E virus (HEV) is an important cause of enterically transmitted viral hepatitis, especially in developing countries. Recently, HEV isolates have been identified in humans also in industrialized countries, where it has been considered nonendemic. OBJECTIVES: To investigate whether HEV is a cause of unexplained hepatitis in humans in Finland. STUDY DESIGN: The prevalence of anti-HEV IgM and IgG and HEV RNA was determined in 105 serum samples from 97 patients diagnosed with acute unexplained hepatitis. Partial nucleotide sequences of the HEV isolates obtained were compared with reported sequences in GenBank. RESULTS: Anti-HEV IgM and/or IgG by both ELISA test and immunoblotting were detected in 29 serum samples (27.6%) from 22 patients (22.7%). HEV RNA was detected in eight patient samples (8.2%) and partial nucleotide sequences were present in five of these. All five viruses belonged to HEV genotype 1, and three of them were from patients who had traveled to Asia. CONCLUSION: These preliminary results indicate that HEV must be considered a possible cause of acute hepatitis in Finland.

Survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and caliciviruses in drinking water-associated biofilms grown under high-shear turbulent flow.

Most of the bacteria in drinking water distribution systems are associated with biofilms. In biofilms, their nutrient supply is better than in water, and biofilms can provide shelter against disinfection. We used a Propella biofilm reactor for studying the survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and canine calicivirus (CaCV) (as a surrogate for human norovirus) in drinking water biofilms grown under high-shear turbulent-flow conditions. The numbers of M. avium and L. pneumophila were analyzed with both culture methods and with peptide nucleic acid fluorescence in situ hybridization (FISH) methods. Even though the numbers of pathogens in biofilms decreased during the experiments, M. avium and L. pneumophila survived in biofilms for more than 2 to 4 weeks in culturable forms. CaCV was detectable with a reverse transcription-PCR method in biofilms for more than 3 weeks. E. coli was detectable by culture for only 4 days in biofilms and 8 days in water, suggesting that it is a poor indicator of the presence of certain waterborne pathogens. With L. pneumophila and M. avium, culture methods underestimated the numbers of bacteria present compared to the FISH results. This study clearly proved that pathogenic bacteria entering water distribution systems can survive in biofilms for at least several weeks, even under conditions of high-shear turbulent flow, and may be a risk to water consumers. Also, considering the low number of virus particles needed to result in an infection, their extended survival in biofilms must be taken into account as a risk for the consumer.

Norovirus outbreaks from drinking water.

As part of an intensified monitoring program for foodborne disease outbreaks in Finland, waterborne outbreaks were investigated for viruses. The diagnostic procedure included analysis of patients' stool samples by electron microscopy and reverse transcription-polymerase chain reaction (RT-PCR) for noroviruses and astroviruses. When these test results were positive for a virus, the water sample was analyzed. Virus concentration was based on positively charged filters from 1-L samples. Of the total 41 waterborne outbreaks reported during the observation period (1998-2003), samples from 28 outbreaks were available for analysis. As judged by RT-PCR results from patient samples, noroviruses caused 18 outbreaks. In 10 outbreaks, the water sample also yielded a norovirus. In all but 1 instance, the amplicon sequence was identical to that recovered from the patients. The ubiquity of waterborne norovirus outbreaks calls for measures to monitor water for viruses.

Round-robin comparison of methods for the detection of human enteric viruses in lettuce.

Five methods that detect human enteric virus contamination in lettuce were compared. To mimic multiple contaminations as observed after sewage contamination, artificial contamination was with human calicivirus and poliovirus and animal calicivirus strains at different concentrations. Nucleic acid extractions were done at the same time in the same laboratory to reduce assay-to-assay variability. Results showed that the two critical steps are the washing step and removal of inhibitors. The more reliable methods (sensitivity, simplicity, low cost) included an elution/concentration step and a commercial kit. Such development of sensitive methods for viral detection in foods other than shellfish is important to improve food safety.

Internet use and epidemiologic investigation of gastroenteritis outbreak.

In March 2000, a large outbreak of gastroenteritis occurred in a community where a regional computer network provides free Internet access for 42% of the households. We conducted an epidemiologic investigation using the Internet for data collection. Norovirus was identified in stool samples of nine patients but not in the municipal water supply. Of households with access to the network, 19% participated in the survey. The overall attack rate by household was 63%. Drinking water from the nonchlorinated community water system was associated with illness (relative risk [RR] 1.6; 95% confidence interval [CI] 1.1 to 2.2); drinking water only from a private well was associated with decreased likelihood of illness (RR 0.3; 95% CI 0.1 to 0.8). Data collection through the Internet was efficient. Internet surveys may become more common in epidemiologic investigations and have the potential to provide data rapidly, enabling appropriate public health action. However, methods should be developed to increase response rates and minimize bias.

Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of new norovirus variant.

BACKGROUND: Highly publicised outbreaks of norovirus gastroenteritis in hospitals in the UK and Ireland and cruise ships in the USA sparked speculation about whether this reported activity was unusual. METHODS: We analysed data collected through a collaborative research and surveillance network of viral gastroenteritis in ten European countries (England and Wales were analysed as one region). We compiled data on total number of outbreaks by month, and compared genetic sequences from the isolated viruses. Data were compared with historic data from a systematic retrospective review of surveillance systems and with a central database of viral sequences. FINDINGS: Three regions (England and Wales, Germany, and the Netherlands) had sustained epidemiological and viral characterisation data from 1995 to 2002. In all three, we noted a striking increase in norovirus outbreaks in 2002 that coincided with the detection and emergence of a new predominant norovirus variant of genogroup II4, which had a consistent mutation in the polymerase gene. Eight of nine regions had an annual peak in 2002 and the new genogroup II4 variant was detected in nine countries. Also, the detection of the new variant preceded an atypical spring and summer peak of outbreaks in three countries. INTERPRETATION: Our data from ten European countries show a striking increase and unusual seasonal pattern of norovirus gastroenteritis in 2002 that occurred concurrently with the emergence of a novel genetic variant. In addition to showing the added value of an international network for viral gastroenteritis outbreaks, these observations raise questions about the biological properties of the variant and the mechanisms for its rapid dissemination.

Campylobacter spp., Giardia spp., Cryptosporidium spp., noroviruses, and indicator organisms in surface water in southwestern Finland, 2000-2001.

A total of 139 surface water samples from seven lakes and 15 rivers in southwestern Finland were analyzed during five consecutive seasons from autumn 2000 to autumn 2001 for the presence of various enteropathogens (Campylobacter spp., Giardia spp., Cryptosporidium spp., and noroviruses) and fecal indicators (thermotolerant coliforms, Escherichia coli, Clostridium perfringens, and F-RNA bacteriophages) and for physicochemical parameters (turbidity and temperature); this was the first such systematic study. Altogether, 41.0% (57 of 139) of the samples were positive for at least one of the pathogens; 17.3% were positive for Campylobacter spp. (45.8% of the positive samples contained Campylobacter jejuni, 25.0% contained Campylobacter lari, 4.2% contained Campylobacter coli, and 25.0% contained Campylobacter isolates that were not identified), 13.7% were positive for Giardia spp., 10.1% were positive for Cryptosporidium spp., and 9.4% were positive for noroviruses (23.0% of the positive samples contained genogroup I and 77.0% contained genogroup II). The samples were positive for enteropathogens significantly (P < 0.05) less frequently during the winter season than during the other sampling seasons. No significant differences in the prevalence of enteropathogens were found when rivers and lakes were compared. The presence of thermotolerant coliforms, E. coli, and C. perfringens had significant bivariate nonparametric Spearman's rank order correlation coefficients (P < 0.001) with samples that were positive for one or more of the pathogens analyzed. The absence of these indicators in a logistic regression model was found to have significant predictive value (odds ratios, 1.15 x 10(8), 7.57, and 2.74, respectively; P < 0.05) for a sample that was negative for the pathogens analyzed. There were no significant correlations between counts or count levels for thermotolerant coliforms or E. coli or the presence of F-RNA phages and pathogens in the samples analyzed.

International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of noroviruses.

To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n = 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies.

Frequent reassortments may explain the genetic heterogeneity of rotaviruses: analysis of Finnish rotavirus strains.

The predominant rotavirus electropherotypes (e-types) during 17 epidemic seasons (1980 through 1997) in Finland were established, and representative virus isolates were studied by nucleotide sequencing and phylogenetic analysis. The virus isolates were either P[8]G1 or P[8]G4 types. The G1 and G4 strains formed one G1 lineage (VP7-G1-1) and one G4 lineage, respectively. Otherwise, they belonged to two P[8] lineages (VP4-P[8]-1 and -2) unrelated to their G types. Phylogenetic analysis of partial sequences of all 11 RNA segments obtained from the strains also revealed genetic diversity among gene segments other than those defining P and G types. With the exception of segments 1, 3, and 10, the sequences of the other segments could be assigned to 2 to 4 different genetic clusters. The results of this study suggest that, in addition to the RNA segments encoding VP4 and VP7, the other RNA segments may segregate independently as well. In total, the 9 predominant e-types represented 7 different RNA segment combinations when the phylogenetic clusters of their 11 genes were determined. The extensive genetic diversity and number of e-types among rotaviruses are best explained by frequent genetic reassortment.

Foodborne viruses.

Foodborne and waterborne viral infections are increasingly recognized as causes of illness in humans. This increase is partly explained by changes in food processing and consumption patterns that lead to the worldwide availability of high-risk food. As a result, vast outbreaks may occur due to contamination of food by a single foodhandler or at a single source. Although there are numerous fecal-orally transmitted viruses, most reports of foodborne transmission describe infections with Norwalk-like caliciviruses (NLV) and hepatitis A virus (HAV), suggesting that these viruses are associated with the greatest risk of foodborne transmission. NLV and HAV can be transmitted from person to person, or indirectly via food, water, or fomites contaminated with virus-containing feces or vomit. People can be infected without showing symptoms. The high frequency of secondary cases of NLV illness and - to a lesser extent - of hepatitis A following a foodborne outbreak results in amplification of the problem. The burden of illness is highest in the elderly, and therefore is likely to increase due to the aging population. For HAV, the burden of illness may increase following hygienic control measures, due to a decreasing population of naturally immune individuals and a concurrent increase in the population at risk. Recent advances in the research of NLV and HAV have led to the development of molecular methods which can be used for molecular tracing of virus strains. These methods can be and have been used for the detection of common source outbreaks. While traditionally certain foods have been implicated in virus outbreaks, it is clear that almost any food item can be involved, provided it has been handled by an infected person. There are no established methods for detection of viruses in foods other than shellfish. Little information is available on disinfection and preventive measures specifically for these viruses. Studies addressing this issue are hampered by the lack of culture systems. As currently available routine monitoring systems exclusively focus on bacterial pathogens, efforts should be made to combine epidemiological and virological information for a combined laboratory-based rapid detection system for foodborne viruses. With better surveillance, including typing information, outbreaks of foodborne infections could be reported faster to prevent further spread.