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Detection of norovirus (NoV) and hepatitis A virus (HAV) on fruits and vegetables using current standard methodologies can be inefficient. Method optimisation focussing on ease, rapidity and increased viral RNA recovery is needed for efficient reverse transcription (RT)-qPCR detection of viruses. A simple and quick direct lysis method for RNA extraction was optimised (method A) to achieve increased viral RNA recovery and minimised RT-qPCR inhibition by increasing the volume of lysis buffer and inclusion of pectinase, Plant RNA Isolation Aid and OneStep PCR Inhibitor Removal Kit. Method A and an internal method structurally comparable to the ISO 15216 standard (method B) were compared for their efficiencies to recover viral RNA from the process controls, mengovirus (MC0) and murine norovirus (MNV), spiked in 13 types of fruits, vegetables, compound foods or seeds/nuts. All extracts (> 61) were also analysed for RT-qPCR inhibition and for natural contamination of NoV and HAV. The overall mean extraction efficiencies of MC0 and MNV were 36 ± 31 and 44 ± 38%, respectively, for method A and 9 ± 16 and 5 ± 11%, respectively, for method B. Inhibition of RT-qPCR amplification of RNA from NoV genogroup (G)I, NoV GII, and HAV ranged from 5 ± 10 to 13 ± 14% for method A and 34 ± 36 to 48 ± 40% for method B. NoV GII was detected in samples of strawberries and seaweed processed by both methods. In conclusion, the new direct lysis method showed an overall better performance compared to the modified ISO 15216 standard and should be validated for implementation in analysis of viruses in foods of plant origin.

In a recent report by risk assessment experts on the identification of food safety priorities using the Delphi technique, foodborne viruses were recognized among the top rated food safety priorities and have become a greater concern to the food industry over the past few years. Food safety experts agreed that control measures for viruses throughout the food chain are required. However, much still needs to be understood with regard to the effectiveness of these controls and how to properly validate their performance, whether it is personal hygiene of food handlers or the effects of processing of at risk foods or the interpretation and action required on positive virus test result. This manuscript provides a description of foodborne viruses and their characteristics, their responses to stress and technologies developed for viral detection and control. In addition, the gaps in knowledge and understanding, and future perspectives on the application of viral detection and control strategies for the food industry, along with suggestions on how the food industry could implement effective control strategies for viruses in foods. The current state of the science on epidemiology, public health burden, risk assessment and management options for viruses in food processing environments will be highlighted in this review., Highlights • Foodborne virus outbreaks carry both a heavy public health and economic burden • Reliable detection of viruses in food matrixes remains a challenge • Current process validations are hampered by difficulty in cultivating viruses. • Classical approaches to risk assessment are possible, if the appropriate methodologies are developed. • Risk assessments are based on general principles and research is needed to support these assessments. • Research effort, needs to be undertaken to understand the ecology, behaviour and transmission of foodborne viruses from the farm and to the consumer.

Human viral pathogens are a major public health threat. Reliable information that accurately describes and characterizes the global occurrence and transmission of human viruses is essential to support national and global priority setting, public health actions, and treatment decisions. However, large areas of the globe are currently without surveillance due to limited health care infrastructure and lack of international cooperation. We propose a novel surveillance strategy, using metagenomic analysis of toilet material from international air flights as a method for worldwide viral disease surveillance. The aim of this study was to design, implement, and evaluate a method for viral analysis of airplane toilet waste enabling simultaneous detection and quantification of a wide range of human viral pathogens. Toilet waste from 19 international airplanes was analyzed for viral content, using viral capture probes followed by high-throughput sequencing. Numerous human pathogens were detected including enteric and respiratory viruses. Several geographic trends were observed with samples originating from South Asia having significantly higher viral species richness as well as higher abundances of salivirus A, aichivirus A and enterovirus B, compared to samples originating from North Asia and North America. In addition, certain city specific trends were observed, including high numbers of rotaviruses in airplanes departing from Islamabad. Based on this study we believe that central sampling and analysis at international airports could be a useful supplement for global viral surveillance, valuable for outbreak detection and for guiding public health resources.

Summary Background Viruses cause a major proportion of human infections, especially gastroenteritis and respiratory infections in children and adults. Indirect transmission between humans via environmental surfaces may play a role in infections, but methods to investigate this have been sparse. Aim To validate and test efficient and reliable procedures to detect multiple human pathogenic viruses on surfaces. Methods The study was divided into two parts. In Part A, six combinations of three different swabs (consisting of cotton, foamed cotton, or polyester head) and two different elution methods (direct lysis or immersion in alkaline glycine buffer before lysis) were tested for efficient recovery of human norovirus GII.7 and mengovirus from artificially contaminated surfaces. In Part B we determined the detection limit for norovirus GI.1 and GII.3 using the best procedure found in Part A linked with a commercial multiplex real-time quantitative polymerase chain reaction detection assay. Findings Combining the polyester swab with direct lysis allowed recovery down to 100 and 10 genome copies/cm 2 of norovirus GI.1 and GII.3, respectively. This procedure resulted in the significant highest recovery of both norovirus and mengovirus, whereas no differences in amplification efficiencies were observed between the different procedures. Conclusion The results indicate that it is possible to detect low concentrations of virus on environmental surfaces. We therefore suggest that a polyester swab, followed by direct lysis, combined with a multiplex qPCR detection assay is an efficient screening tool that merits study of different respiratory and gastrointestinal viruses on environment surfaces.

Introduction: In early April 2016, an unusual high number of point-source outbreaks of gastrointestinal disease were reported to occur in Denmark. Methods: Outbreaks were individually investigated. Two analytical studies were performed. Patient stool samples collected and analysed; positive stool samples were sequenced over the polymerase and/or capsid gene areas. Implicated lettuce heads were collected and analysed for the presence of norovirus. Foods were traced-back and traced-forward and international alert systems applied. Results: A total of 23 linked point-source outbreaks occurred over the course of one week. Fresh green coral lettuce (Lollo Bionda lettuce) had been consumed in all settings. In a cohort study including 234 participants a dish containing green lettuce was associated with illness. Norovirus of Genogroup I (GI) was detected in samples from 28 patients comprising eight of the outbreaks. Sequencing showed GI.P2-GI.2. GI norovirus was detected in one of 20 examined lettuce heads. All lettuce consumed was supplied by the same packer who in turn had bought the lettuce from a wholesaler in France. The two lots of lettuce came from two different growers in different parts of France. Discussion: Green coral lettuce produced in France was found to have caused a large series of linked norovirus outbreaks in Denmark as established by a number of lines of evidence. A similar incidence occurred in 2010. Fresh lettuce increasingly appear to be a risk food for norovirus infections.

Summary Background The rising number of children in daycare nurseries increases opportunities for the transmission of infectious diseases. Pathogens may be transmitted directly from child to child via sneezing, coughing and touching, or indirectly via the environment. Toys are among the fomites with the highest pathogen load, but their role in disease transmission is unknown. Aim To determine if washing and disinfection of toys can reduce sickness absence and microbial pathogen load in the nursery environment. Methods Twelve nurseries (caring for 587 children) were randomized to intervention and control groups. The intervention consisted of washing and disinfection of toys and linen every two weeks for three months by a commercial cleaning company. The extent and causes of sickness absence among the children were recorded in both groups before and after introduction of the intervention. Ten sampling points in each nursery were examined for bacteria and respiratory viruses. Results The presence of respiratory virus DNA/RNA was widespread, but very few pathogenic bacteria were found in the environment. The intervention reduced the presence of adenovirus [odds ratio (OR) 2.4, 95% confidence interval (CI) 1.1–5.0], rhinovirus (OR 5.3, 95% CI 2.3–12.4) and respiratory syncytial virus (OR 4.1, 95% CI 1.5–11.2) compared with the control group, but the intervention had no effect on sickness absence or disease patterns in the nurseries. Conclusion Although cleaning and disinfection of toys every two weeks can decrease the microbial load in nurseries, it does not appear to reduce sickness absence among nursery children.

Exposure to bioaerosols associated with wastewater treatment processes may represent an occupational health risk for workers at wastewater treatment plants (WWTPs). A high frequency of acute symptoms in the gastrointestinal tract among the wastewater workers at a Danish WWTP has been reported. The objective of the study was therefore to examine the exposure of the workers to aerosolised microorganisms. Sampling of inhalable endotoxin, bacteria, moulds and viruses was performed on one occasion using personal samplers. Noroviruses (NoVs) and endotoxin were detected at concentrations that could pose an occupational health risk and possibly contribute to the increased frequency of gastrointestinal illness among the workers and should therefore be investigated further. In addition, positive correlations between exposure to endotoxin, bacteria, moulds and NoVs were found and indicate that the exposure to bioaerosols may be related to work tasks. This is the first study directly showing an occupational exposure to airborne NoVs by its detection in airborne dust.

Foodborne outbreaks caused by noroviruses (NoVs) and hepatitis A virus (HAV) are often linked to consumption of contaminated shellfish. The objective of this study was to identify an appropriate virus recovery method for real-time reverse transcriptase (RT)-PCR detection and subsequently to evaluate this method on shellfish bioaccumulated with virus in a collaborative study. Five methods were compared for recovery of NoV GII.7 and feline calicivirus from spiked digestive tissue of oysters and mussels. A method based on proteinase K digestion followed by NucliSENS miniMAG extraction was found to be the most efficient with a 50% limit of detection (LOD(50)) of 62 and 12 RT-PCR U/1.5 g digestive tissue for NoV GII.7 in oysters and mussels, respectively. Evaluation of the method in four laboratories found the percentage of sensitivity, based on low/high levels of virus bioaccumulated in oysters, to be 33/80 for NoV GI.3b, 13/92 for NoV GII.4 and 50/42 for HAV. A specificity of 100% was found for all three viruses in non-bioaccumulated oysters. As process control Mengovirus (vMC(0)) showed an average recovery of 1.8% from oysters and 1.2% from mussels. The study demonstrates that this recovery method can be useful for harmonized data generation and routine viral analyses of shellfish.

BACKGROUND: Norovirus (NoV) is the predominant cause of foodborne disease outbreaks. Virus contamination may occur during all steps of food processing, from production to preparation and serving. The relative importance of these different routes of contamination is unknown.METHODS: The purpose of this study was to estimate the proportions of outbreaks caused by asymptomatic and symptomatic food handlers (FHs). Reports of foodborne NoV and sapovirus outbreaks (n = 191) that occurred over a 7-year period were extracted, reviewed, and categorized according to the available evidence for source of contamination.RESULTS: In 64 (34%) of the outbreaks, contamination from FHs took place during preparation or serving of food. In the majority of these outbreaks (n = 41; 64%), the FHs were asymptomatic during food handling. Some had been in contact with ill household members before handling the food and remained asymptomatic; others developed symptoms shortly after or were post-symptomatic. In 51 (27%) of the outbreaks, contamination occurred during production of the food, and in 55 (29%) of the outbreaks, contamination had supposedly occurred after serving a guest at a self-serve buffet.CONCLUSIONS: Guidelines regarding exclusion of FHs where household members suffer from gastroenteritis could limit the number of outbreaks.

To the Editor: Norovirus is the world’s leading cause of nonbacterial acute gastroenteritis (1). Since their emergence, GII.P17-GII.17 noroviruses have replaced the GII.4 Sydney 2012 variant as the dominating norovirus genotype in parts of Asia (2), although they have been detected only sporadically, in a limited number, on other continents (3).

SUMMARYNorovirus outbreaks occur frequently in Denmark and it can be difficult to establish whether apparently independent outbreaks have the same origin. Here we report on six outbreaks linked to frozen raspberries, investigated separately over a period of 3 months. Norovirus from stools were sequence-typed; including extended sequencing of 1138 bp encompassing the hypervariable P2 region of the capsid gene. Norovirus was detected in 27 stool samples. Genotyping showed genotype GI.Pb_GI.6 (polymerase/capsid) with 100% identical sequences. Samples from five outbreaks were furthermore identical over the variable capsid P2 region. In one outbreak at a hospital canteen, frozen raspberries was associated with illness by cohort investigation (relative risk 6·1, 95% confidence interval 3·2–11). Bags of raspberries suspected to be the source were positive for genogroup I and II noroviruses, one typable virus was genotype GI.6 (capsid). These molecular investigations showed that the apparently independent outbreaks were the result of one contamination event of frozen raspberries. The contaminated raspberries originated from a single producer in Serbia and were originally not considered to belong to the same batch. The outbreaks led to consultations and mutual visits between producers, investigators and authorities. Further, Danish legislation was changed to make heat-treatment of frozen raspberries compulsory in professional catering establishments.

In December 2012, an outbreak of acute gastrointestinal illness occurred in a geographical distinct area in Denmark covering 368 households. A combined microbiological, epidemiological and environmental investigation was initiated to understand the outbreak magnitude, pathogen(s) and vehicle in order to control the outbreak. Norovirus GII.4 New Orleans 2009 variant was detected in 15 of 17 individual stool samples from 14 households. Norovirus genomic material from water samples was detected and quantified and sequencing of longer parts of the viral capsid region (>1000 nt) were applied to patient and water samples. All five purposely selected water samples tested positive for norovirus GII in levels up to 1.8×104 genomic units per 200 ml. Identical norovirus sequences were found in all 5 sequenced stool samples and 1 sequenced water sample, a second sequenced water sample showed 1 nt (

Bivalve shellfish are at constant risk of being exposed to pathogens as a consequence of contamination of the shellfish beds with human or animal waste originating from sewage treatment plants or slurry fertilized fields. Consumption of contaminated oysters and mussels are frequently reported as causes of disease outbreaks caused by norovirus or hepatitis A virus. Other zoonotic pathogens such as hepatitis E virus (HEV), rotavirus (RV) and Salmonella from livestock may also be transmitted to shellfish via this route. In this study, 29 pooled samples from commercial Danish blue mussels were tested for porcine pathogens and indicator bacteria Escherichia coli (E. coli). All samples tested negative for HEV, RV and Salmonella, whereas E. coli and the highly stable porcine circovirus type 2 (PCV2) were detected in eight and 12 samples, respectively. This is the first study to report the detection of PCV2 in commercial mussels. Based on the detection of PCV2 in clean areas with low prevalence of the normally applied fecal indicator E. coli, testing for PCV2 may be a more sensitive and robust specific porcine waste indicator in shellfish harvesting areas.

Human disease outbreaks caused by norovirus (NoV) following consumption of contaminated raspberries are an increasing problem. An efficient method to decontaminate the fragile raspberries and the equipment used for processing would be an important step in ensuring food safety. A potential surface treatment that combines pressurized steam and high-power ultrasound (steam-ultrasound) was assessed for its efficacy to inactivate human NoV surrogates: coliphage (MS2), feline calicivirus (FCV), and murine norovirus (MNV) inoculated on plastic surfaces and MS2 inoculated on fresh raspberries. The amounts of infectious virus and viral genomes were determined by plaque assay and reverse transcription-real time quantitative PCR (RT-qPCR), respectively. On plastic surfaces, an inactivation of >99.99% was obtained for both MS2 and FCV, corresponding to a 9.1-log and >4.8-log reduction after 1 or 3 s of treatment, respectively; while a 3.7-log (99.9%) reduction of MNV was reached after 3 s of treatment. However, on fresh raspberries only a 1-log reduction (∼89%) of MS2 could be achieved after 1 s of treatment, at which point damage to the texture of the fresh raspberries was evident. Increasing treatment time (0 to 3 s) resulted in negligible reductions of viral genome titers of MS2, FCV, and MNV on plastic surfaces as well as of MS2 inoculated on raspberries. Steam-ultrasound treatment in its current format does not appear to be an appropriate method to achieve sufficient decontamination of NoV-contaminated raspberries. However, steam-ultrasound may be used to decontaminate smooth surface areas and utensils in food production and processing environments.

Viruses are increasingly recognized as pathogens involved in foodborne infections. The viruses most adapted and likely to be carried in this way are those transmitted by the faecal-oral route. These include viral agents causing gastro-intestinal disease in humans such as noroviruses (NoV), but also agents such as hepatitis A virus (HAV), which, although being transmitted by the faecal-oral route, exhibit their classical clinical symptoms elsewhere in the body. Data from a Europe-wide study of NoV outbreaks illustrate that outbreaks are commonly detected in all countries that have sufficient diagnostic capacity and undertake systematic evaluation of outbreaks of gastro-enteritis other than for bacterial infections. Experience with other pathogen/food matrix combinations has demonstrated the necessity to establish sampling strategies in order to undertake effective monitoring of foodstuffs for risk management and control purposes. The ultimate goal in source tracing is the possibility to rapidly type the isolates. This is particularly relevant for the highly variable NoV, which together with HAV, is the main target for detection in food.

BACKGROUND: Current detection and genotyping methods of genogroup (G) I and II noroviruses (NoVs) consist of a 2-step approach including detection of viral RNA by TaqMan realtime RT-PCR (RT-qPCR) followed by conventional RT-PCR and sequencing of partial regions of ORF1 or ORF2. OBJECTIVE: To develop novel long-template one-step TaqMan assays (L-RT-qPCR) for the rapid detection and direct genotyping of GI and GII NoVs and to evaluate the sensitivity and specificity of the assays. STUDY DESIGN: GI and GII-specific broadly reactive L-RT-qPCR assays were developed by combining existing NoV primers and probes targeting the open reading frame (ORF)1–ORF2 junction as well as region C at the 5′–ORF2. The assays were validated using GI and GII RNA transcripts and a coded panel of 75 stool samples containing NoV strains representing 9 GI genotypes and 12 GII genotypes, as well as sapoviruses, astroviruses, polioviruses, and rotaviruses. L-RT-qPCR products were typed by sequencing. RESULTS: The novel GI and GII L-RT-qPCR assays detected and typed all but one of the NoV positive panel samples. As few as 5–500 RNA copies could be accurately typed by sequencing of amplicons. CONCLUSIONS: We developed novel one-step TaqMan RT-qPCR assays for the sensitive detection and direct genotyping of GI and GII NoVs from clinical and environmental matrices.

Enteric viruses, including norovirus (NoV) and hepatitis A virus (HAV), have emerged as a major cause of waterborne outbreaks worldwide. Due to their low infectious doses and low concentrations in water samples, an efficient and rapid virus concentration method is required for routine control. Three newly developed methods, A, B and C, for virus concentration in bottled water were compared against the reference method D: (A) Convective Interaction Media (CIM) monolithic chromatography; filtration of viruses followed by (B) direct lysis of viruses on membrane; (C) concentration of viruses by ultracentrifugation; and (D) concentration of viruses by ultrafiltration, for each methods' (A, B and C) efficacy to recover 10-fold dilutions of HAV and feline calicivirus (FCV) spiked in bottles of 1.5L of mineral water. Within the tested characteristics, all the new methods showed better performance than method D. Methods A, B and C shared a limit of detection (LOD(50)) of nine 50%-tissue culture infectious dose (TCID(50)) of FCV/1.5L, but differed with regard to the LOD(50)'s of HAV with 45, 361 and 3607 TCID(50)/1.5L, respectively, and the percentage of recoveries of HAV/FCV with 34/6, 32/25 and 0.3/0.5, respectively. Method B resulted in significantly (p

Human enteric viruses are detected frequently in various types of environmental water samples, such as irrigation water, wastewater, recreational water, ground or subsurface water and even drinking water, constituting a primary source of gastroenteritis or hepatitis outbreaks. Only a few, but still infective number of viral particles are normally present in water samples, therefore an efficient virus concentration procedure is essential prior to molecular detection of the viral nucleic acid. In this study, a novel chromatographic technology, Convective Interaction Media (CIM) monolithic supports, were optimized and applied to the concentration of hepatitis A virus (HAV) and feline calicivirus (FCV), a surrogate of norovirus (NoV), from water samples. Two-step real-time RT-qPCR was used for quantitation of the virus concentration in the chromatographic fractions. Positively charged CIM QA (quaternary amine) monolithic columns were used for binding of HAV and FCV present in previously inoculated 1.5 l bottled water samples. Column bound viruses were eluted from the monolith using 1M NaCl to a final volume of 15 ml. Elution volume was concentrated further by ultracentrifugation. When the CIM/ultracentrifugation method was compared with another concentration method employing positively charged membranes and ultrafiltration, the recovery of HAV was improved by approximately 20%.

Virus-contaminated soft fruits or vegetables are increasingly identified as causes of foodborne viral illness. Noroviruses and hepatitis A virus are the most common pathogens in viral infections transmitted by these kinds of foods. To improve microbiological detection and monitoring and to increase insights into the contribution of fruits and vegetables to foodborne viral transmission, sensitive, reliable, and standardized methods are needed. More studies on virus detection methods for foods are being published, but validated consensus protocols are not yet available. In this paper, different methodologies are reviewed critically. The use of process controls and internal amplification controls is discussed.

In the absence of culture methods for noroviruses, detection in foods relies on molecular techniques such as Reverse Transcription-Polymerase Chain Reaction (RT-PCR) on extracted viral RNA followed by PCR product confirmation by hybridisation and/or sequencing. However, in order to obtain a successful detection it is of great importance to remove the tissue inhibitors during the viral RNA extraction. To select the most efficient extraction procedure of oysters we have compared four protocols. A pool of digestive gland material from oyster samples was divided into 1.5 g portions and spiked with 10-fold dilutions of human faecal samples containing norovirus genogroup II. The samples were tested on three different occasions using four different sample treatment protocols. The protocols were assessed with regard to their ability to recover viral RNA and detect norovirus in spiked oysters and for their in-house reproducibility. One method using viral elution by a Mixer Mill Cell Disrupter resulted in a 10-fold better recovery than the other three protocols when an RT-seminested PCR (G2SKR/COG2F and G2SKR/G2SKF) detection approach was applied. Although less distinctive this was also the case when NoV was detected by a single round RT-PCR approach using the primers JV13i and JV12y. The second most efficient method was a method using chloroform extraction and polyethylene precipitation.

At least 11 linked outbreaks of gastroenteritis with a total of 260 cases have occurred in Denmark in mid January 2010. Investigations showed that the outbreaks were caused by norovirus of several genotypes and by enterotoxigenic Escherichia coli. Lettuce of the lollo bionda type grown in France was found to be the vehicle.

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 calici-virus 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.

The amount of animal manure used in modern agriculture is increasing due to the increase in global animal production. Pig slurry is known to contain zoonotic bacteria such as E. coli, Salmonella spp. and Campylobacter spp., and viruses such as hepatitis E virus and group A rotavirus. Coliform bacteria, present in manure, have previously been shown to leach into tile drains. This poses a potential threat to aquatic environments and may also influence the quality of drinking water. As knowledge is especially scarce about the fate of viruses when applied to fields in natural settings, this project sets out to investigate the leaching potential of six different microorganisms: E. coli and Enterococcus spp. (detected by colony assay), somatic coliphages (using plaque assays), and hepatitis E virus, porcine circovirus type 2, and group A rotavirus (by real-time polymerase chain reaction). All six microorganisms leached through the soil entering the tile drains situated at 1-m depth the first day following pig slurry application. The leaching pattern of group A rotavirus differed substantially from the pattern for somatic coliphages, which are otherwise used as indicators for virus contamination. Furthermore, group A rotavirus was detected in monitoring wells at 3.5-m depth up to 2 months after pig slurry application. The detection of viral genomic material in drainage water and shallow groundwater signifies a potential hazard to human health that needs to be investigated further, as water reservoirs used for recreational use and drinking water are potentially contaminated with zoonotic pathogens.