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Bivalve molluscan shellfish (BMS) contamination with gastroenteritis viruses such as norovirus (NoV) is recognized as a significant public health risk worldwide. These foodborne epidemics are characterized by very low viral concentrations in the implicated foods, and by diverse viruses or multiple NoV strains originating from human sewage, resulting in different strains (co-)infecting the consumers. Next-generation sequencing (NGS) offers promising means to describe the diversity of strains present in BMS or to retrace transmission chains in outbreak settings, but their sensitivity and reproducibility remained to be assessed for this application. In this work, we evaluated the ability of three promising NGS methods to sequence diverse NoV in environmental or food BMS and human stool samples. Using laboratory-prepared samples of known NoV composition, we evaluated the sensitivity, reproducibility, repeatability and selectivity of metabarcoding, capture-based metagenomics and long amplicon sequencing, considering representative NoV strains from genogroup I and II, and the impact of the BMS matrix. The metabarcoding, with separate amplification of polymerase and capsid gene segments followed by Illumina sequencing, was the most sensitive method. It was applied to a selection of 212 BMS samples collected through the European Commission’s NoV baseline survey (BLS), demonstrating a high diversity of NoV sequences found in the BMS which reflect the diversity of NoV strains circulating in the European human population. Besides, a capture-based metagenomics with enrichment of vertebrate viruses was applied on 20 of these BLS samples as well as 20 BMS linked to outbreaks and 10 related human stool samples. In BMS, it yielded NoV sequences compatible with the genomes identified in stool samples, but they were too short to allow definitive confirmation of the infection source. The present report describes NGS methods, including the bioinformatic pipelines, applicable to molecular epidemiology of NoV in BMS, their current limitations and expected outcomes

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.

Viral sewage metagenomics is a novel field of study used for surveillance, epidemiological studies, and evaluation of waste water treatment efficiency. In raw sewage human waste is mixed with household, industrial and drainage water, and virus particles are, therefore, only found in low concentrations. This necessitates a step of sample concentration to allow for sensitive virus detection. Additionally, viruses harbor a large diversity of both surface and genome structures, which makes universal viral genomic extraction difficult. Current studies have tackled these challenges in many different ways employing a wide range of viral concentration and extraction procedures. However, there is limited knowledge of the efficacy and inherent biases associated with these methods in respect to viral sewage metagenomics, hampering the development of this field. By the use of next generation sequencing this study aimed to evaluate the efficiency of four commonly applied viral concentrations techniques (precipitation with polyethylene glycol, organic flocculation with skim milk, monolithic adsorption filtration and glass wool filtration) and extraction methods (Nucleospin RNA XS, QIAamp Viral RNA Mini Kit, NucliSENS® miniMAG®, or PowerViral® Environmental RNA/DNA Isolation Kit) to determine the viriome in a sewage sample. We found a significant influence of concentration and extraction protocols on the detected viriome. The viral richness was largest in samples extracted with QIAamp Viral RNA Mini Kit or PowerViral® Environmental RNA/DNA Isolation Kit. Highest viral specificity were found in samples concentrated by precipitation with polyethylene glycol or extracted with Nucleospin RNA XS. Detection of viral pathogens depended on the method used. These results contribute to the understanding of method associated biases, within the field of viral sewage metagenomics, making evaluation of the current literature easier and helping with the design of future studies.

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 can pose a health risk to workers at wastewater treatment plants (WWTPs) and to habitants of their surroundings. The main objective of this study was to examine the presence of harmful microorganisms in the air emission from a new type of hospital WWTP employing advanced wastewater treatment technologies. Air particle measurements and sampling of inhalable bacteria, endotoxin and noroviruses (NoVs) were performed indoor at the WWTP and outside at the WWTP ventilation air exhaust, downwind of the air exhaust, and upwind of the WWTP. No significant differences were seen in particle and endotoxin concentrations between locations. Bacterial concentrations were comparable or significantly lower in the exhaust air than inside the WWTP and in the upwind reference. Bacterial isolates were identified using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In total, 35 different bacterial genera and 64 bacterial species were identified in the air samples. Significantly higher genus and species richness was found with an Andersen Cascade Impactor compared with filter-based sampling. No pathogenic bacteria were found in the exhaust air. Streptomyces was the only bacterium found in the air both inside the WWTP and at the air emission, but not in the upwind reference. NoV genomes were detected in the air inside the WWTP and at the air exhaust, albeit in low concentrations. As only traces of NoV genomes could be detected in the exhaust air they are unlikely to pose a health risk to surroundings. Hence, we assess the risk of airborne exposure to pathogenic bacteria and NoVs from the WWTP air emission to surroundings to be negligible. However, as a slightly higher NoV concentration was detected inside the WWTP, we cannot exclude the possibility that exposure to airborne NoVs can pose a health risk to susceptible to workers inside the WWTP, although the risk may be low.

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.

Viral contamination of drinking water is frequently reported as the primary source of gastroenteritis or hepatitis outbreaks. The presence of viruses at low concentration levels in most environmental water poses major analytical problems when determining their concentration. To evaluate the efficiency of different recovery methods of viral RNA from bottled water, a comparison was made of 2 positively and 2 negatively charged membranes that were used for absorbing and releasing HAV virus particles during the filtration of viral spiked bottled water. All the 4 membranes, regardless of charge and pore size, had low level viral recovery. The results show that a considerable number of the virus particles passed through the pores of the membranes instead of being trapped by the electrostatic charges. Two different procedures were then compared using 1.5L polyethylene bottles spiked with 10-fold serial dilutions of HAV and FCV. The first procedure included an ultrafiltration-based method followed by MiniMag RNA extraction, and the second an ultracentrifugation-based method followed by RNA extraction using QIAamp viral RNA mini kit. The ultracentrifugation-based method resulted in a better recovery of HAV and FCV when compared to the ultrafiltration-based method.

The number of children in daycare centers (DCCs) is rising. This increases exposure to microorganisms and infectious diseases. Little is known about which bacteria and viruses are present in the DCC environment and where they are located. In the study described in this article, the authors set out to determine the prevalence of pathogenic bacteria and viruses and to find the most contaminated fomites in DCCs. Fifteen locations in each DCC were sampled for bacteria, respiratory viruses, and gastrointestinal viruses. The locations were in the toilet, kitchen, and playroom areas and included nursery pillows, toys, and tables, among other things. Coliform bacteria were primarily found in the toilet and kitchen areas whereas nasopharyngeal bacteria were found mostly on toys and fabric surfaces in the playroom. Respiratory viruses were omnipresent in the DCC environment, especially on the toys.

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.

This chapter is devoted to sample processing, which plays a key role in the ultimate success of any detection technology. Many of the techniques discussed in the following chapters process from microliter to a few milliliter, whereas it may be necessary to sample hundreds of milliliter to thousands of liters. In this chapter the need for sample processing is justified, and different methods of sampling are presented. Subsequently, the chapter introduces different methods of sampling and gives an introduction to the basic processes behind different sample processing techniques. The chapter is organized according to process order. First, initial methods of concentration are presented followed by secondary concentration steps, which further reduce the volume and in some cases isolate the pathogen of interest. There is some overlap between techniques used in these stages. Next, a section is devoted to the extraction of nucleic acids for detection by molecular methods. Analytical controls are important to determine how well a process is working, in terms of recovery rate, and different approaches to this are discussed. The chapter concludes with a summary identifying what has thus far been achieved in the area of sample processing, what has so far been neglected, and what needs to be done.

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.

A next frontier of the global food safety agenda has to consider a broad spectrum of bio-risks, such as accidental and intentional contaminations in the food and feed chain. In this article, the background for the research needs related to biotraceability and response to bioterrorism incidents are outlined. Given the current scale of international trade any response need to be considered in an international context. Biotraceability (e.g. the ability to use downstream information to point to processes or within a particular food chain that can be identified as the source of undesirable agents) is crucial in any food-born outbreak and particular in the response to bioterrorism events. In the later case, tested and proven biotraceability improves the following: (i) international collaboration of validated tracing tools and detection methods, (ii) multi-disciplinary expertise and collaboration in the field of food microbiology and conceptual modeling of the food chain, (iii) sampling as a key step in biotracing (iv) optimized sample preparation procedures, including laboratory work in Biosafety level 3 (BSL-3) laboratories, (v) biomarker discovery for relevant tracing and tracking applications, and (vi) high-throughput sequencing using bio-informatic platforms to speed up the characterization of the biological agent. By applying biotraceability, the response phase during a bioterrorism event may be shortened and is facilitated for tracing the origin of biological agent contamination.

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.

Why are there no standardised methods for the detection of enteric viruses in food and water?This is a repeated question raised by the industry and food authority in particular in connection with the recent norovirus outbreaks associated with imported oysters and raspberries. The food industry feels abandoned, as without standardised methods they have no tools to convincingly demonstrate that the food prepared at their factories will be safe for the consumer. Because currently no methods are available to measure the infectivity of norovirus, the food authority lacks the needed data for performing appropriate risk assessment and risk analysis studies.In the last couple of years, laboratory methods to detect noroviruses in foods have been greatly improved resulting in increased knowledge on which steps in the concentration, detection and typing of noroviruses need to be further optimized before international validated methods can be routinely used. This report presents the most important issues regarding the challenges of detecting enteric viruses (including noroviruses) in food and water that were discussed at the 4th Nordic Workshop. At this meeting researchers from Nordic countries and leading international experts concluded that it is important to not only focus on the epidemiology of foodborne and waterborne viral infections but also increase efforts to improve, harmonize and standardize laboratory methods for the detection of enteric viruses direct in potentially contaminated food such as oysters and raspberries.

The aim of this study was to compare the distribution of VTEC O157 subtypes isolated from human sporadic infections with those in the Danish bovine reservoir, and to correlate the subtypes with the severity of the clinical symptoms in humans. The study included a total of 149 Danish eae-positive VTEC O157 isolates (63 of bovine origin and 86 from human clinical cases) isolated between 1987 and 2001. All were analysed by vtx-PCR-RFLP and phage typing. The vtx-PCR-RFLP showed that isolates carrying the vtx2 gene was more than four times as prevalent among the human clinical isolates (55%) as compared to the bovine isolates (13%). Furthermore, a significant correlation between the presence of the vtx2 gene and development of haemolytic-uraemic syndrome was found. The 149 isolates encompassed 16 different phage types (PTs). The majority (87%) of the human clinical isolates were identified, as PT2, PT4, PT8 or PT14 while only 46% of the bovine isolates belonged to these PTs. PT8 and PT14 were found at similar rates among bovine (36%) and human isolates (40%). However, the predominant PTs in the human isolates, PT2 (19%) and PT4 (28%), were only identified in 2% and 8%, respectively, of the bovine isolates. All but one PT2 and PT4 isolate carried either vtx2 alone or in combination with vtx2c, whereas none of the PT8 and PT14 isolates carried vtx2. The significant overlap between vtx/phage type combinations in bovine and human clinical isolates indicate that cattle are an important reservoir for human VTEC O157 infections in Denmark. However, the vtx2-carrying isolates, causing the most severe clinical symptoms, constitute only a minor fraction of the isolates from the Danish bovine reservoir.

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.

The Nordic countries faced a food-borne outbreak of hepatitis A that started in October 2012 and was ongoing with 103 reported cases as of 27 June 2013. A case–control study in Denmark, Finland, Norway and Sweden, combined with trace-back investigations, has identified frozen strawberries as the likely cause of the outbreak. The origin of the berries is still being investigated.