Clustered Cases of Waterborne Hepatitis E Virus Infection, France.

The identification of seven cases of hepatitis E virus infection in a French rural hamlet in April 2015 led to investigations confirming the clustering and identifying the source of the infection. Laboratories and general practitioners in the area actively searched for other cases based on RT-PCR and serological tests. The environment, including water sources, was also checked for HEV RNA. Phylogenetic analyses were performed to compare HEV sequences. No other cases were found. Six of the seven patients lived in the same hamlet, and the seventh used to visit his family who lived there. All HEV strains were very similar and belonged to the HEV3f subgenotype, confirming the clustering of these cases. All the patients drank water from the public network. A break in the water supply to the hamlet was identified at the time the infection probably occurred; HEV RNA was also detected in a private water source that was connected to the public water network. The water flowing from the taps was quite turbid during the break. The private water supply containing HEV RNA was the likely source of the contamination. Private water supplies not disconnected from the public network are still frequent in rural areas, where they may contribute to public water pollution.

Evaluation of a Proteinase K-Based Extraction Method to Detect Hepatitis A Virus, Hepatitis E Virus and Norovirus in Artificially Contaminated Dairy Products.

Human norovirus and hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) are leading causes of foodborne disease worldwide. Among the various food products, different types of dairy products can be implicated in viral foodborne outbreaks and contamination can occur at different stages, such as preparation, contact with contaminated equipment or via other foods. The aim of this study was to characterise a proteinase K method adapted from the ISO 15216 method for the detection of HAV, HEV and norovirus in artificially contaminated dairy products, based on the recent international standard of ISO 16140-4. Results showed that the recovery yields obtained from pure RNA in dairy products ranged from 5.76% to 76.40% for HAV, from 35.09% to 100.00% for HEV, from 25.09% to 100.00% for norovirus GI and from 47.83% to 100.00% for norovirus GII. The process control MNV-1 was detected in all RNA extracts, with recovery yields between 36.83% and 100.00%. The limit of detection (LOD) of the method was between 184 and 642 genome copies/mL (or/g) for the LOD50 and 802 and 2800 genome copies/mL or/g for the LOD95 according to the virus analysed. This method proved to be suitable for detecting viruses in dairy products for routine diagnostic needs.

Development of an Extraction Method to Detect Hepatitis A Virus, Hepatitis E Virus, and Noroviruses in Fish Products.

Viruses are a leading cause of foodborne disease worldwide. Hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) and human norovirus are recognized as the main viruses of public health concern in food hygiene. ISO 15216 approved procedures are not validated for detection of HAV and human norovirus in foodstuffs, such as fishes, leading to an inability to ensure the safety of these products. This study aimed to provide a rapid and sensitive method for detecting these targets in fish products. An existing method that includes proteinase K treatment was selected for further validation using artificially contaminated fish products, according to the recent international standard ISO 16140-4. Recovery efficiencies in pure RNA extracts of viruses ranged from 0.2% to 66.2% for HAV, 4.0% to 100.0% for HEV, 2.2% to 100.0% for norovirus GI, and 0.2% to 12.5% for norovirus GII. LOD50 values were between 144 and 8.4 × 104 genome copies/g for HAV and HEV, and 104 and 2.0 × 103 copies/g for norovirus GI and norovirus GII, respectively. LOD95 values were between 3.2 × 103 and 3.6 × 105 genome copies/g for HAV and HEV, and between 8.8 × 103 and 4.4 × 104 genome copies/g for norovirus GI and norovirus GII, respectively. The method developed here was successfully validated in various fish products and can be applied for routine diagnostic needs.

Method for detecting norovirus, hepatitis A and hepatitis E viruses in tap and bottled drinking water.

Viruses are a leading cause of foodborne disease worldwide. Human norovirus and hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) are recognised to be the main viruses of importance to public health. The ISO 15216 procedure describes molecular methods for detecting HAV and norovirus in bottled water by using an electropositive filter to concentrate viruses. The aim of this study was to validate the Zeta Plus 1MDS membrane (1MDS) for detecting enteric viruses from tap and bottled water using the recent international standard ISO/DIS/16140-4:2018, which describes the protocol for validating methods for microbiology in the food chain. Method with direct lysis of viruses from the 1MDS filter, and RNA extraction was used for detecting noroviruses, HAV and HEV from different tap and bottled drinking water. By taking into account virus's inoculation levels above the LOD, the recovery rates of noroviruses and HAV obtained from pure RNA extracts ranged from 2.50% to 14.31% and for HEV from 27.87% to 53.54% according to the water samples analysed. The virus recovery rates did not differ according to the operator or drinking water analysed but did according to the virus inoculated. The LOD95 values were respectively 50 genome copies/mL for HAV and 2.8 genome copies/mL for HEV, 420 genome copies/mL for norovirus GI and 134 genome copies/mL of water sample for norovirus GII. LOQs were determined for HAV and HEV by the total error approach and were 15.8 genome copies/mL for HAV and 2.8 genome copies/mL of water sample for HEV. The described method could be used for detecting viruses from tap and bottled water for routine diagnosis needs.

A One-Health Approach to Investigating an Outbreak of Alimentary Tick-Borne Encephalitis in a Non-endemic Area in France (Ain, Eastern France): A Longitudinal Serological Study in Livestock, Detection in Ticks, and the First Tick-Borne Encephalitis Virus Isolation and Molecular Characterisation.

Tick-borne encephalitis virus' (TBEV) geographic range and the human incidence are increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of tick-born encephalitis (TBE) in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurised raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and witness in real-time the seroconversion of recently exposed individuals and excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for the first alimentary-transmitted TBE in France, obtained its full-length genome sequence, and found that it belongs to the European subtype of TBEV. TBEV is now a notifiable human disease in France, which should facilitate surveillance of its incidence and distribution throughout France.

Method for tick-borne encephalitis virus detection in raw milk products.

The transmission of tick-borne encephalitis virus (TBEV) through food is rare, but can occur through the consumption of raw milk products from animals infected by tick bites. In 2020, France faced a TBEV outbreak linked to the consumption of unpasteurized goat cheese. The aim of this study was to develop and characterize a molecular method for the detection of TBEV in raw milk products based on the recent international standard PR ISO/DIS 16140-4. The TBEV recovery rates varied with the inoculation level and settings. The LOD50 and LOD95 of TBEV were 6.40 × 103 genome copies per g or per mL and 2.84 × 104 genome copies per g or per mL, respectively. The percentages of RT-qPCR inhibitions were lower than 75% and the murine norovirus (MNV-1), used as process control, was detected in all samples with a recovery rate higher than 1%, as recommended in ISO 15216. We conclude that the described method is appropriate to detect TBEV in raw milk products for routine diagnosis, and to assess potential health risks.

Detection of norovirus, hepatitis A and hepatitis E viruses in multicomponent foodstuffs.

Among the enteric viruses implicated in foodborne outbreaks, the human norovirus and hepatitis viruses A and E (HAV and HEV) represent a serious public health concern. International standard ISO 15216 proposes methods for detecting HAV and norovirus (genogroups I and II) RNA from soft fruit, leaf, stem and bulb vegetables, bottled water or food surfaces. These methods had not previously been validated for detecting the targeted viruses in other foodstuffs such as multicomponent foods, nor for detecting other viruses in foodstuffs. The aim of this study was to characterise a method derived from the vegetable method described in ISO 15216 to detect HAV, HEV and norovirus in artificially-contaminated multicomponent foodstuffs according to the recent international standard ISO 16140-4. Results showed that the mean recovery rates for all settings did not differ according to the operator. The mean extraction yields ranged from 0.35% to 40.44% for HAV, 5.19% to 100% for HEV, 0.10% to 40.61% for norovirus GI and 0.88% to 69.16% for norovirus GII. The LOD95 was 102 genome copies/g for HAV, HEV and norovirus GII and 103 genome copies/g for norovirus GI. The LOQ was 2.90 × 104, 1.40 × 103, 1.60 × 104 and 1.30 × 104 genome copies/g for HAV, HEV, norovirus GI and norovirus GII respectively. The MNV-1 process control was detected in 120 out of 128 RNA extracts analysed and was recovered with an efficiency of between 3.83% and 50.22%. The mean inhibition rates of quantitative real-time RT-PCR reaction ranged from 3.25% to 28.70% and varied significantly with the type of food matrix. The described method could be used to detect viruses in composite food products for routine diagnosis needs.

Evaluation of three different filters and two methods for recovering viruses from drinking water.

Among the enteric viruses implicated in waterborne outbreaks, human norovirus and hepatitis A virus (HAV) are a serious public health issue. Most foodborne viruses are difficult or currently unlikely to cultivate. Because of the lack of a cell culture method, real-time reverse transcriptase PCR is commonly used for the detection of norovirus in foodstuffs and environmental samples. Due to low infectious doses in humans and low virus concentration in water sample, filter adsorption methods were used for concentrating viruses from water. The ISO (Anonymous, ISO 15216-1, 2017) describes standardized molecular methods for detecting HAV and norovirus in bottled water. This method includes a two-step procedure: concentrating the virus using a microporous electropositive filter (47 mm diameter, 0.45 µm pore size) then molecular detection. The Zetapor filter, which had a charged membrane with a pore size of 0.45 µm, was commonly used in the past to concentrate viruses from water or from salad leaves following virus elution. But, unfortunately, the Zetapor filter is no longer marketed and it is therefore necessary to assess an alternative filter. The aim of this study was to compare the ability of two electropositive filters with a pore size of 0.45 µm or 0.22 µm and one uncharged filter (0.45 µm) to recover norovirus and HAV from two different types of drinking water (bottled water and tap water) with the adsorption-elution method proposed by ISO (Anonymous, ISO 15216-1, 2017) (method A) and with direct viral extraction using filters (method B). The mean extraction yields for norovirus and HAV calculated with RNA extracts ranged from 0.2 % - 4.81 % with method A and from 5.05 % - 53.58 % with method B, and did not differ significantly between the two types of drinking water tested. For method B, the mean extraction yields for HAV and norovirus were evaluated according to results from the three filters used. The recovery rate of HAV and norovirus ranged between 3.47 % and 62.41 % with the 0.45 µm electropositive filter and were higher than the other filters. The 0.45 µm electropositive filter could be used to concentrate viruses for routine viral monitoring of drinking water for researchers who want to adopt the method in their lab routine.

Virological analyses in collective catering outbreaks in France between 2012 and 2017.

Enteric viruses cause the majority of foodborne illnesses and common symptoms of many foodborne illnesses include vomiting, diarrhea, abdominal pain, and fever. Among the enteric viruses, human Norovirus (NoV) and hepatitis virus (HAV and HEV) are the main viruses suspected to cause foodborne outbreaks and represent a serious public health. The study presents survey tools of viruses in a wide variety of foodstuffs and results obtained during 56 foodborne outbreaks investigation in France between 2012 and 2017. 246 suspected foods were examined for the presence of four human enteric viruses (NoV GI and NoV GII, HAV or HEV) either using methods described in the EN ISO 15216-1 or in house methods. All viral analysis of food samples were performed with the implementation of process control and an external amplification controls. Eighteen of 56 foodborne outbreaks investigated included at least one positive food sample (16/18 NoV, 1/18 HAV and 1/18 HEV). The genomic levels of four viruses detected ranged from < 102 to 107 genome copies per g or per L. This study showed the interest to develop methods for the extraction of viruses in different foodstuffs to increase the possibility to identify the association between viral illness and food consumption.

Development of an extraction method to detect enteric viruses in dressed vegetables.

Food-borne viral infections are caused mainly by noroviruses (NoV) and the hepatitis A virus (HAV), which respectively cause gastroenteritis and hepatitis. Various foods have been implicated in viral outbreaks, including vegetables that are consumed in a variety of forms, often with salad dressing. NF EN ISO procedures (15216-1:2017) propose standard methods for quantifying NoV and HAV in high-risk food categories, such as vegetables, based on viral elution and PEG concentration methods, but these methods are not suitable for composite meals like salads dressed with oily, fatty or emulsified food ingredients. The development of sensitive and reliable techniques for the detection of viruses in these products is therefore needed to ensure the safety of these products. The aim of this study was to develop an RT-qPCR based method for the detection and quantification of NoV and HAV in various vegetables with different dressings. Three methods for recovering NoV and HAV from artificially contaminated dressed vegetables were evaluated. The selected method was based on the use of Trizol reagent and, according to the type of dressing, the limit of detection ranged from 104 to 106 genome copies/g for NoV and from 102 to 103 PFU/g for HAV. The described method can be applied for detecting NoV and HAV in food containing salad dressing for routine diagnosis needs.

Evaluation of methods for elution of HEV particles in naturally contaminated sausage, figatellu and pig liver.

Foodborne transmission of HEV is a growing public health concern in industrialised countries, where the disease is mainly autochthonous, caused by zoonotic HEV of either genotype 3 or 4. Foodstuffs containing pig's liver were suspected on several occasions to be the cause of autochthonous cases of HEV infection, while the transmission was associated with animal contact and the ingestion of raw or uncooked meat, especially liver. In assessing the risk related to the presence of HEV in food, detection methods were previously developed but HEV detection rates seem to vary with the type of samples and methods. As foodstuff containing pig liver can be contaminated with HEV internally, an efficient virus extraction procedure is required. The aim of this study was to evaluate six methods for their efficiency in releasing HEV viral particles from figatelli, pig liver sausages and liver samples previously tested positive for the presence of HEV. The ratio weight to volume of elution buffer (1:5) and the FastPrep®-24 homogeniser showed to significantly improve the quantity of HEV genomes released per gram of figatelli and pig liver sausages. To our knowledge, this study is the first to evaluate several methods for elution of HEV particles from naturally contaminated pig liver products, and may be extended for quantifying other viral genomes from food of animal origin.

Development of a Real-Time Cell Analysis (RTCA) Method as a Fast and Accurate Method for Detecting Infectious Particles of the Adapted Strain of Hepatitis A Virus.

Hepatitis A virus (HAV) is one of the most common agents causing acute liver disease worldwide. HAV has been increasingly reported as the cause of foodborne disease outbreaks. The standard method currently available for detection of the genome of HAV in vulnerable foodstuffs is by RT-qPCR (ISO 15216). Despite its usefulness in the investigation of foodborne viruses, the use of RT-qPCR in food virology has been shown to overestimate the quantity of infectious virus or to highly underestimate the effect of the treatment on virus inactivation. The gold standard methods currently used for evaluating the efficacy of inactivation treatments on the adapted strain of HAV (HM175/18f) are either the plaque assay or the end-point dilution assay (TCID50). However, both assays are labor-intensive and time-consuming. The aim of this study was to evaluate the use of the xCELLigence real-time cell analysis (RTCA) system for detecting the infectivity of the adapted strain of HAV. Kinetics of cell impedance showed that HAV induced a decrease in cell index (CI) correlated with the onset of HAV-induced cell death. In addition, the time to which the HAV-induced CI drop occurred was dependent on the viral concentration. An inverse linear relation could be established over a range of 5 log10 between the concentration of HAV and the time to reach 50% of CI decrease (TCI50), showing that the RTCA assay could be used as a titration method for HAV. In addition, the RTCA-based assay could be performed in less than 6 days instead of 12 to 14 days with the gold standard methods. Therefore, the RTCA-based titration method is a powerful and suitable tool for high-throughput screening of anti-viral treatments. Its usefulness in HAV inactivation studies will improve the assessment of viral risk in food virology, as controlling transmission of viruses through their removal from foodstuffs is also an important challenge in reducing the burden of viral foodborne illnesses.

Discrimination of infectious and heat-treated norovirus by combining platinum compounds and real-time RT-PCR.

Human noroviruses (NoV) are major agents of foodborne outbreaks. Because of the lack of a standardized cell culture method, real-time reverse transcriptase PCR is now commonly used for the detection of NoV in foodstuffs and environmental samples. However, this approach detects the viral nucleic acids of both infectious and non-infectious viruses and needs to be optimized to predict infectivity for public health risk assessment. The aim of this study was to develop a viability PCR method to discriminate between native and heat-treated virus, for both NoV and its surrogate, murine norovirus (MNV). To this end, screening of viability markers (monoazide dyes, platinum and palladium compounds) was performed on viral RNA, native virus or heat-treated virus, and incubation conditions were optimized with PtCl4, the most efficient viability marker. Multiple MNV molecular models were designed: no impact of amplicon length was observed on inactivated MNV genomic titer; but the 5'NTR, ORF1 and 3'UTR regions resulted in higher reductions than central genomic regions. The optimal viability PCR conditions developed (incubation with 2.5 mM PtCl4 in PBS for 10 min at 5 °C) were finally applied to MNV by performing heat inactivation studies and to native and heat-treated NoV clinical strains. The viability PCR discriminated efficiently between native and heat-inactivated MNV at 72 °C and 80 °C, and efficiently reduced the genomic titer of heat-treated NoV strains. This viability PCR method could be useful to study heat inactivation kinetics of NoV and MNV. It could also be evaluated for the identification of infectious enteric viruses in foodstuffs and environmental samples.

Comparison of three extraction methods to detect noroviruses in dairy products.

Noroviruses (NoV) are currently the most common cause of viral foodborne diseases and RT-qPCR is widely used for their detection in food because of its sensitivity, specificity and rapidity. The ISO/TS (15216-1, 15216-2) procedures for detecting NoV and HAV in high-risk food categories such as shellfish, bottled water and vegetables were published in 2013. Milk products are less implicated in foodborne viral outbreaks but they can be contaminated with fruit added to these products or by the food handler. Thus, the development of sensitive and reliable techniques for the detection of NoV in dairy products is needed to ensure the safety of these products. The aim of this study was to develop a RT-qPCR based method for the detection of NoV in milk products. Three methods were tested to recover NoV from artificially contaminated milk and cottage cheese. The selected method was based on the use of proteinase K and the recovery efficiencies ranged from 54.87% to 98.87% for NoV GI, 61.16%-96.50% for NoV GII. Murine norovirus and mengovirus were used as process controls and their recovery efficiencies were respectively 60.59% and 79.23%. The described method could be applied for detecting NoV in milk products for routine diagnosis needs.

Digital RT-PCR method for hepatitis A virus and norovirus quantification in soft berries.

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

Quantification of Hepatitis E Virus in Naturally-Contaminated Pig Liver Products.

Hepatitis E virus (HEV), the cause of self-limiting acute hepatitis in humans, is widespread and endemic in many parts of the world. The foodborne transmission of HEV has become of concern due to the identification of undercooked pork products as a risk factor for infection. Foodborne enteric viruses are conventionally processed by quantitative RT-PCR (RT-qPCR), which gives sensitive and quantitative detection results. Recently, digital PCR (dPCR) has been described as a novel approach to genome quantification with no need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR when detecting HEV in pig liver products. The sensitivity of the RT-dPCR assay was similar to that of RT-qPCR, and quantitative data obtained by both detection methods were not significantly different for almost all samples. This absolute quantification approach may be useful for standardizing quantification of HEV in food samples and may be extended to quantifying other human pathogens in food samples.

Determination of which virus to use as a process control when testing for the presence of hepatitis A virus and norovirus in food and water.

Noroviruses (genogroup I (NoV GI) and genogroup II (NoV GII)) and the hepatitis A virus (HAV) are frequently involved in foodborne infections worldwide. They are mainly transmitted via the fecal-oral route, direct person-to-person contact or consumption of contaminated water and foods. In food virology, detection methods are currently based on identifying viral genomes using real-time reverse transcriptase PCR (RT-qPCR). One of the general requirements for detecting these viruses in food involves the use of a process control virus to monitor the quality of the entire viral extraction procedure as described in the ISO/TS 15216-1 and 15216-2 standards published in 2013. The selected process control virus should have similar morphological and physicochemical properties as the screened pathogenic virus and thus have the potential to provide comparable extraction efficiency. The aim of this study was to determine which virus should be used for process control, murine norovirus (MNV-1) or Mengovirus, when testing for the presence of HAV, NoV GI and NoV GII in bottled water, lettuce and semi-dried tomatoes. Food samples were spiked with HAV, NoV GI or NoV GII alone or in the presence of MNV-1 or Mengovirus. Recovery rates of each pathogenic virus were compared to those of both process control viruses using a multiple comparison procedure. Neither process control virus influenced the recovery of pathogenic virus regardless of the type of food matrix. MNV-1 was the most appropriate virus for validating the detection of HAV and NoV GII in all three food matrices as well as NoV GI in lettuce. Mengovirus proved to be the most appropriate control for NoV GI detection in bottled water and semi-dried tomatoes. The process control virus is essential for validating viral detection in food and the choice of virus depends on food type and the screened pathogenic virus.

Method for HEV detection in raw pig liver products and its implementation for naturally contaminated food.

It is now recognized that Hepatitis E virus (HEV) infection is not confined to developing countries. HEV infection is a growing public health concern in industrialized countries where the disease is mainly autochthonous, caused by HEV genotypes 3 and 4 and is today considered to be zoonotic. HEV causes acute hepatitis in humans, predominantly through contamination of food and water. Due to the low concentrations found in food and water samples, an efficient and rapid virus concentration method is required for routine control. Because of the absence of a reliable cell culture method for the main enteric viruses most commonly involved in the outbreaks, reverse transcription quantitative real time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in all types of samples. The aim of this study was to provide a rapid and sensitive method for detecting HEV in pig liver products. A method which includes a virus concentration step by PEG has been chosen from 9 protocols to be further validated. We used a one-step duplex RT-qPCR for detecting HEV and the murine norovirus (MNV-1) used as a process control for monitoring the quality of the whole extraction procedure. The mean recovery rates of the HEV and MNV-1 obtained from pig liver sausages were respectively 3.94% and 2.92%, increasing in figatelli to 18.38% and 13.11% respectively. This method also proved to be effective for HEV detection in naturally contaminated foodstuffs containing raw pig liver.

Duplex RT-qPCR for the detection of hepatitis E virus in water, using a process control.

Human hepatitis E virus (HEV) causes acute hepatitis in humans, predominantly by contamination of food and water. HEV, in particular genotype III, is currently considered to be an emerging pathogen in industrialized countries. Because of the low infectious dose, an efficient and rapid virus concentration method is required to detect low amounts of HEV in food and water samples for routine control. Because of the absence of a reliable cell culture method for the main enteric viruses most involved in the outbreaks, reverse transcription quantitative real time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in food and water samples. One of the general requirements for viral diagnosis concerns the use of a process control to monitor the efficiency of the concentration of viral particles, the extraction of nucleic acid and the presence of the potential inhibitors of the RT-qPCR reaction. The aim of this study was to provide a rapid and sensitive method for detecting HEV in water. The method is based on viral concentration by filtration on membrane filters and direct lysis of adsorbed viruses from filters before RNA extraction and RT-qPCR amplification. We developed a one-step duplex RT-qPCR for detecting HEV and the murine norovirus (MNV-1) was used as a process control. The data show that MNV-1 offers a very reliable and simple way of monitoring false-negative results and is a valuable tool in the routine diagnostic laboratory. The limit of detection (LOD) was in the range of 700 to 3500 HEV genome copies/0.5L bottled water and 3500 HEV genome copies/0.5L tap water.

Comparison of two extraction methods for the detection of hepatitis A virus in semi-dried tomatoes and murine norovirus as a process control by duplex RT-qPCR.

Enteric viruses are important agents of foodborne diseases. Due to their low infectious doses and low concentrations in food samples, an efficient and rapid virus concentration method is required for routine control. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, reverse transcription quantitative real-time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in food samples. One of the general requirements for viral diagnosis concerns the use of a process control to monitor the efficiency of viral particle concentration, nucleic acid extraction and the presence of potential inhibitors of the RT-PCR reaction. Recent epidemiological studies have linked hepatitis A outbreaks to the consumption of semi-dried tomatoes (SDT) in Australia, the Netherlands and France. In this study, the virus concentration reference method proposed by the CEN/TC275/WG6/TAG4 working group for samples of soft fruit and salad vegetables was compared to a method including an ultracentrifugation step to recover hepatitis A virus (HAV) in SDT. Murine norovirus (MNV-1) was used as a process control and detected simultaneously with HAV in a one-step duplex RT-qPCR in both procedures. The LOD of HAV was 10 PFU and 1 PFU of HAV/25 g of SDT in the presence or absence of MNV-1 respectively, whatever the method used. We conclude that both methods achieved an identical limit of detection and that the MNV-1 offers a very reliable and simple way to monitor the quality of the extraction procedures and the presence of RT-qPCR inhibitors.