Maisons-Alfort, France
Maisons-Alfort, France

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Fraisse A.,Food and Water Virology Unit | Temmam S.,ADRIA NORMANDIE | Deboosere N.,Institute Pasteur Of Lille Ipl | Guillier L.,Modelling of Bacterial Behaviour Unit | And 5 more authors.
International Journal of Food Microbiology | Year: 2011

In recent years, raw fruits and vegetables have frequently been involved in foodborne transmission to humans of enteric viruses, particularly noroviruses and hepatitis A virus (HAV). Although viral contamination can occur during all steps of food processing, primary production is a critical stage on which prevention measures must be focused to minimize the risk of infection to consumers. Postharvest sanitation may be a valid technological solution for decreasing the bacterial load on fresh raw material, but there is a lack of data concerning the effectiveness of this process on enteric viruses. In this study, we compared the survival of two human norovirus surrogates, the feline calicivirus (FCV), and the murine norovirus (MNV-1), and of HAV on lettuce after water washing with bubbles and with or without ultrasound, and washing with bubbles in the presence of active chlorine (15 ppm) or peroxyacetic acid-based disinfectant (100 ppm). Cell culture and quantitative RT-PCR assays were used to detect and quantify the viruses on the surface of the lettuce after the sanitizing treatments. Levels of viral inactivation on the lettuce leaves were not significantly different between washing with bubbles and washing with bubbles plus ultrasound and were not dependant on the quantification method. A simple washing without disinfectant resulted in a decrease of approximately 0.7 log units in the quantity of virus detected for HAV and FCV and of 1.0 log unit for MNV-1.In the experimental set-up including a washing step (with or without ultrasound) followed by washing for 2 min in the presence of disinfectants, 15 ppm of active chlorine was found more effective for inactivating FCV (2.9 log units) than HAV and MNV-1 (1.9 log units and 1.4 log units, respectively) whereas 100 ppm of peroxyacetic-based biocide was found effective for inactivating FCV (3.2 log units) and MNV-1 (2.3 log units), but not HAV (0.7 log units). Quantitative RT-PCR results indicated that the presence of viral RNA did not correlate with the presence of infectious viruses on disinfected lettuce, except for MNV-1 processed with chlorine (15 ppm). In comparison with water washing, a substantial additional decrease of genomic FCV titer (1.1 log units) but no significant reduction of the genomic titers of HAV and MNV-1 were found on lettuce treated with chlorine (15 ppm). No significant effect of the disinfection step of lettuce with peroxyacetic-based biocide (100 ppm peracetic acid) was found by qRT-PCR on all genomic viral titers tested. This study illustrates the necessity of determining the effectiveness of technological processes against enteric viruses, using a relevant reference such as HAV, in order to reduce the risk of hepatitis and gastroenteritis by exposure to vegetables. © 2011.


Deboosere N.,Institute Pasteur Of Lille Ipl | Pinon A.,Institute Pasteur Of Lille Ipl | Caudrelier Y.,Institute Pasteur Of Lille Ipl | Delobel A.,Institute Pasteur Of Lille Ipl | And 10 more authors.
Food Microbiology | Year: 2012

Enteric viruses, particularly human Noroviruses (NoV) and hepatitis A virus (HAV), are key food-borne pathogens. The attachment of these pathogens to foodstuff and food-contact surfaces is an important mechanism in the human contamination process. Studies were done to investigate the nature of the physicochemical forces, such as hydrophobic and electrostatic ones, involved in the interaction virus/matrix but, at this day, only few data are available concerning surface properties of viruses and prediction of the adhesion capacity of one specific virus onto matrices is still very difficult. The purpose of this study was to propose a reference system, including a representative virus surrogate, able to predict as close as possible behaviour of pathogenic viruses in term of adhesion on inert (stainless steel and polypropylene) and food surfaces (lettuce leaves, strawberries and raspberries). The adhesion of human pathogenic enteric viruses, cultivable strain of HAV and non-cultivable strains of human NoV (genogroups I and II), have been quantified and compared to these of human enteric viruses surrogates, included the MNV-1 and three F-specific RNA bacteriophages (MS2, GA and Qβ). A standardized approach was developed to assess and quantify viral adhesion on tested matrices after a contact time with each virus using real-time RT-PCR. Methods used for virus recovery were in accordance with the CEN recommendations, including a bovine Enterovirus type 1 as control to monitor the efficiency of the extraction process and amplification procedure from directly extracted or eluted samples. The adhesion of human pathogenic viruses, ranging from 0.1 to 2%, could be comparable for all matrices studied, except for NoV GII on soft fruits. Adhesion percentages obtained for the studied surrogate virus and phages were shown to be comparable to those of HAV and NoV on inert and lettuce surfaces. The MNV-1 appeared as the best candidate to simulate adhesion phenomena of all human pathogenic enteric viruses on all studied surfaces, while MS2 and GA bacteriophages could be a good alternative as model of viral adhesion on inert and lettuce surfaces. These results will be usable to design relevant experimental systems integrating adhesion behaviour of enteric viruses in the assessment of the efficiency of a technological or hygienic industrial process. © 2012 Elsevier Ltd.


Coudray C.,Food and Water Virology Unit | Merle G.,Food and Water Virology Unit | Martin-Latil S.,Food and Water Virology Unit | Guillier L.,Modelling of Bacterial Behaviour Unit | Perelle S.,Food and Water Virology Unit
Journal of Virological Methods | Year: 2013

Enteric viruses are important agents of foodborne diseases. In recent years, raw fruits and vegetables have frequently been involved in foodborne transmission of enteric viruses to humans, particularly noroviruses and hepatitis A virus (HAV). Although viral contamination can occur at any stage of food processing, primary production is a critical stage in which prevention measures are essential to minimise the risk of infection to consumers. Due to the low infectious doses and low concentrations of enteric viruses in food samples, an efficient and rapid virus concentration method is required for routine control and risk assessment. In this study, the virus concentration reference method proposed by the CEN/TC275/WG6/TAG4 working group for samples of soft fruits and salad vegetables was compared with a method including a filtration step in order to recover hepatitis A virus (HAV) on lettuces. Murine norovirus (MNV-1) was used as a process control and detected simultaneously with HAV in a one-step duplex RT-qPCR following both procedures. The HAV LOD ranged from 10 to 100 PFU/25. g of lettuce in the presence or absence of MNV-1, regardless of method used. In conclusion, MNV-1 offers a very reliable and simple way to monitor the quality of the detection procedures. Although it has been found that both methods achieved an identical limit of detection, the method including a filtration step requires less processing and could be proposed as an alternative method. © 2013 Elsevier B.V.


Coudray-Meunier C.,Food and Water Virology Unit | Fraisse A.,Food and Water Virology Unit | Martin-Latil S.,Food and Water Virology Unit | Guillier L.,Modelling of Bacterial Behaviour Unit | Perelle S.,Food and Water Virology Unit
BMC Microbiology | Year: 2013

Background: Human enteric viruses are major agents of foodborne diseases. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, real-time reverse transcriptase PCR is now widely used for the detection of RNA viruses in food samples. However this approach detects viral nucleic acids of both infectious and non infectious viruses, which limits the impact of conclusions with regard to public health concern. The aim of the study was to develop a method to discriminate between infectious and non-infectious particles of hepatitis A virus (HAV) and two strains of rotavirus (RV) following thermal inactivation by using intercalating dyes combined with RT-qPCR. Results: Once the binding of propidium monoazide (PMA) or ethidium monoazide (EMA) was shown to be effective on the viral ssRNA of HAV and dsRNA of two strains of RV (SA11 and Wa), their use in conjunction with three surfactants (IGEPAL CA-630, Tween 20, Triton X-100) prior to RT-qPCR assays was evaluated to quantify the infectious particles remaining following heat treatment. The most promising conditions were EMA (20 μM) and IGEPAL CA-630 (0.5%) for HAV, EMA (20 μM) for RV (WA) and PMA (50 μM) for RV (SA11). The effectiveness of the pre-treatment RT-qPCR developed for each virus was evaluated with three RT-qPCR assays (A, B, C) during thermal inactivation kinetics (at 37°C, 68 C, 72°C, 80°C) through comparison with data obtained by RT-qPCR and by infectious titration in cell culture. At 37°C, the quantity of virus (RV, HAV) remained constant regardless of the method used. The genomic titers following heat treatment at 68°C to 80°C became similar to the infectious titers only when a pre-treatment RT-qPCR was used. Moreover, the most effective decrease was obtained by RT-qPCR assay A or B for HAV and RT-qPCR assay B or C for RV. Conclusions: We concluded that effectiveness of the pre-treatment RT-qPCR is influenced by the viral target and by the choice of the RT-qPCR assay. Currently, it would be appropriate to further develop this approach under specific conditions of inactivation for the identification of infectious viruses in food and environmental samples. © 2013 Coudray-Meunier et al.; licensee BioMed Central Ltd.


Hennechart-Collette C.,Food and Water Virology Unit | Martin-Latil S.,Food and Water Virology Unit | Guillier L.,Modelling of bacterial behaviour Unit | Perelle S.,Food and Water Virology Unit
Journal of Applied Microbiology | Year: 2014

Aims: To provide a rapid and sensitive method for detecting NoV GI and NoV GII in water and to evaluate the use of the murine norovirus (MNV-1) as a process control. Methods and Results: The method is based on viral concentration by filtration on electropositive filters and direct lysis of adsorbed viruses from filters before RNA extraction and RT-qPCR amplification. An one-step multiplex RT-qPCR assay was developed for the simultaneous detection of NoV GI, NoV GII and MNV-1. Then, water samples were artificially contaminated to determine mean virus recoveries and method sensitivity. The method showed a higher sensitivity for detecting NoV GII (103 genome copies per 0·5 l) than for NoV GI (104 genome copies per 0·5 l) in the presence of MNV-1 regardless of the type of water. The data also showed that MNV-1 is a robust option as process control. Conclusions: The method described provides a valuable tool for the monitoring of potential public health risks associated with NoV contamination in drinkable water. Significance and Impact of Study: Given the increasing evidence for NoV involvement in food outbreaks, the one-step multiplex RT-qPCR assay we used in this study would be a very useful tool to investigate NoV contamination in other food products. © 2013 The Society for Applied Microbiology.


Martin-Latil S.,Food and Water Virology Unit | Hennechart-Collette C.,Food and Water Virology Unit | Guillier L.,Modelling of Bacterial Behaviour Unit | Perelle S.,Food and Water Virology Unit
Food Microbiology | Year: 2012

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. © 2012 Elsevier Ltd.


Martin-Latil S.,Food and Water Virology Unit | Hennechart-Collette C.,Food and Water Virology Unit | Guillier L.,Modelling of bacterial behaviour Unit | Perelle S.,Food and Water Virology Unit
International Journal of Food Microbiology | Year: 2012

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/0.5. L bottled water and 3500 HEV genome copies/0.5. L tap water. © 2012 Elsevier B.V.


PubMed | Food and Water Virology Unit
Type: Journal Article | Journal: Journal of applied microbiology | Year: 2013

To provide a rapid and sensitive method for detecting NoV GI and NoV GII in water and to evaluate the use of the murine norovirus (MNV-1) as a process control.The method is based on viral concentration by filtration on electropositive filters and direct lysis of adsorbed viruses from filters before RNA extraction and RT-qPCR amplification. An one-step multiplex RT-qPCR assay was developed for the simultaneous detection of NoV GI, NoV GII and MNV-1. Then, water samples were artificially contaminated to determine mean virus recoveries and method sensitivity. The method showed a higher sensitivity for detecting NoV GII (10(3) genome copies per 05 l) than for NoV GI (10(4) genome copies per 05 l) in the presence of MNV-1 regardless of the type of water. The data also showed that MNV-1 is a robust option as process control.The method described provides a valuable tool for the monitoring of potential public health risks associated with NoV contamination in drinkable water.Given the increasing evidence for NoV involvement in food outbreaks, the one-step multiplex RT-qPCR assay we used in this study would be a very useful tool to investigate NoV contamination in other food products.


PubMed | Food and Water Virology Unit
Type: | Journal: BMC microbiology | Year: 2013

Human enteric viruses are major agents of foodborne diseases. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, real-time reverse transcriptase PCR is now widely used for the detection of RNA viruses in food samples. However this approach detects viral nucleic acids of both infectious and non infectious viruses, which limits the impact of conclusions with regard to public health concern. The aim of the study was to develop a method to discriminate between infectious and non-infectious particles of hepatitis A virus (HAV) and two strains of rotavirus (RV) following thermal inactivation by using intercalating dyes combined with RT-qPCR.Once the binding of propidium monoazide (PMA) or ethidium monoazide (EMA) was shown to be effective on the viral ssRNA of HAV and dsRNA of two strains of RV (SA11 and Wa), their use in conjunction with three surfactants (IGEPAL CA-630, Tween 20, Triton X-100) prior to RT-qPCR assays was evaluated to quantify the infectious particles remaining following heat treatment. The most promising conditions were EMA (20M) and IGEPAL CA-630 (0.5%) for HAV, EMA (20M) for RV (WA) and PMA (50M) for RV (SA11). The effectiveness of the pre-treatment RT-qPCR developed for each virus was evaluated with three RT-qPCR assays (A, B, C) during thermal inactivation kinetics (at 37C, 68 C, 72C, 80C) through comparison with data obtained by RT-qPCR and by infectious titration in cell culture. At 37C, the quantity of virus (RV, HAV) remained constant regardless of the method used. The genomic titers following heat treatment at 68C to 80C became similar to the infectious titers only when a pre-treatment RT-qPCR was used. Moreover, the most effective decrease was obtained by RT-qPCR assay A or B for HAV and RT-qPCR assay B or C for RV.We concluded that effectiveness of the pre-treatment RT-qPCR is influenced by the viral target and by the choice of the RT-qPCR assay. Currently, it would be appropriate to further develop this approach under specific conditions of inactivation for the identification of infectious viruses in food and environmental samples.


PubMed | Food and Water Virology Unit
Type: Journal Article | Journal: International journal of food microbiology | Year: 2012

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.

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