Leen Baert

Detection of Murine Norovirus 1 by Using Plaque Assay, Transfection Assay, and Real-Time Reverse Transcription-PCR before and after Heat Exposure

ABSTRACT The correlation between the detection of murine norovirus 1 RNA by real-time reverse transcription-PCR and the infectivity by plaque assay before and after heat exposure (80°C) was examined. No correlation was found in the current study. Moreover, heat inactivation had a much stronger detrimental effect on virus infectivity than on the integrity of the viral genome.

The efficacy of preservation methods to inactivate foodborne viruses

During the last decade an increased incidence of infections and outbreaks attributed to foodborne viruses, in particular noroviruses (NoV), was observed world wide. The awareness of the presence of viruses on food emphasized the need to acquire knowledge regarding the effect of preservation methods upon viruses. Most foodborne viruses cannot be cultured in the laboratory, which hinders studies of their stability in food. Cultivable surrogate viruses, genetically related to the human infecting strains, are taken as a substitute to define inactivation rates. The last years, the number of survival and inactivation studies using various surrogate viruses increased. In this review, state-of-the-art information regarding the efficacy of preservation methods to reduce the level of viruses on food is compiled. In the first place, the effect of preservation methods establishing microbial growth inhibition (chilling, freezing, acidification, reduced water activity and modified atmosphere packaging) upon foodborne viruses is described. Secondly, the use of preservation methods establishing microbial inactivation such as heat treatment, high hydrostatic pressure processing and irradiation to eliminate viruses is discussed. In the third place, the efficacy of decontamination methods on fresh produce and purification procedures applied on live bivalve shellfish to reduce the viral load is included. These studies indicate that viruses persist well on chilled, acidified, frozen foods and foods packed under modified atmosphere or in dried conditions. Intervention strategies inducing microbial inactivation are required to achieve a 3 log reduction of the level of viruses. Decontamination of fresh produce reduces viruses with a maximum of 1 to 2 log while purification of live bivalves is not adequate to prevent viral outbreaks. It was noted that the effect of a particular food preservation method is dependent upon the virus tested and type of food.

Review: Norovirus prevalence in Belgian, Canadian and French fresh produce: a threat to human health?

Foodborne viruses, especially noroviruses (NoV), are increasingly reported as the cause of foodborne outbreaks. NoV outbreaks have been reported linked to fresh soft red fruits and leafy greens. Belgium, Canada and France were the first countries to provide data about the prevalence of NoV on fresh produce. In total, 867 samples of leafy greens, 180 samples of fresh soft red fruits and 57 samples of other types of fresh produce (tomatoes, cucumber and fruit salads) were analyzed. Firstly, the NoV detection methodology, including virus and RNA extraction, real-time RT-PCR and quality controls were compared among the three countries. In addition, confirmation and genotyping of the NoV strains was attempted for a subset of NoV positive samples using conventional RT-PCR targeting an alternative region followed by sequencing. Analysis of the process control showed that 653, 179 and 18 samples of the leafy greens, soft red fruits and other fresh produce types were valid for analysis based on the recovery of the process control. NoV was detected by real-time RT-PCR in 28.2% (N=641), 33.3% (N=6) and 50% (N=6) of leafy greens tested in Canada, Belgium and France, respectively. Soft red fruits were found positive by real-time RT-PCR in 34.5% (N=29) and 6.7% (N=150) of the samples tested in Belgium and France, respectively. 55.5% (N=18) of the other fresh produce types, analyzed in Belgium, were found NoV positive by real-time RT-PCR. Conventional RT-PCR resulted in an amplicon of the expected size in 19.5% (52/266) of the NoV positive samples where this assay was attempted. Subsequent sequencing was only successful in 34.6% (18/52) of the suspected amplicons obtained by conventional RT-PCR. From this study, using the described methodology, NoV genomes were frequently detected in fresh produce however sequence confirmation was not successful for the majority of the samples tested. Infection or outbreaks were rarely or not known to be related to the NoV positive samples. With the increase in sensitivity of the detection methodology, there is an increasing concern about the interpretation of positive NoV results by real-time amplification. Strategies to confirm the results by real-time RT-PCR should be developed in analogy with the detection of microbial pathogens in foods. Detection might indicate contact with NoV in the fresh produce chain. Consequently, a potential risk for infection cannot be excluded but the actual risk from RT-PCR NoV positive produce is still unknown. Studies should be designed determining the probability of infection related to the presence or levels of NoV genomic copies.

Evaluation of viral extraction methods on a broad range of Ready-To-Eat foods with conventional and real-time RT-PCR for Norovirus GII detection

Noroviruses (NoV) are a common cause of foodborne outbreaks. In spite of that, no standard viral detection method is available for food products. Therefore, three viral elution-concentration methods and one direct RNA isolation method were evaluated on a broad range of Ready-To-Eat (RTE) food products (mixed lettuce, fruit salad, raspberries and two RTE dishes) artificially seeded with a diluted stool sample contaminated with NoV genogroup II. These seeding experiments revealed two categories of RTE products, fruits and vegetables grouped together and RTE dishes (penne and tagliatelle salads) which are rich in proteins and fat formed another category. The RNA extracts were amplified and detected with two conventional RT-PCR systems (Booster and Semi-nested GII) and one real-time RT-PCR (Real-time GII) assay. A fast direct RNA isolation method detected 10(2) RT-PCRU on 10 g penne and tagliatelle salads with the conventional RT-PCR assays. However real-time RT-PCR was less sensitive for penne salad. A viral elution-concentration method, including a buffer solution for the elution step and one polyethylene glycol (PEG) precipitation step, was able to detect 10(2) RT-PCRU on 50 g frozen raspberries with conventional and real-time RT-PCR assays. Moreover the latter extraction method used no environmental hazardous chemical reagents and was easy to perform.

Survival and Transfer of Murine Norovirus 1, a Surrogate for Human Noroviruses, during the Production Process of Deep-Frozen Onions and Spinach

The reduction of murine norovirus 1 (MNV-1) on onions and spinach by washing was investigated as was the risk of contamination during the washing procedure. To decontaminate wash water, the industrial sanitizer peracetic acid (PAA) was added to the water, and the survival of MNV-1 was determined. In contrast to onions, spinach undergoes a heat treatment before freezing. Therefore, the resistance of MNV-1 to blanching of spinach was examined. MNV-1 genomic copies were detected with a real-time reverse transcription PCR assay in PAA-treated water and blanched spinach, and PFUs (representing infectious MNV-1 units) were determined with a plaque assay. A < or = 1-log reduction in MNV-1 PFUs was achieved by washing onion bulbs and spinach leaves. More than 3 log PFU of MNV-1 was transmitted to onion bulbs and spinach leaves when these vegetables were washed in water containing approximately 5 log PFU/ml. No decline of MNV-1 occurred in used industrial spinach wash water after 6 days at room temperature. A concentration of 20 ppm of PAA in demineralized water (pH 4.13) and in potable water (pH 7.70) resulted in reductions of 2.88 +/- 0.25 and 2.41 +/- 0.18 log PFU, respectively, after 5 min of exposure, but no decrease in number of genomic copies was observed. No reduction of MNV-1 PFUs was observed on frozen onions or spinach during storage for 6 months. Blanching spinach (80 degrees C for 1 min) resulted in at least 2.44-log reductions of infectious MNV-1, but many genomic copies were still present.

Efficacy of sodium hypochlorite and peroxyacetic acid to reduce murine norovirus 1, B40-8, Listeria monocytogenes, and Escherichia coli O157:H7 on shredded iceberg lettuce and in residual wash water.

The efficiency of sodium hypochlorite (NaOCl) and peroxyacetic acid (PAA) to reduce murine norovirus 1 (MNV-1), a surrogate for human norovirus, and Bacteroides fragilis HSP40-infecting phage B40-8 on shredded iceberg lettuce was investigated. The levels of removal of viruses MNV-1 and B40-8 were compared with the reductions observed for bacterial pathogens Listeria monocytogenes and Escherichia coli O157:H7. Two inoculation levels, one with a high organic load and the other containing a 10-fold lower number of pathogens and organic matter, showed that the effectiveness of NaOCl was greatly influenced by the presence of organic material, which was not observed for PAA. Moreover, the present study showed that 200 mg/liter NaOCl or 250 mg/liter PAA is needed to obtain an additional reduction of 1 log (compared with tap water) of MNV-1 on shredded iceberg lettuce, whereas only 250 mg/liter PAA achieved this for bacterial pathogens. None of the treatments resulted in a supplementary 1-log PFU/g reduction of B40-8 compared with tap water. B40-8 could therefore be useful as an indicator of decontamination processes of shredded iceberg lettuce based on NaOCl or PAA. Neither MNV-1, B40-8, nor bacterial pathogens could be detected in residual wash water after shredded iceberg lettuce was treated with NaOCl and PAA, whereas considerable numbers of all these microorganisms were found in residual wash water consisting solely of tap water. This study illustrates the usefulness of PAA and NaOCl in preventing cross-contamination during the washing process rather than in causing a reduction of the number of pathogens present on lettuce.

Biogenic silver for disinfection of water contaminated with viruses.

ABSTRACT The presence of enteric viruses in drinking water is a potential health risk. Growing interest has arisen in nanometals for water disinfection, in particular the use of silver-based nanotechnology. In this study, Lactobacillus fermentum served as a reducing agent and bacterial carrier matrix for zerovalent silver nanoparticles, referred to as biogenic Ag0. The antiviral action of biogenic Ag0 was examined in water spiked with an Enterobacter aerogenes-infecting bacteriophage (UZ1). Addition of 5.4 mg liter−1 biogenic Ag0 caused a 4.0-log decrease of the phage after 1 h, whereas the use of chemically produced silver nanoparticles (nAg0) showed no inactivation within the same time frame. A control experiment with 5.4 mg liter−1 ionic Ag+ resulted in a similar inactivation after 5 h only. The antiviral properties of biogenic Ag0 were also demonstrated on the murine norovirus 1 (MNV-1), a model organism for human noroviruses. Biogenic Ag0 was applied to an electropositive cartridge filter (NanoCeram) to evaluate its capacity for continuous disinfection. Addition of 31.25 mg biogenic Ag0 m−2 on the filter (135 mg biogenic Ag0 kg−1 filter medium) caused a 3.8-log decline of the virus. In contrast, only a 1.5-log decrease could be obtained with the original filter. This is the first report to demonstrate the antiviral efficacy of extracellular biogenic Ag0 and its promising opportunities for continuous water disinfection.

Multiplex real-time RT-PCR for simultaneous detection of GI/GII noroviruses and murine norovirus 1

A quantitative two-step multiplex real-time reverse transcriptase (RT-) PCR assay for the simultaneous detection of genogroup I (GI) and genogroup II (GII) noroviruses (NoVs) is described below. A murine norovirus 1 (MNV-1) real-time PCR detection assay described recently was integrated successfully into the multiplex assay, making it possible to detect GI and GII NoVs and MNV-1 in one reaction tube with MNV-1 plasmid DNA as real-time PCR internal amplification control (IAC). The results showed a nearly complete concordance between the multiplex assay and the corresponding single-target PCRs. Analysis of competition between the individual reactions within the multiplex real-time PCR assay showed that GI and GII NoV plasmid DNAs mixed at equimolar concentrations were detected reproducibly and quantitatively, while a 4 log excess between GI and GII plasmid DNAs hindered amplification of the target with the lowest concentration. High concentrations of the real-time PCR IAC (MNV-1 plasmid DNA) also interfered with the possibility of the developed multiplex real-time RT-PCR assay to detect quantitatively and simultaneously the presence of GI and GII NoVs within one sample. The specificity of the multiplex assay was evaluated by testing a NoV RNA reference panel containing nine GI, eight GII, and one GIV in vitro synthesized RNA fragment, plus 16 clinical samples found positive for GI and GII NoVs previously. In addition, a collection of bovine NoVs and other (non-NoV) enteric viruses were found to be negative, and no cross-amplification between genogroups was observed.

Reported foodborne outbreaks due to noroviruses in Belgium: the link between food and patient investigations in an international context

The Belgian data for foodborne norovirus (NoV) outbreaks became available for the first time with the introduction of an extraction and detection protocol for NoV in the National Reference Laboratory for foodborne outbreaks in September 2006. In 2007, 10 NoV foodborne outbreaks were reported affecting 392 persons in Belgium. NoV became the most detected agent in foodborne outbreaks followed by Salmonella (eight foodborne outbreaks). The major implicated foods were sandwiches (4/10), where food handlers reported a history of gastroenteritis in two outbreaks. A food handler was implicated in the limited number of Belgian NoV outbreaks which is in accord with internationally recorded data. Forty foodborne and waterborne outbreak events due to NoV, epidemiological and/or laboratory confirmed, from 2000 to 2007 revealed that in 42.5% of the cases the food handler was responsible for the outbreak, followed by water (27.5%), bivalve shellfish (17.5%) and raspberries (10.0%).

Extraction of food-borne viruses from food samples: A review

Detection of food-borne viruses such as noroviruses, rotaviruses and hepatitis A virus in food products differs from detection of most food-borne bacteria, as most of these viruses cannot be cultivated in cell culture to date. Therefore, detection of food-borne viruses in food products requires multiple steps: first, virus extraction; second, purification of the viral genomic material (RNA for the majority of food-borne viruses); and last, molecular detection. This review is focused on the first step, the virus extraction. All of the numerous published protocols for virus extraction from food samples are based on 3 main approaches: 1) (acid adsorption-) elution-concentration; 2) direct RNA extraction; and 3) proteinase K treatment. This review summarizes these virus extraction approaches and the results obtained from published protocols. The use of process controls is also briefly described.