Norovirus In Oysters Research Articles

Inhibition of Norovirus GII.4 binding to HBGAs by Sargassum fusiforme polysaccharide.

Norovirus (NoV) is the main pathogen that causes acute gastroenteritis and brings a heavy socio-economic burden worldwide. In this study, five polysaccharide fractions, labeled pSFP-1-5, were isolated and purified from Sargassum fusiforme (S. fusiforme). In vitro experiments demonstrated that pSFP-5 significantly prevented the binding of type A, B and H histo-blood group antigens (HBGAs) to NoV GII.4 virus-like particles (NoV GII.4 VLPs). In addition, in vivo experiments revealed that pSFP-5 was effective in reducing the accumulation of NoV in oysters, indicating that pSFP-5 could reduce the risk of NoV infection from oyster consumption. The results of transmission electron microscopy showed that the appearance of NoV GII.4 VLPs changed after pSFP-5 treatment, indicating that pSFP-5 may achieve antiviral ability by altering the morphological structure of the viral particles so that they could not bind to HBGAs. The results of the present study indicate that pSFP-5 may be an effective anti-NoV substance and can be used as a potential anti-NoV drug component.

Spatial Distribution and Enrichment Dynamics of Foodborne Norovirus in Oyster Tissues.

The prevalence of norovirus in oysters poses a significant threat to food safety, necessitating a comprehensive understanding of contamination patterns. This study explores the temporal dynamics of norovirus distribution in various oyster tissues over a contamination period ranging from 6 to 96 h. Four tissues-the gill, palp, digestive gland, and stomach-were subjected to systematic monitoring using RT-qPCR for absolute quantification. Results revealed rapid norovirus detection in all tissues six hours post-contamination, with subsequent variations in detection rates. Gill and digestive gland tissues exhibited a peak in detection at 12-24 h, aligning with the oyster's gastrointestinal circulatory system. The digestive gland, distinguished by specific enrichment and adsorption capabilities, demonstrated the highest virus concentration at 48 h. In contrast, the stomach displayed a reemergence of norovirus. Beyond 72 h, detection remained exclusive to the digestive gland, with Ct values comparable to earlier time points. At 96 h, a limited amount of norovirus was detected in the digestive gland, emphasizing the importance for timely monitoring. In addition to providing critical insights into optimal detection strategies, these findings highlight the time-related characteristics of norovirus contamination in oysters. The study identifies the digestive gland as a key target for reliable monitoring, providing valuable data to improve protocols for reducing hazards associated with oyster consumption and foodborne norovirus infections. This research contributes to the understanding of norovirus dynamics in oyster tissues and reinforces current efforts aimed at ensuring food safety and public health.

Research Progress on Biological Accumulation, Detection and Inactivation Technologies of Norovirus in Oysters.

Noroviruses (NoVs) are major foodborne pathogens that cause acute gastroenteritis. Oysters are significant carriers of this pathogen, and disease transmission from the consumption of NoVs-infected oysters occurs worldwide. The review discusses the mechanism of NoVs bioaccumulation in oysters, particularly the binding of histo-blood group antigen-like (HBGA-like) molecules to NoVs in oysters. The review explores the factors that influence NoVs bioaccumulation in oysters, including temperature, precipitation and water contamination. The review also discusses the detection methods of NoVs in live oysters and analyzes the inactivation effects of high hydrostatic pressure, irradiation treatment and plasma treatment on NoVs. These non-thermal processing treatments can remove NoVs efficiently while retaining the original flavor of oysters. However, further research is needed to reduce the cost of these technologies to achieve large-scale commercial applications. The review aims to provide novel insights to reduce the bioaccumulation of NoVs in oysters and serve as a reference for the development of new, rapid and effective methods for detecting and inactivating NoVs in live oysters.

Amplicon-Based High-Throughput Sequencing Method for Genotypic Characterization of Norovirus in Oysters.

Norovirus is a highly diverse RNA virus often implicated in foodborne outbreaks, particularly those associated with shellfish. Shellfish are filter feeders, and when harvested in bays exposed to wastewater overflow or storm overflows, they can harbor various pathogens, including human-pathogenic viruses. The application of Sanger or amplicon-based high-throughput sequencing (HTS) technologies to identify human pathogens in shellfish faces two main challenges: (i) distinguishing multiple genotypes/variants in a single sample and (ii) low concentrations of norovirus RNA. Here, we assessed the performance of a novel norovirus capsid amplicon HTS method. We generated a panel of spiked oysters containing various norovirus concentrations with different genotypic compositions. Several DNA polymerases and reverse transcriptases (RTs) were compared, and performance was evaluated based on (i) the number of reads passing quality filters per sample, (ii) the number of correct genotypes identified, and (iii) the sequence identity of outputs compared to Sanger-derived sequences. A combination of the reverse transcriptase LunaScript and the DNA polymerase AmpliTaq Gold provided the best results. The method was then employed, and compared with Sanger sequencing, to characterize norovirus populations in naturally contaminated oysters. IMPORTANCE While foodborne outbreaks account for approximately 14% of norovirus cases (L. Verhoef, J. Hewitt, L. Barclay, S. Ahmed, R. Lake, A. J. Hall, B. Lopman, A. Kroneman, H. Vennema, J. Vinjé, and M. Koopmans, Emerg Infect Dis 21:592-599, 2015), we do not have standardized high-throughput sequencing methods for genotypic characterization in foodstuffs. Here, we present an optimized amplicon high-throughput sequencing method for the genotypic characterization of norovirus in oysters. This method can accurately detect and characterize norovirus at concentrations found in oysters grown in production areas impacted by human wastewater discharges. It will permit the investigation of norovirus genetic diversity in complex matrices and contribute to ongoing surveillance of norovirus in the environment.

A quantitative exposure assessment model for norovirus in oysters harvested from a classified production area

Norovirus is a significant hazard to consumers of shellfish, in particular oysters. Oysters grown in waters contaminated with wastewater filter and accumulate norovirus particles, causing infection in humans when the product is consumed raw or lightly cooked. In the European Union (EU) and the United States, bacterial detection criteria are used to assess and manage microbial risk in shellfish. This regulatory framework is effective in managing the bacterial risks associated with microbiological contamination of bivalve shellfish but not for viruses. Although a standard detection method for norovirus in oyster exists (ISO 15,216–1:2017), no quantitative microbial risk assessment has been published that links the concentration of norovirus in oysters from a classified production area with consumer exposure. This study shows the successful development of a two-dimensional Monte Carlo exposure assessment model, taking an ISO 15,216–1:2017 detection result and producing an estimate of the resulting per-serving consumer exposure distribution. In contrast to previous oyster virus risk assessments, consumption is modelled using individual oysters as the unit, rather than total flesh weight. The variation in copies per oyster is modelled using a Poisson-lognormal distribution. The results show the boundaries for potential exposure following a given ISO detection result, and the relative importance of mean concentration, serving size, and oyster grade. This is directly relevant to potential regulatory thresholds being considered in the EU.

Development of a Reverse Transcription-Recombinase Polymerase Amplification Assay for Duplex Detection of Foodborne Viruses in Oysters

Abstract Viral contamination may occur at any stage of food processing. The study aim was to develop a two-step reverse transcription (RT)-recombinase polymerase amplification (RPA) assay and evaluate for in-field duplex detection of hepatitis A virus (HAV) and norovirus in oysters. The RNA expression plasmids were generated by amplifying a fragment of the VP1 gene of HAV and norovirus through PCR and cloning it into an expression vector. The RNAs were transcribed in vitro from the plasmids and further used for reverse transcription. The resulting single-stranded cDNAs were used as the purified or spiking templates to determine the sensitivities of simplex and duplex RT-RPA compared with RT-PCR, and RT-qPCR assays. The reproducibility and application of duplex RT-RPA in the field were also evaluated. Our results showed that simplex RT-RPA was at least 100 times more sensitive than RT-PCR and RT-qPCR and even more than duplex detection using purified targets. Unlike RT-PCR, the RT-RPA reaction was unaffected by inhibitors found in food, allowing simple sample preparation methods for detection within a fraction of the time. The duplex assay detected HAV, norovirus, or both in 12/30 (40%) oyster samples tested. Duplex RT-RPA proved to be a rapid, accurate, and reproducible method in a field test for detecting HAV and norovirus. Thus, duplex RT-RPA should be suitable for use in minimally equipped laboratories and field settings. If in-field RT-RPA services are provided to oyster farmers, the technique can minimize the risk of infection to consumers, thereby improving food safety. Keywords: Food safety, Hepatitis A virus, Direct extraction, Norovirus, Nucleic acid amplification

Differences in the effectiveness of the high-efficient concentrated pretreatment method on the norovirus detection in oysters and mussels

Oysters and mussels are important vectors for norovirus (NoV). An efficient pretreatment method for NoV detection in oysters based on ISO 15216-2:2019 was established in our previous work, but its effectiveness for other types of shellfish remains unknown. Therefore, this study systematically compared the differences between the standard and modified ISO methods in detecting NoV for oysters and mussels. Using the standard ISO method, the recovery rates of NoV in oysters (2.10 ± 0.80 %) and mussels (2.39 ± 0.56 %) were comparable (p > 0.05, unpaired t-test). In contrast, the virus recovery rates in oysters (19.83 ± 3.64 %) and mussels (46.96 ± 3.55 %) were both significantly improved by the modified method. Also, a significant difference was found between the virus recovery rates in two shellfish (p < 0.05, unpaired t-test), resulting in a 2.09-fold difference in their virus concentrations. Additionally, the limits of detection at 95 % probability of the modified ISO method for oysters and mussels could both reach 3.33 × 103 copies/g of digestive glands. Finally, the modified ISO method has been successfully applied in commercial oysters (14/27, 51.85 %) and mussels (15/23, 65.22 %), and the results indicated a significant difference in NoV recovery rates between two shellfish (p < 0.05, one-way analysis of variance). In summary, the modified ISO method showed higher virus recovery rates than the standard ISO method, which would be used as an essential tool for NoV detection in oysters and mussels.

Performance Comparison of Domestic and International Methods for Detection and Quantification of Norovirus in Oysters

Performance Comparison of Domestic and International Methods for Detection and Quantification of Norovirus in Oysters

Detection and Genetic Correlation Analysis of Diarrhea Cases and Norovirus in Oysters in Yantai, China.

BackgroundThis study aimed to assess the correlation between Norovirus (NoV), diarrhea, and raw oysters from the eastern coastal areas of Yantai, Shandong, China.MethodsMarine oysters were selected from the three aquatic markets in Laishan district, Yantai City, in March 2019. Meanwhile, 100 fecal samples were collected from patients with diarrhea from the same areas during the same period. Nucleic acids were extracted from these samples and detected by employing reverse transcription polymerase chain reaction (RT-PCR) for NoV GI/GII. The VP1 gene of the coat protein of NoV was amplified by semi-nested RT-PCR and sequenced. Sequence comparison of VP1 was performed with BioEdit software, and the evolutionary tree was constructed with Mega7.0 software.ResultsOf the 151 oysters, 42 (27.8%) were positive for NoV. Among them, 32 (21.2%) were GII-positive, 10 (6.6%) were GI-positive, and one GI VP1 sequence was obtained in the oyster samples. Of 100 fecal samples from patients with diarrhea, 38 were GII-positive and 17 were GI-positive. Totally, 19 GII VP 1 sequences and eight GI VP 1 sequences were obtained. Two G1 VP 1 sequences in two fecal samples showed 98.7% nucleotide sequence identity and 99.1% amino acid sequence identity G1 VP 1 acquired in the oyster sample.ConclusionsThe results suggest that oysters may be responsible for the spread of NoV in Yantai, Shandong province, China.

Updating a New Semi-nested PCR Primer Pair for the Specific Detection of GII Norovirus in Oysters

Oysters are major transmission vectors of noroviruses (NoVs) in the environment. Outbreaks of NoVs are often associated with the consumption of NoV-contaminated oysters. Laboratory confirmation of suspected oyster samples is a critical step in the surveillance and control of NoVs. Because of non-specific amplification, false-positive results are frequently obtained by semi-nested RT-PCR with the presently widely used primer set (G2SKF/G2SKR). Here, a novel universal PCR primer set N (NG2OF/NG2OR) specific for genogroup II (GII) NoVs was designed based on all GII NoV sequences available in public databases. Specific products were obtained with the primer set N when the NoV-positive oysters, spiked with each of five representative genotypes of GII NoVs (GII.17, GII.13, GII.4, GII.3, and GII.12), were subjected to analyzing. No products were detected with the primer set N for the NoV-negative oysters, while the primer set C gave various non-specific bands. Twenty-three out of 156 fresh oyster samples were NoV-positive with both the primer set N and the classic primer set, while eight were NoV-positive solely with the primer set N. Compared with the classic primer set, the newly designed primer set N had a higher detection rate and improved specificity for GII NoVs in oyster samples. These results show that the novel PCR primer pair is specific and applicable for the detection of GII NoVs in oysters.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12560-022-09511-6.

Application of two RT-PCR methods for detection of norovirus in market-sold oysters and norovirus genetic characteristic analysis, a survey conducted in Beijing

Objective: To evaluate the application of real-time RT-PCR and semi-nested RT-PCR in the detection of norovirus in oysters and analyzing the genetic characteristics of the isolates. Methods: Real-time fluorescent RT-PCR and semi-nested RT-PCR were used to detect norovirus GⅠ/GⅡ in fresh oysters collected from the markets in Beijing from November 2014 to October 2015. The detection rate of the parallel test was also analyzed. In addition, the reliability of semi-nested RT-PCR was evaluated by agreement rate and consistency test (Kappa value). The positive products of norovirus GⅠ/GⅡ capsid protein region gene by semi-nested RT-PCR were sequenced. Software BioEdit 7.0.9.0 was used for sequence alignment, and software Mega 6.0 was used to construct the evolutionary tree. Results: In 72 samples, the detection rate of norovirus was 31.94% (23/72) by real-time RT-PCR, 38.89% (28/72) by semi-nested RT-PCR and 48.61% (35/72) by parallel test. The coincidence rate of the two methods was 73.61%, a moderate degree (Kappa value =0.43). A total of 13 norovirus strains were successfully sequenced, and 11 strains (7 GⅡ.17 strains, 2 GⅡ. 4 Sydney_ 2012 strains, 1 GⅡ. 1 strain and 1 GⅡ. 21 strain) were obtained from norovirus positive samples by two RT-PCR methods, two strains (1 GⅡ. 17 strain and 1 GⅡ. 3 strain) were obtained from real-time RT-PCR negative samples which were positive for norovirus by semi-nested RT-PCR. The similarity between these strains and reference strains from diarrhea patients, environmental sewage, and shellfish products were 84.4% - 100.0%. Conclusions: The parallel test of norovirus in oysters by two RT-PCR methods can improve the detection rate and detect more genotypes. Norovirus strains in oysters were highly homologous with reference strains from diarrheal patients, environmental sewage, and shellfish products. Therefore, surveillance, prevention and control for norovirus should be carried out in people who have frequent contacts with oysters and related environments.

Assessment of Microbiological Quality of Fresh Vegetables and Oysters Produced in Buenos Aires Province, Argentina.

Fresh vegetables and shellfish are prone to microbial contamination through irrigation or breeding with sewage-polluted waters, as well as by infected food handlers. In this work, we studied the presence of human and bovine polyomaviruses and human norovirus in fresh lettuces, strawberries and oysters produced in Buenos Aires province, Argentina. In oysters, we also investigated F-specific RNA bacteriophages, indicator Escherichia coli (E. coli) and pathogen bacteria of concern (Salmonella spp., Vibrio spp.). Within vegetables, we found viral contamination of human origin given the presence of human-associated polyomaviruses -MCPyV, HPyV6, JCPyV, and SV40- in lettuce and strawberry samples (16 and 10%, respectively), probably coming from irrigation waters and food handling. Among oysters, human (MCPyV, 4.2%) and bovine (BPyV1, 8.4%) polyomaviruses were detected even with low counts of E. coli. Bacteriophages (n = 3) and Salmonella spp. (n = 1) were also found, while Vibrio spp. was not detected. These results may indicate that the contamination in oysters comes from human and animal excreta, probably present in breeding waters. Norovirus was not detected in any food sample. To our knowledge, this is the first description of SV40 in lettuces and MCPyV and BPyV1 in oysters. The detection of different viral contaminants encourages further studies to evaluate the need for including viral indicators in microbiological standards. The identification of possible sources and routes of contamination using viral markers during routine microbiological controls, such as the polyomaviruses used in this work, would be useful to focus attention on the most hazardous stages of the food production chain.

Norovirus Persistence in Oysters to Prolonged Commercial Purification.

Depuration is generally the main treatment employed for bivalve mollusks harvested from contaminated sites. Commercial depuration has demonstrated to be effective for removal of bacterial pathogens, although it probably provides only limited efficacy against human enteric viruses. We evaluated the quantitative reduction of norovirus (NoV) genogroups I and II in naturally contaminated oysters after 1, 4, and 9 days of depuration. The process was conducted in an authorized depuration plant, and NoV concentration was determined by RT-qPCR according to ISO 15216-1:2017 method. Regardless of the NoV genogroup, our results showed no significant reduction in NoV concentration after 1 day of depuration. Higher mean reduction (68%) was obtained after 4 days of treatment, while no further increase was observed after 9 days. Overall, reduction was highly variable, and none of the trials showed statistically significant reduction in NoV RNA concentration at the end of each depuration period. Indeed, NoV concentration remained high in 70% of samples even after 9 days of depuration, with values ranging between 4.0 × 102 and 2.3 × 104 g.c./g. These results indicate that an extension of commercial depuration time does not appear to be effective for reducing or eliminating NoV in oysters.

Development of a High-Efficiency Immunomagnetic Enrichment Method for Detection of Human Norovirus via PAMAM Dendrimer/SA-Biotin Mediated Cascade-Amplification.

Human norovirus is a common cause of acute gastroenteritis worldwide, and oysters have been found to be the main carriers for its spread. The lack of efficient pre-treatment methods has been a major bottleneck limiting the detection of viruses in oysters. In this study, we established a novel immunomagnetic enrichment method using polyamidoamine (PAMAM) dendrimer/SA-biotin-mediated cascade amplification for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) detection. We compared the capture efficiency of traditional immunomagnetic enrichment, biotin-amplified immunomagnetic enrichment, and PAMAM dendrimer/SA-biotin-mediated cascade-amplification immunomagnetic enrichment. The optimal capture efficiency of the novel method was 44.26 ± 1.45%, which increased by 183.17% (P < 0.01) and 18.09% (P < 0.05) compared with the first two methods, respectively. Three methods were all applied in detecting norovirus in 44 retail oysters, the detection rate of the PAMAM dendrimer/SA-biotin-mediated method was 25.0%, which was higher than those of traditional IME (15.90%) and SA-biotin-amplified IME (18.80%) by 9.1 and 6.2%, respectively. In conclusion, the novel method can be applied for the rapid detection of norovirus in oysters, which can help reduce the cost and time of detection and improve detection rates.

Evaluation of Norovirus Reduction in Environmentally Contaminated Pacific Oysters During Laboratory Controlled and Commercial Depuration

Norovirus contamination of oysters is the lead cause of non-bacterial gastroenteritis and a significant food safety concern for the oyster industry. Here, norovirus reduction from Pacific oysters (Crassostrea gigas), contaminated in the marine environment, was studied in laboratory depuration trials and in two commercial settings. Norovirus concentrations were measured in oyster digestive tissue before, during and post-depuration using the ISO 15216-1 quantitative real-time RT-PCR method. Results of the laboratory-based studies demonstrate that statistically significant reductions of up to 74% of the initial norovirus GII concentration was achieved after 3 days at 17–21 °C and after 4 days at 11–15 °C, compared to 44% reduction at 7–9 °C. In many trials norovirus GII concentrations were reduced to levels below 100 genome copies per gram (gcg−1; limit of quantitation; LOQ). Virus reduction was also assessed in commercial depuration systems, routinely used by two Irish oyster producers. Up to 68% reduction was recorded for norovirus GI and up to 90% for norovirus GII reducing the geometric mean virus concentration close to or below the LOQ. In both commercial settings there was a significant difference between the levels of reduction of norovirus GI compared to GII (p < 0.05). Additionally, the ability to reduce the norovirus concentration in oysters to < LOQ differed when contaminated with concentrations below and above 1000 gcg−1. These results indicate that depuration, carried out at elevated (> 11 °C) water temperatures for at least 3 days, can reduce the concentration of norovirus in oysters and therefore consumer exposure providing a practical risk management tool for the shellfish industry.

Interlaboratory Evaluation of a Method for Quantification of Norovirus RNA as an Alternative Use for ISO 15216-1:2017 to Conduct Japan Baseline Survey of Oysters.

In this study, we aimed to investigate the standard method used for quantification of norovirus in oysters in Japan for the provisional adaptation of the method as an alternative to ISO 15216-1:2017, to conduct a Japan baseline survey of norovirus in oysters. For this purpose, the method provided by the Japan Committee for Standardization of Virus Detection in Food was subjected to an interlaboratory study to determine the performance characteristics of the standard method used in Japan. As a result, the theoretical limit of quantification for norovirus GI and GII in oysters by the standard method used in Japan was expected to be 1.92 and 1.85 log10 copies/g, respectively. The repeatability standard deviations (Sr) were 0.26 and 0.30 log10 copies/g for GI and GII, respectively, and the reproducibility standard deviations (SR) were 0.47 and 0.44 log10 copies/g for GI and GII, respectively. Through the interlaboratory study, we specified several critical points to obtain scientifically reliable results by using the standard method used in Japan. Especially, necessity for application of using process control virus was the most crucial point that needed to be improved. In addition, there are many participating laboratories that could not handle dilution of standard and quantify or detect the viruses in the test samples. To ensure scientifically reliable test result, capacity building of laboratories and implementation of proficiency testing should be considered for future tasks in combination with an application of process control materials in the method. On the assumption that the problems revealed in this study will be solved, the standard method used in Japan would be suitable for use in Japan baseline survey of norovirus in oysters, which will contribute to the international action against norovirus in oysters, led by the EU.

Occurrence and persistence of enteric viruses, arsenic and biotoxins in Pacific oysters farmed in an Italian production site

The presence of Norovirus (NoV) and Hepatitis E virus (HEV) in non-depurated and depurated oysters raised in the north-west Italian coast was investigated by quantitative real-time RT-PCR. Total and inorganic arsenic (As) and the presence of marine biotoxins (DSP, ASP, PSP) by LC-MS were also investigated. NoV was detected through all the sampling period in non depurated and depurated oysters with highest levels during wintertime (>104 genome copies per gram, gc/g) and minimum values in summer below the LOQ (<130/140 gc/g). HEV has never been found as well as biotoxins. Total As concentration was found in oysters in the range 0.45–3.0 mg/kg, while inorganic As was found in all samples in concentrations below the LOQ (<0.020 mg/kg). The study highlights how the 24 h depuration process didn't reduce significantly NoV levels and therefore the high concentration of NoV in oysters could represent a risk for consumers especially during winter and spring months.

Surveillance of human norovirus in oysters collected from production area in Shandong Province, China during 2017–2018

Human norovirus (HuNoV) is the leading cause of non-bacterial gastroenteritis worldwide. Oysters are known food-related vehicles for HuNoV transmission. However, there are few data available on the viral contamination of shellfish from production areas in China. A one-year monitoring program was carried out to detect and characterize HuNoV contamination in oysters, collected from two shellfish commercial harvesting areas in Shandong province, China. Oysters were sampled every month from July 2017 to August 2018. From the 356 oyster samples, HuNoV was detected in 16.9% (60/356) using reverse transcription-qPCR. Of the 60 positive samples, 43.3% (26/60) were positive for HuNoV genogroup I (GI) only and 31.7% (19/60) were positive for genogroup II (GII) only. Both GI and GII were detected in 25.0% (15/60) samples. The majority (65.0%, 39/60) of positive samples were collected in winter months (November–December). There was no significant correlation between the detection of HuNoV in shellfish and the pH or fecal coliforms levels in seawater. Using next-generation sequencing, several HuNoV genotypes were identified including capsid genotypes GI.5, GI.6, GII.2, GII.3, GII.4_Sydney 2012, GII.13 and GII.14. These Results indicate that monitoring of HuNoV in oyster production area is necessary to ensure the safety of shellfish prior to distribution to consumers.

Human norovirus disease burden of consuming Crassostrea gigas oysters: A case-study from Japan

Shellfish borne viral gastroenteritis outbreaks are most commonly attributed to human norovirus. Minimizing the norovirus bioaccumulation in oysters and improved norovirus removal/inactivation techniques are paramount in reducing the shellfish-borne norovirus outbreaks. Genotype-dependent bioaccumulation of human norovirus in oysters can affect the norovirus removal/inactivation efficiency and ultimately the quantity of human norovirus ingested by the consumer. To evaluate this phenomenon, Crassostrea gigas oysters collected from a shellfisheries area in Eastern Japan were artificially contaminated for 72 h in a sea water bath with only human norovirus GII.2 strain, GII.17 strain and a bath containing a mixture of GII.2 + GII.17. After 72-h contamination, only GII.17 was bioaccumulated in the oysters. Then depuration of oysters was conducted for 48 h, as customary in commercial shellfisheries areas in Japan. However, depuration didn't contribute to significantly reduce the norovirus bioaccumulated in the oyster digestive tract. Based on the norovirus concentration in the depurated oyster digestive tract, human norovirus disease burden by consuming raw oysters was calculated and it was above the 10−4 DALYpppy, which is the acceptable disease burden. Since all the norovirus particles are not infectious, infectivity ratio; the ratio between infectious particles and total virus particles was introduced and the disease burden was recalculated. Even after incorporating infectivity ratio, disease burden value was higher than 10−4 DALYpppy. These results emphasize that depuration doesn't contribute to significantly reduce the human norovirus accumulated in the oyster digestive tract and therefore the disease burden. Minimizing the exposure of shellfisheries areas to human norovirus can be a suitable strategy to reduce the human norovirus disease burden of consuming raw oysters.

Estimating the distribution of norovirus in individual oysters

Norovirus in oysters is a significant food safety risk. A recent ISO detection method allows for reliable and repeatable estimates of norovirus concentrations in pooled samples, but there is insufficient data to estimate a distribution of copies per animal from this. The spread of norovirus accumulated across individual oysters is useful for risk assessment models. Six sets of thirty individual Crassostrea gigas oysters were tested for norovirus concentration levels by reverse-transcription quantitative PCR (RT-qPCR): three from a commercial harvest site, and three post-depuration. Five sets had norovirus GII means above the limit of quantification (LOQ), and one below the LOQ, but above the limit of detection. No norovirus GI was detected in pooled tests, and individual oysters were not tested for norovirus GI. Depuration was shown to reduce the mean concentration of GII copies, but not to affect the shape of the distribution around the mean. Deconvoluting the uncertainty of the method, the coefficient of variation was stationary (0.45 ± 0.2). The best fit distribution was either a lognormal distribution or a gamma. Multiplying these distributions by the weight of oyster digestive tissues gave an estimate for the count mean. This was used as the parameter λ in three compound Poisson distributions: Poisson-lognormal, Poisson-gamma, and Poisson-K. No model was found to fit better than the others, with advantages for each. All three could be used in future risk assessments. Preliminary validation of sampling uncertainty using repeated testing data from a previous study suggests that these results have predictive power.