Shellfish-Borne Viral Outbreaks: A Systematic Review

Investigations of disease outbreaks linked to shellfish consumption have been reported in the scientific literature; however, only few countries systematically collate and report such data through a disease surveillance system. We conducted a systematic review to investigate shellfish-borne viral outbreaks and to explore their distribution in different countries, and to determine if different types of shellfish and viruses are implicated. Six databases (Medline, Embase, Scopus, PubMed, Eurosurveillance Journal and Spingerlink electronic Journal) and a global electronic reporting system (ProMED) were searched from 1980 to July 2012. About 359 shellfish-borne viral outbreaks, alongside with nine ProMED reports, involving shellfish consumption, were identified. The majority of the reported outbreaks were located in East Asia, followed by Europe, America, Oceania, Australia and Africa. More than half of the outbreaks (63.6 %) were reported from Japan. The most common viral pathogens involved were norovirus (83.7 %) and hepatitis A virus (12.8 %). The most frequent type of consumed shellfish which was involved in outbreaks was oysters (58.4 %). Outbreaks following shellfish consumption were often attributed to water contamination by sewage and/or undercooking. Differences in reporting of outbreaks were seen between the scientific literature and ProMED. Consumption of contaminated shellfish represents a risk to public health in both developed and developing countries, but impact will be disproportionate and likely to compound existing health disparities.     

Prevalence and Molecular Genotyping of Noroviruses in Market Oysters,Mussels, and Cockles in Bangkok,Thailand

Noroviruses are the most common cause of acute gastroenteritis associated with bivalve shellfish consumption. This study aimed to detect and characterize noroviruses in three bivalve shellfish species: oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The virus concentration procedure (adsorption-twice elution-extraction) and a molecular method were employed to identify noroviruses in shellfish. RT-nested PCR was able to detect known norovirus GII.4 of 8.8 × 10^?2 genome copies/g of digestive tissues from oyster and cockle concentrates, whereas in mussel concentrates, the positive result was seen at 8.8 × 10² copies/g of digestive tissues. From August 2011 to July 2012, a total of 300 shellfish samples, including each of 100 samples from oysters, cockles, and mussels were collected and tested for noroviruses. Norovirus RNA was detected in 12.3 % of shellfish samples. Of the noroviruses, 7.7 % were of the genogroup (G) I, 2.6 % GII, and 2.0 % were mixed GI and GII. The detection rate of norovirus GI was 2.1 times higher than GII. With regards to the different shellfish species, 17 % of the oyster samples were positive, while 14.0 and 6.0 % were positive for noroviruses found in mussels and cockles, respectively. Norovirus contamination in the shellfish occurred throughout the year with the highest peak in September. Seventeen norovirus-positive PCR products were characterized upon a partial sequence analysis of the capsid gene. Based on phylogenetic analysis, five different genotypes of norovirus GI (GI.2, GI.3, GI.4, GI.5, and GI.9) and four different genotypes of GII (GII.1, GII.2, GII.3, and GII.4) were identified. These findings indicate the prevalence and distribution of noroviruses in three shellfish species. The high prevalence of noroviruses in oysters contributes to the optimization of monitoring plans to improve the preventive strategies of acute gastroenteritis.     

Environmental Conditions Leading to Shellfish Contamination and Related Outbreaks

Human fecal wastes contain a large variety of viruses that can enter the environment through discharge of waste materials from infected individuals. Despite the high diversity of viruses that are introduced into the environment by human fecal pollution, only a few have been recognized to cause disease in association with consumption of contaminated shellfish. To explain bivalve mollusks contamination, several factors including human epidemiology, virus persistence through sewage treatment plant, and shellfish uptake may be suggested. Considering different outbreaks described in the literature, the most common route for transmission is accidental contamination after heavy rainfall, when extra loads cause an overflow, and release of untreated sewage into the aquatic environment. Outbreak analysis also demonstrates the impact on shellfish consumption of some viral strain transmission and thus their impact on molecular epidemiology, especially for norovirus. To limit shellfish contamination and thus to protect the consumer, the most desirable and effective option is to reduce the viral input.     

Norovirus and Other Human Enteric Viruses in Moroccan Shellfish

The aim of this study was to evaluate the presence of human enteric viruses in shellfish collected along the Mediterranean Sea and Atlantic Coast of Morocco. A total of 77 samples were collected from areas potentially contaminated by human sewage. Noroviruses were detected in 30 % of samples, with an equal representation of GI and GII strains, but were much more frequently found in cockles or clams than in oysters. The method used, including extraction efficiency controls, allowed the quantification of virus concentration. As in previous reports, results showed levels of contamination between 100 and 1,000 copies/g of digestive tissues. Sapoviruses were detected in 13 % of samples mainly in oyster and clam samples. Hepatitis A virus was detected in two samples, with concentrations around 100 RNA copies/g of digestive tissues. Only two samples were contaminated with enterovirus and none with norovirus GIV or Aichi virus. This study highlights the interest of studying shellfish samples from different countries and different production areas. A better knowledge of shellfish contamination helps us to understand virus levels in shellfish and to improve shellfish safety, thus protecting consumers.     

Follow-Up of Norovirus Contamination in an Oyster Production Area Linked to Repeated Outbreaks

A production area repeatedly implicated in oyster-related gastroenteritis in France was studied for several months over 2 years. Outbreaks and field samples were analyzed by undertaking triplicate extractions, followed by norovirus (NoV) detection using triplicate wells for genomic amplification. This approach allowed us to demonstrate that some variabilities can be observed for samples with a low level of contamination, but most samples analyzed gave reproducible results. At the first outbreak, implicated oysters were collected at the beginning of the contamination event, which was reflected by the higher NoV levels during the first month of the study. During the second year, NoV concentrations in samples implicated in outbreaks and collected from the production area were similar, confirming the failure of the shellfish depuration process. Contamination was detected mainly during winter-spring months, and a high prevalence of NoV GI contamination was observed. A half-life of 18 days was calculated from NoV concentrations detected in oysters during this study, showing a very slow decrease of the contamination in the production area. Preventing the contamination of coastal waters should be a priority.     

Norovirus Detection at Oualidia Lagoon,a Moroccan Shellfish Harvesting Area,by Reverse Transcription PCR Analysis

Norovirus (NoV) is the leading cause of acute viral gastroenteritis outbreaks in the world. These outbreaks are frequently associated with bivalve shellfish consumption, particularly because these products are often eaten raw or only slightly cooked. In Morocco, regulations concerning the acceptable levels of enteric bacteria indicator organisms in these products have been put in place. However, these regulations do not take into account the risk of viral contamination, and many gastroenteritis outbreaks have been linked to the ingestion of bivalve shellfish from areas that comply with the current food safety criteria. The aim of this study was to investigate NoV presence in shellfish samples (n = 104) collected at four sites owcff Oualidia lagoon (Moroccan Atlantic coast) from November 2015 to February 2017. Samples were analysed using real-time RT-PCR in accordance with the ISO 15216-2 method. NoVs of the genogroup II were detected in 7% of samples that were all collected during the winter months. Moreover, 71% of NoV-positive samples were harvested at sites upstream of the lagoon. These results highlight the need of regularly monitoring viral contamination in bivalve shellfish to limit the risk of viral gastroenteritis outbreaks.

     

Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels

Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log₁₀ between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.

     

Human Enteric Viruses in Groundwater

Waterborne outbreaks of enteric viruses are a major public health concern. The present study has been carried out to assess the presence of enteric viruses responsible for human acute gastroenteritis (AGE) in groundwater intended for drinking and produce washing. In total, 62 samples from groundwater for drinking and produce washing collected from Dec 2007 to Dec 2008 in Seoul were tested for enteric viruses using conventional RT–PCR, ELISA, and real-time RT–PCR. Our results showed that enteric viruses were detected in 7 (8.8%) groundwater samples. Rotaviruses were detected in 3 (4.8%) of the samples by ELISA; human adenoviruses were detected in 2 (3.2%) of the samples by ELISA; and nested RT–PCR detected noroviruses in 2 (3.2%) of the samples. In one of the groundwater sample, the norovirus RNA was detected by conventional RT–PCR which was confirmed positive by real-time RT–PCR. Additionally, real-time RT–PCR successfully detected norovirus RNA in five out of 62 water samples (8.1%). The data demonstrate that real-time RT–PCR will be useful as a rapid and sensitive method for detecting norovirus in water samples. Phylogenetic analysis revealed that the noroviruses detected in two of the groundwater samples belonged to GII-4. These studies can provide important information for the prevalence of enteric viruses in Korean groundwater.     

Effect of the Shellfish Proteinase K Digestion Method on Norovirus Capsid Integrity

Norovirus outbreaks are associated with the consumption of contaminated shellfish, and so efficient methods to recover and detect infectious norovirus in shellfish are important. The Proteinase K digestion method used to recover norovirus from shellfish, as described in the ISO 15216, would be a good candidate but its impact on the virus capsid integrity and thus infectivity was never examined. The aim of this study was to assess the impact of the Proteinase K digestion method, and of the heat treatment component of the method alone, on norovirus (genogroups I and II) and MS2 bacteriophage capsid integrity. A slightly modified version of the ISO method was used. RT-qPCR was used for virus detection following digestion of accessible viral RNA using RNases. MS2 phage infectivity was measured using a plaque assay. The effect of shellfish digestive glands (DG) on recovery was evaluated. In the presence of shellfish DG, a reduction in MS2 phage infectivity of about 1 log₁₀ was observed after the Proteinase K digestion method and after heat treatment component alone. For norovirus GII and MS2 phage, there was no significant loss of genome following the Proteinase K digestion method but there was a significant 0.24 log₁₀ loss of norovirus GI. In the absence of shellfish DG, the reduction in MS2 phage infectivity was about 2 log₁₀, with the addition of RNases resulting in a significant loss of genome for all tested viruses following complete Proteinase K digestion method and the heat treatment alone. While some protective effect from the shellfish DG on viruses was observed, the impact on capsid integrity and infectivity suggests that this method, while suitable for norovirus genome detection, may not completely preserve virus infectivity.     

Critical Review on the Public Health Impact of Norovirus Contamination in Shellfish and the Environment: A UK Perspective

We review the risk of norovirus (NoV) infection to the human population from consumption of contaminated shellfish. From a UK perspective, risk is apportioned for different vectors of NoV infection within the population. NoV spreads mainly by person-to-person contact or via unsanitary food handling. NoV also enters the coastal zone via wastewater discharges resulting in contamination of shellfish waters. Typically, NoV persists in the marine environment for several days, with its presence strongly linked to human population density, wastewater discharge rate, and efficacy of wastewater treatment. Shellfish bioaccumulate NoV and current post-harvest depuration is inefficient in its removal. While NoV can be inactivated by cooking (e.g. mussels), consumption of contaminated raw shellfish (e.g. oysters) represents a risk to human health. Consumption of contaminated food accounts for 3–11% of NoV cases in the UK (~74,000 cases/year), of which 16% are attributable to oyster consumption (11,800 cases/year). However, environmental and human factors influencing NoV infectivity remain poorly understood. Lack of standard methods for accurate quantification of infective and non-infective (damaged) NoV particles represent a major barrier, hampering identification of an appropriate lower NoV contamination limit for shellfish. Future management strategies may include shellfish quality assessment (at point of harvest or at point of supply) or harvesting controls. However, poor understanding of NoV inactivation in shellfish and the environment currently limits accurate apportionment and risk assessment for NoV and hence the identification of appropriate shellfish or environmental quality standards.     

江苏省某市部分农村河道水体和污水处理厂主要工艺环节病毒污染状况调查

为研究江苏省某市农村河道水体和污水处理厂主要工艺环节病毒的污染状况,分析常见理化因素与病毒污染之间的相关性,对江苏省某市部分农村河道水体和污水处理厂主要工艺环节7个水样、1个污泥和2个沉积物样点进行为期9个月的病毒(包括诺如病毒G Ⅰ、诺如病毒G Ⅱ、轮状病毒、札如病毒、腺病毒、星状病毒、肠道病毒)污染状况监测采样(共90份样本),通过阴离子膜吸附-洗脱法进行富集,荧光定量RT-PCR检测法对病毒进行检测,分析病毒污染状况;同时,监测水温、pH、COD(化学耗氧量)、TP(总磷)、TN(总氮)、EC(电导率)和DO(溶解氧)等7个理化指标的变化情况,探讨这些常见理化因素与病毒污染之间的相关性. 结果表明:90份样本中,诺如病毒G Ⅱ检出率为36.67%,肠道病毒检出率为30%,其他病毒检出率均低于诺如病毒G Ⅱ和肠道病毒. 污水处理后出水中有诺如病毒G Ⅱ、肠道病毒和轮状病毒检出,相关性分析显示,水温与诺如病毒G Ⅱ检出率呈显著负相关(P < 0.05). 研究显示,江苏省某市农村河道水体和污水处理厂主要工艺环节存在病毒污染,诺如病毒G Ⅱ为优势毒株;目前的污水消毒处理工艺并不能完全去除病毒,人群在接触外环境水体时,有潜在病毒暴露风险.      

Detection and Typing of Norovirus from Frozen Strawberries Involved in a Large-Scale Gastroenteritis Outbreak in Germany

During September/October 2012, a norovirus gastroenteritis outbreak affecting about 11,000 people occurred in Germany. Epidemiological studies suggested that frozen strawberries represented the vehicle of infection. We describe here the analysis of frozen strawberries for the presence of norovirus. Samples were taken by applying a stratified subsampling scheme. Two different methods for virus extraction from strawberries were compared. First, viruses were eluted from strawberries under alkaline conditions and concentrated using a polyethylene glycol precipitation. Second, ultrafiltration was applied for concentration of viruses rinsed off of the berries. In both cases, RNA was extracted and analyzed by real-time RT-PCR. Application of the ultrafiltration method generally resulted in a lower detection rate. Noroviruses were detected in 7/11 samples derived from the lot of strawberries implicated in the outbreak using the precipitation method. Typing of norovirus revealed three different genotypes including a combination of norovirus genotype II.16 (viral polymerase) and II.13 (viral capsid). This genotype combination was also found in some of the patients that were involved in the outbreak, but that had not been reported in Germany so far. In conclusion, heterogeneously distributed noroviruses in frozen strawberries can be detected by applying an optimized combination of sampling procedures, virus extraction methods, and real-time RT-PCR protocols. The detection of several different genotypes in the strawberries may suggest contamination from sewage rather than from a single infected food handler.     

Detection and Sequencing of Multiple Human Norovirus Genotypes from Imported Frozen Raspberries Linked to Outbreaks in the Province of Quebec,Canada, in 2017

Food and Environmental Virology - Human noroviruses are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several norovirus food-related outbreaks....     

Use of F-Specific RNA Bacteriophage to Estimate Infectious Norovirus Levels in Oysters

Food and Environmental Virology - Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a significant food safety risk. Methods for quantification of norovirus...     

A One-Year Survey of Norovirus in UK Oysters Collected at the Point of Sale

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a food safety risk and a potential contributor to the overall burden of gastroenteritis in the community. The United Kingdom (UK) has comprehensive national baseline data on the prevalence, levels, and seasonality of norovirus in oysters in production areas resulting from a previous two-year study (2009–2011). However, previously, data on final product as sold to the consumer have been lacking. As part of a wider project to establish the overall burden of foodborne norovirus in the UK, this study aimed to address this data gap. A one-year survey of oysters collected from the point-of-sale to the consumer was carried out from March 2015 to March 2016. A total of 630 samples, originating in five different European Union Member States, were collected from 21 regions across the UK using a randomised sampling plan, and tested for norovirus using a method compliant with ISO 15216-1, in addition to Escherichia coli as the statutory indicator of hygiene status. As in the previous production area study, norovirus RNA was detected in a high proportion of samples (68.7%), with a strong winter seasonality noted. Some statistically significant differences in prevalences and levels in oysters from different countries were noted, with samples originating in the Netherlands showing lower prevalences and levels than those from either the UK or Ireland. Overall, levels detected in positive samples were considerably lower than seen previously. Investigation of potential contributing factors to this pattern of results was carried out. Application of normalisation factors to the data from the two studies based on both the numbers of norovirus illness reports received by national surveillance systems, and the national average environmental temperatures during the two study periods resulted in a much closer agreement between the two data sets, with the notably different numbers of illness reports making the major contribution to the differences observed in norovirus levels in oysters. The large majority of samples (76.5%) contained no detectable E. coli; however, in a small number of samples (2.4%) levels above the statutory end product standard (230 MPN/100 g) were detected. This study both revealed the high prevalence of norovirus RNA in oysters directly available to the UK consumer, despite the high level of compliance with the existing E. coli-based health standards, while also highlighting the difficulty in comparing the results of surveys carried out in different time periods, due to variability in risk factors.