Viruses In Shellfish Research Articles

Quantitative Risk Assessment of Five Foodborne Viruses in Shellfish Based on Multiplex qPCR.

Foodborne diseases are currently the most critical food safety issue in the world. There are not many hazard identification and exposure assessments for foodborne viruses (Norovirus GI, GII, Hepatitis A Virus, Rotavirus, Adenovirus) in shellfish. Multiplex qPCR for the simultaneous detection of five foodborne viruses was established and used to assess infection risk based on a 1-year pathogenesis study. The sensitivity, specificity and reproducibility of the multiplex qPCR method are consistent with that of conventional qPCR, which saves more time and effort. Overall, 37.86% of shellfish samples had one or more foodborne viruses. Risk assessment formulae and matrices were used to develop risk assessments for different age groups, different seasons and different shellfish. The annual probability of contracting a foodborne virus infection from shellfish is greater than 1.6 × 10-1 for all populations, and even for infants aged 0-4 years, it is greater than 1.5 × 10-2, which is much higher than the risk thresholds recommended by WHO (10-6) and the US EPA (10-4). High risk (level IV) is associated with springtime, and medium risk (level III) is associated with Mussel consumption. This study provides a basis for the risk of foodborne viral infections in people of different ages, in different seasons, and by consuming different shellfish.

Viruses in Shellfish — Food Safety Risks

Viruses in Shellfish — Food Safety Risks

Molecular Detection of Rotavirus in Mollusks from the Oued El Maleh Estuary of Mohammedia, Morocco

Viral outbreaks can result from the consumption of contaminated bivalve mollusks. However, despite the regulation related to enteric bacteria in food products, the consumption of raw and undercooked mollusks remains linked to viral epidemics in human populations. Real-time RT-PCR is a highly sensitive approach for detecting and quantifying enteric viruses, and after eliminating enzymatic amplification inhibitors from samples of interest, sensitive and specific tests, like real-time RT-PCR, can facilitate the detection and quantification of a wide range of viruses that are concentrated in mollusk digestive tissues. The aim of the present study was to evaluate the prevalence of Group-A rotaviruses in mussel (Mytilus edulis Linnaeus, 1758) specimens (n=576) collected downstream of the Oued El Maleh Estuary, which is along the coast of Mohammedia City in Morocco, using real-time RT-PCR. Rotavirus A RNA was detected in 37.5% (n=18) of the 48 sample batches, and viral loads ranged from 0.42×101 to 1.8603×104 genomic copies per g digestive tissue. Most (72.22%) of the positive samples were collected during the wet season (September-April), and the probability of detecting rotaviruses was significantly greater during the wet season than during the dry season (P<0.001). Monitoring Rotavirus A and similar viruses in shellfish may help prevent viral contamination and preserve public health.

Preliminary Quantitative Risk Assessment of Norovirus in Shellfish in the Yellow Sea and Bohai Sea of China

Norovirus (NoV) is a main foodborne pathogen of acute gastroenteritis in the world. A preliminary quantitative risk assessment (QRA) was conducted to evaluate the health risk caused by this virus in shellfish in the Yellow Sea and Bohai Sea of China. The QRA framework was established from the process of shellfish at retail through cooking at home to consumer consumption. The prevalence and quantity of NoVs in shellfish, cooking methods, internal temperature and time of shellfish in different cooking conditions, shellfish consumption frequency, and consumption amount were analyzed in the exposure assessment. The results of exposure assessment were introduced into the beta-Poisson dose-response model, and Monte Carlo analysis was used to calculate the risk of gastroenteritis caused by shellfish consumption in the cities around the Yellow Sea and Bohai Sea of China. The results showed that the probability of illness caused by NoVs due to shellfish consumption per year (Pill,yr) was 1.86 × 10-5. It was estimated that the annual number of patients with gastroenteritis per 1,000,000 general population (Nexp,mil) was 0.10, 1.23, 16.90, and 0.38 for population aged 0-4, 5-18, 19-64, and >65 years, respectively. This assessment provides valuable information such as the probability of illness associated with the consumption of shellfish and it also provides a reference for further in-depth QRA of NoVs in shellfish or other foods.

Occurrence of Human Enteric Viruses in Shellfish along the Production and Distribution Chain in Sicily, Italy.

Contamination of bivalve mollusks with human pathogenic viruses represents a recognized food safety risk. Thus, monitoring programs for shellfish quality along the entire food chain could help to finally preserve the health of consumers. The aim of the present study was to provide up-to-date data on the prevalence of enteric virus contamination along the shellfish production and distribution chain in Sicily. To this end, 162 batches of mollusks were collected between 2017 and 2019 from harvesting areas, depuration and dispatch centers (n = 63), restaurants (n = 6) and retail stores (n = 93) distributed all over the island. Samples were processed according to ISO 15216 standard method, and the presence of genogroup GI and GII norovirus (NoV), hepatitis A and E viruses (HAV, HEV), rotavirus and adenovirus was investigated by real-time reverse transcription polymerase chain reaction (real-time-RT PCR), nested (RT)-PCR and molecular genotyping. Our findings show that 5.56% of samples were contaminated with at least one NoV, HAV and/or HEV. Contaminated shellfish were sampled at production sites and retail stores and their origin was traced back to Spain and several municipalities in Italy. In conclusion, our study highlights the need to implement routine monitoring programs along the whole food chain as an effective measure to prevent foodborne transmission of enteric viruses.

First Report of Hepatitis E Virus in Shellfish in Southeast Italy

Hepatitis E virus (HEV) represents one of the principal causative agents of hepatitis globally. Among the five HEV genotypes affecting humans, genotypes 3 and 4 are zoonotic and are the main source of hepatitis E in developed countries. HEV has been detected in several foods. The present work investigated the presence of this virus in shellfish sold at retail in the Apulia region of Italy. The presence of HEV RNA was assessed by real-time RT-PCR in 225 shellfish samples collected during 2018. Overall, two (0.89%) of these samples tested positive for HEV RNA. To our knowledge, this is the first notification of the detection of HEV in mussels sold at retail in the Apulia region. These data highlight the potential role of shellfish as a vehicle for the transmission of viral pathogens.

Detection of Hepatitis A Virus and Other Enteric Viruses in Shellfish Collected in the Gulf of Naples, Italy.

To assess the quality of shellfish harvest areas, bivalve mollusk samples from three coastal areas of the Campania region in Southwest Italy were evaluated for viruses over a three-year period (2015–2017). Screening of 289 samples from shellfish farms and other locations by qPCR and RT-qPCR identified hepatitis A virus (HAV; 8.9%), norovirus GI (NoVGI; 10.8%) and GII (NoVGII; 39.7%), rotavirus (RV; 9.0%), astrovirus (AsV; 20.8%), sapovirus (SaV; 18.8%), aichivirus-1 (AiV-1; 5.6%), and adenovirus (AdV, 5.6%). Hepatitis E virus (HEV) was never detected. Sequence analysis identified HAV as genotype IA and AdV as type 41. This study demonstrates the presence of different enteric viruses within bivalve mollusks, highlighting the limitations of the current EU classification system for shellfish growing waters.

Molecular characterization of human enteric viruses in food, water samples, and surface swabs in Sicily

ObjectivesEnteric viruses are responsible for foodborne and waterborne infections affecting a large number of people. Data on food and water viral contamination in the south of Italy (Sicily) are scarce and fragmentary. The aim of this study was to evaluate the presence of viral contamination in food, water samples, and surface swabs collected in Sicily MethodsThe survey was conducted on 108 shellfish, 23 water samples (seawater, pipe water, and torrent water), 52 vegetables, one peach and 17 berries, 11 gastronomic preparations containing fish products and/or raw vegetables, and 28 surface swabs. Hepatitis A virus (HAV), genogroup GI, GII, and GIV norovirus (NoV), enterovirus (EV), rotavirus (RoV), hepatitis E virus (HEV), adenovirus (AdV), and bocavirus (BoV) were detected by nested (RT) PCR, real-time PCR, and sequence analysis. ResultsThe most frequently detected viruses in shellfish were HAV (13%), NoV (18.5%), and EV (7.4%). Bocavirus was found in 3.7%, HEV in 0.9%, and AdV in 1.9% of the molluscs. Of the 23 water samples, 21.7% were positive for GII NoV and 4.3% for RoV and HEV genotype 3. Of the 70 vegetable samples, 2.9% were positive for NoV GI (GI.5 and GI.6), 2.9% for EV, and 1.4% for HEV. In the gastronomic preparations, only one EV (9%) was detected. No enteric viruses were detected in the berries, fruit, or swabs analyzed. ConclusionsMolecular surveillance of water and food samples clearly demonstrated that human pathogenic viruses are widely found in aquatic environments and on vegetables, and confirmed the role of vegetables and bivalve molluscs as the main reservoirs.

F-Specific RNA Bacteriophages, Especially Members of Subgroup II, Should Be Reconsidered as Good Indicators of Viral Pollution of Oysters.

Norovirus (NoV) is the leading cause of gastroenteritis outbreaks linked to oyster consumption. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as indicators of viral contamination in oysters by focusing especially on FRNAPH subgroup II (FRNAPH-II). These viral indicators have been neglected because their behavior is sometimes different from that of NoV in shellfish, especially during the depuration processes usually performed before marketing. However, a significant bias needs to be taken into account. This bias is that, in the absence of routine culture methods, NoV is targeted by genome detection, while the presence of FRNAPH is usually investigated by isolation of infectious particles. In this study, by targeting both viruses using genome detection, a significant correlation between the presence of FRNAPH-II and that of NoV in shellfish collected from various European harvesting areas impacted by fecal pollution was observed. Moreover, during their depuration, while the long period of persistence of NoV was confirmed, a similar or even longer period of persistence of the FRNAPH-II genome, which was over 30 days, was observed. Such a striking genome persistence calls into question the relevance of molecular methods for assessing viral hazards. Targeting the same virus (i.e., FRNAPH-II) by culture and genome detection in specimens from harvesting areas as well as during depuration, we concluded that the presence of genomes in shellfish does not provide any information on the presence of the corresponding infectious particles. In view of these results, infectious FRNAPH detection should be reconsidered as a valuable indicator in oysters, and its potential for use in assessing viral hazard needs to be investigated.IMPORTANCE This work brings new data about the behavior of viruses in shellfish, as well as about the relevance of molecular methods for their detection and evaluation of the viral hazard. First, a strong correlation between the presence of F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and that of norovirus (NoV) in shellfish impacted by fecal contamination has been observed when both viruses are detected using molecular approaches. Second, when reverse transcription-PCR and culture are used to detect FRNAPH-II in shellfish, it appears that the genomes of the viruses present a longer period of persistence than infectious virus, and thus, virus genome detection fails to give information about the concomitant presence of infectious viruses. Finally, this study shows that FRNAPH persist at least as long as NoV does. These data are major arguments to reconsider the potential of FRNAPH as indicators of shellfish viral quality.

An overview of 20years of studies on the prevalence of human enteric viruses in shellfish from Galicia, Spain.

Galicia (NW Spain) has 1490km of coastline, and its particular topography, characterized by the presence of fiord-like inlets, called rías, with an important primary production, makes this region very favourable for shellfish growth and culture. In fact, Galicia is one of the most important mussel producers in the world. Due to its proximity to cities and villages and the anthropogenic activities in these estuaries, and despite the routine official controls on the bivalve harvesting areas, contamination with material of faecal origin is sometimes possible but, current regulation based on Escherichia coli as an indicator micro-organism has been revealed as useful for bacterial contaminants, this is not the case for enteric viruses. The aim of this review is to offer a picture on the situation of different harvesting areas in Galicia, from a virological standpoint. A recompilation of results obtained in the last 20years is presented, including not only the data for the well-known agents norovirus (NoV) and hepatitis A virus (HAV) but also data on emerging viral hazards, including sapovirus (SaV), hepatitis E virus (HEV) and aichivirus (AiV). Epidemiological differences related to diverse characteristics of the harvesting areas, viral genotype distribution or epidemiological links between environmental and clinical strains will also be presented and discussed. The presentation of these historical data all together could be useful for future decisions by competent authorities for a better management of shellfish growing areas.

High prevalence of betanodavirus barfin flounder nervous necrosis virus as well as red-spotted grouper nervous necrosis virus genotype in shellfish.

Using two serially executed PCRs, the discriminative multiplex two-step RT-PCR (DMT-2 RT-PCR) following the detection seminested two-step RT-PCR (DSN-2 RT-PCR), we found a high frequency presence of BFNNV genotype as well as RGNNV in various domestic and imported shellfish. This was definitely different from the previous reports of outbreaks and asymptomatic infection only by the RGNNV genotype in cultured finfish in Korea. Cultivation of NNV entrapped in shellfish was performed successfully by a blind passage. Thus, in an attempt to elucidate the epidemiology of betanodavirus, experiments conducted on 969 shellfish samples concluded that (i) distribution of NNV genotype, especially BFNNV, in shellfish is clearly different from that found in finfish of the world; (ii) unlike RGNNV, which showed a high rate in summer, BFNNV showed no seasonal variation and this result suggests BFNNVs in the marine environment remain fairly constant throughout the year; and (iii) the entrapped virus in shellfish was alive and culturable invitro. These results are the first report of high level prevalence of invitro culturable NNV in shellfish, for both BFNNV and RGNNV, which may present a potential risk in transmitting nodaviruses to host species in a marine environment.

First Detection of Hepatitis E Virus in Shellfish and in Seawater from Production Areas in Southern Italy.

Shellfish samples (n=384) from production areas, water samples from the same areas (n=39) and from nearby sewage discharge points (n=29) were analyzed for hepatitis E virus (HEV) by real-time and nested RT-PCR. Ten shellfish samples (2.6%) and five seawater samples (12.8%) tested positive for HEV; all characterized strains were G3 and showed high degree of sequence identity. An integrated surveillance in seafood and waters is relevant to reduce the risk of shellfish-associated illnesses.

Food-Borne Viruses in Shellfish: Investigation on Norovirus and HAV Presence in Apulia (SE Italy)

Shellfish are an important vehicle for transmission of food-borne pathogens including norovirus (NoV) and hepatitis A virus (HAV). The risks related with consumption of shellfish are greater if these products are eaten raw or slightly cooked. As molluscs are filter-feeding organisms, they are able to concentrate pathogens dispersed in the water. Data on shellfish viral contamination are therefore useful to obtain a background information on the presence of contamination in the environment, chiefly in shellfish production areas and to generate a picture of the epidemiology of viral pathogens in local populations. From January 2013 to July 2015, 253 samples of bivalve molluscs collected in harvesting areas from a large coastal tract (860 km) of Southern Italy were screened for HAV and NoV of genogroups GI and GII, using real-time reverse transcription qualitative PCR. The RNA of HAV was not detected in any of the analyzed samples. In contrast, the RNA of NoV was identified in 14.2% of the samples with a higher prevalence of NoVs of genogroup GII (12.2%) than genogroup GI (1.6%). Upon sequence analysis of a short diagnostic region located in capsid region, the NoV strains were characterized as GII.2, GII.4 Sydney 2012, GII.6, GII.13, GI.4, and GI.6, all which were circulating in local populations in the same time span. These data confirm that consumption of mussels can expose consumers to relevant risks of infection. Also, matching between the NoV genotypes circulating in local population and detected in molluscs confirms the diffusion in the environment of NoVs.

Human Sapovirus in Mussels from Ría do Burgo, A Coruña (Spain).

The prevalence and genetical diversity of human Sapovirus were studied during an 18-month study in Ría do Burgo, an estuary nearby the city of A Coruña in Galicia (NW Spain). Sapovirus was detected using RT-qPCR procedure in 30 out of 80 mussel samples (37.5%). Quantifications ranged from 2.2×10(3) to 2.1×10(5) RNA copies per gram of digestive tissue. Detection occurred mainly during the cold and rainy seasons of the period studied. Sequences obtained could be distributed into 5 genotypes being the most abundant GI.1 and GI.3. Results obtained indicate that the hydrodynamic characteristics of the harvesting area and the proximity of population density clearly influence the presence of the virus in shellfish.

Surveillance of aquatic animal viruses in seawater and shellfish in Korea

Surveillance of aquatic animal viruses in hosts and seawater is very important to control and prevent the spread of aquatic animal diseases. And also the investigations for shellfish as play role in vector or carrier species, or a candidate host are required. Primer sets for multiplex nested polymerase chain reaction were developed to simultaneously detect aquatic animal viruses (megalocytivirus and white spot syndrome virus as DNA viruses, and viral hemorrhagic septicemia virus, viral nervous necrosis virus [VNNV], and marine birnavirus as RNA viruses) from seawater and shellfish. The multiplex nested PCR amplified each virus specifically with a detection limit of about 10 copies of viral particles and facilitated detection of several aquatic animal viruses from seawater and shellfish. The detection rates of viruses in shellfish samples were not significantly different between areas, except for VNNV which showed much lower positive results in eastern sea area. But, several viruses co-existed in shellfish regardless of the sampling site and time. These results revealed that shellfish might accumulate several viruses over the long-term and could play a role as a bio-indicator or reservoir of viruses derived from aquatic animals. Statement of relevanceThis article is related to field of aquatic animal disease. In this study, we developed multiplex nested PCR assay for detection of aquatic animal viruses from seawater and shellfish. And we found several viruses co-existed in shellfish for long-term, suggesting that shellfish may act as a bio-indicator or reservoir of viruses derived from aquatic animals.

Prevalence of Rotavirus in shellfish from Southern Kerala

Aim: To study the prevalence of Rotavirus in shellfish from Southern Kerala. Materials and Methods: The shellfish samples after processing was concentrated using proteinase K. RNA was isolated from the concentrated samples using phenol chloroform method. Rota viral RNA was detected using reverse transcriptionpolymerase chain reaction. Results: A low prevalence of 2.5% (5/200) was obtained during the study. Rotavirus was detected in 2 out of 60 brown mussels (3.33%), 2 out of 70 yellow clams (2.86%) and 1 out of 70 black clams (1.43%). Conclusion: Low prevalence of Rotavirus was obtained in our study. A more extensive study need to be conducted to estimate the prevalence of enteric virus in shellfish.

Environmental Transmission of Human Noroviruses in Shellfish Waters

Human noroviruses (NoV) are the most common cause of epidemic gastroenteritis following consumption of bivalve shellfish contaminated with fecal matter. NoV levels can be effectively reduced by some sewage treatment processes such as activated sludge and membrane bioreactors. However, tertiary sewage treatment and substantial sewage dilution are usually required to achieve low concentrations of virus in shellfish. Most outbreaks have been associated with shellfish harvested from waters affected by untreated sewage from, for example, storm overflows or overboard disposal of feces from boats. In coastal waters, NoV can remain in suspension or associate with organic and inorganic matter and be accumulated by shellfish. Shellfish take considerably longer to purge NoV than fecal indicator bacteria when transferred from sewage-polluted estuarine waters to uncontaminated waters. The abundance and distribution of NoV in shellfish waters are influenced by the levels of sewage treatment, proximity of shellfish beds to sewage sources, rainfall, river flows, salinity, and water temperature. Detailed site-specific information on these factors is required to design measures to control the viral risk.

Seasonal and regional prevalence of norovirus, hepatitis A virus, hepatitis E virus, and rotavirus in shellfish harvested from South Korea

Foodborne viruses such as norovirus (NoV), hepatitis A virus (HAV), hepatitis E virus (HEV), and rotavirus (RoV) are transmitted through water and food contaminated with stool. The purpose of this study was to examine the prevalence of foodborne viruses in shellfish collected in South Korea using real-time RT-PCR. Virus was eluted from the stomach and the digestive diverticula of 152 shellfish (51 oysters, 51 Manila clams, and 50 mussels) and concentrated with polyethylene glycol precipitation. The detection rate of NoV genogroup II, NoV genogroup I, HAV, HEV, and RoV was 21.7%, 5.9%, 0.7%, 0%, and 0% of shellfish, respectively. Although the geographic distribution of NoV was statistically significant, prominent seasonal variation in NoV was not observed in this study. In order to reduce norovirus food poisoning in the public, it is important to prevent the contamination of NoV in shellfish in South Korea.

Adenovirus and Norovirus Contaminants in Commercially Distributed Shellfish

Shellfish complying with European Regulations based on quantification of fecal bacterial indicators (FIB) are introduced into markets; however, information on viruses, more stable than FIB, is not available in the literature. To assess the presence of noroviruses (NoVs) GI and GII and human adenoviruses (HAdV) in domestic and imported mussels and clams (n=151) their presence was analyzed during winter seasons (2004-2008) in north-west Spanish markets through a routine surveillance system. All samples tested negative for NoV GI and 13% were positive for NoV GII. The role of HAdV as viral indicator was evaluated in 20 negative and 10 positive NoV GII samples showing an estimated sensitivity and specificity of HAdV to predict the presence of NoV GII of 100 and 74% (cut-off 0.5). The levels of HAdV and NoVs and the efficiency of decontamination in shellfish depuration plants (SDP) were evaluated analyzing pre- and post-depurated mussels collected in May-June 2010 from three different SDP. There were no statistically significant differences in the prevalence and quantification of HAdV between pre- and post-depurated shellfish and between seawater entering and leaving the depuration systems. Moreover, infectious HAdV were detected in depurated mussels. These results confirm previous studies showing that current controls and depuration treatments limiting the number of FIB do not guarantee the absence of viruses in shellfish.

Rotavirus Occurrence in Shellfish with Low Levels of E. coli.

The purpose of this study was to evaluate "in field" the accumulation of virus in shellfish and compare it with the concentration of bacterial indicators. Individuals of Mytilus galloprovincialis were placed in two sampling station located in a contaminated coastal bay and in one control station located one kilometer offshore. The presence of Rotavirus and E. coli was assessed weekly both in seawater and in shellfish samples. The Rotavirus genome was detected in water, preliminarily concentrated by tangential flow ultrafiltration method, and in hepatopancreas of mussels by Real-Time PCR. E. coli was enumerated in water matrices by a filtering method and in mussels by the MPN method. Rotaviruses were not recorded in seawater, while in mussels they were detected since third week after placement. E. coli in mussels were always below the limits set in the Regulation (EC) 854/2004. This study suggests the need for a viral indicator to insure the safety for consumption of shellfish.