Enteric Viruses In Shellfish Research Articles

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.

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.

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.

Prevalence of Human Enteric Viruses and a Potential Indicator of Contamination in Shellfish in China

AbstractThe objectives of this study were to quantify enteric viruses in shellfish along the Chinese coast and find an indicator of viral contamination in shellfish. One hundred sixty‐two samples were collected in August from 10 cities. Hepatitis A virus (HAV), norovirus (NV), rotavirus (RV), poliovirus (PV), astrovirus (AsV) and adenovirus (AdV) were detected by polymerase chain reaction (PCR) and confirmed by sequencing. The percentage of positive samples for each virus was as follows: HAV 5%, NV 12%, RV 7%, PV 15%, AsV 6% and AdV 9%. The six species of shellfish were contaminated with different viruses. PV showed a significant correlation with other viruses, with the exception of RV (P < 0.05). Contamination of shellfish with human enteric viruses was common across a large geographical region of China, indicating a potential public health threat from seafood. The data confirm the potential to monitor the food safety of shellfish by using PV as an index of enteric virus contamination.Practical ApplicationsEnteric viruses are one of foodborne pathogens transmitted by bivalve shellfish, which pose a severe potential risk for consumers, as well as a serious problem facing researchers. China leads the worldwide production of shellfish. Therefore, the safety of shellfish harvested in China is particularly important. Norovirus, hepatitis A virus, rotavirus, adenovirus, poliovirus and astrovirus are introduced to assess the seafood safety by documenting risks for transmission of enteric viruses in shellfish along the Chinese coast. Of which, poliovirus could be one of these potential indicator to present human enteric virus contamination in the countries where oral polio vaccines are used.

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.

Processing Strategies to Inactivate Enteric Viruses in Shellfish

Noroviruses, hepatitis A and E viruses, sapovirus, astrovirus, rotavirus, Aichi virus, enteric adenoviruses, poliovirus, and other enteroviruses enter shellfish through contaminated seawater or by contamination during handling and processing, resulting in outbreaks ranging from isolated to epidemic. Processing and disinfection methods include shellfish depuration and relaying, cooking and heat pasteurization, freezing, irradiation, and high pressure processing. All the methods can improve shellfish safety; however, from a commercial standpoint, none of the methods can guarantee total virus inactivation without impacting the organoleptic qualities of the shellfish. Noroviruses cause the majority of foodborne viral illnesses, yet there is conflicting information on their susceptibility to inactivation by processing. The inability to propagate and quantitatively enumerate some viral pathogens in vitro or in animal models has led to the use of norovirus surrogates, such as feline calicivirus and murine norovirus. During processing, these surrogates may not mimic the inactivation of the viruses they represent and are, therefore, of limited value. Likewise, reverse transcription-PCR has limited usefulness in monitoring processing effectiveness due to its inability to identify infectious from inactivated viruses. This article (a) describes mechanisms of virus uptake and persistence in shellfish, (b) reviews the state-of-the-art in food processing strategies for the inactivation of enteric viruses in shellfish, (c) suggests the use of combined processing procedures to enhance shellfish safety, (d) highlights limitations in research data derived from virus surrogate studies and molecular assay procedures, and (e) recommends enhanced funding for human volunteer studies and the development of assays to detect viable viruses.

Detection of human enteric viruses in shellfish, vegetables, waters and environmental samples: a preliminary study

Detection of human enteric viruses in shellfish, vegetables, waters and environmental samples: a preliminary study

Use of FRNA bacteriophages to indicate the risk of norovirus contamination in Irish oysters.

Male-specific (F) RNA bacteriophages have been proposed as indicators for human enteric viruses in shellfish. This study compared the use of Escherichia coli and FRNA bacteriophages to indicate the presence and level of noroviruses in Crassostrea gigas. A total of 167 samples from category A and B shellfish harvesting areas were analyzed for E. coli and FRNA bacteriophages by standard methods and for noroviruses (NoV) by using a previously described real-time PCR assay. FRNA bacteriophage and NoV levels in shellfish showed a seasonal trend and were elevated during the winter period (October through March). Conversely, E. coli levels did not reflect this seasonal trend. Categorizing samples on the basis of E. coli levels according to European Union regulatory limits failed to indicate the occurrence or level of NoV in shellfish. However, by grouping shellfish samples on the basis of FRNA bacteriophage levels a clear correlation was observed with NoV levels. The use of FRNA bacteriophages to predict the occurrence of NoV in shellfish could provide improved public health protection and should be considered when developing risk management procedures for shellfisheries.

Occurrence of enteric viruses in shellfish and relation to climatic-environmental factors

To investigate the presence of enteric viruses [hepatitis A (HAV) and norovirus (NoV)] in shellfish harvested from the deltaic area of the Po river in relation to environmental factors. Fortnightly sampling of shellfish was carried out in two lagoon areas (category B production areas) and one sea area (category A). Environmental parameters in the lagoon and hydrometric level of the tributary river were monitored throughout the sampling period. Samples (n = 120) were analysed for bacterial (E. coli and Salmonella) and viral (HAV and NoV) contamination; samples from category B areas were analysed before and after purification treatment. All the samples were negative for HAV whereas 10 samples (8.3%), all harvested in the lagoon areas, were positive for NoV. Sequencing identified the strains as genotypes II.4 and II.b. None of the samples was found to be contaminated after depuration. The monitoring showed a low frequency of NoV presence; viral contamination, detected exclusively in shellfish collected from the deltaic area (category B), could be influenced by the flow of the tributary river. The data collected are useful for the design of targeted prevention strategies and for the modulation of control plans after meteorological events.

Development of a method for concentrating and detecting rotavirus in oysters

Identification of enteric viruses in outbreak-implicated bivalve shellfish is difficult because of low levels of contamination and natural inhibitors present in shellfish tissue. In this study, the acid adsorption–alkaline elution method developed in our laboratory was proposed for the detection of rotavirus from oyster samples. The acid adsorption–alkaline elution process included the following steps: acid adsorption at pH 4.8, elution with 2.9% tryptose phosphate broth containing 6% glycine, pH 9.0, two polyethylene glycol precipitations, chloroform extraction and reconcentration using speedVac centrifugation. Oyster concentrates were extracted for RNA and examined for rotavirus using reverse transcription-nested polymerase chain reaction (RT-nested PCR). A comparison of SuperScript™ One-Step RT-PCR system and RT followed by PCR before the nested PCR reaction showed the former detecting four-fold lower concentration of rotavirus (78.12 plaque forming units [PFU]/ml or 0.26 PFU/assay) than the latter (3.12 × 10 2 PFU/ml or 1.04 PFU/assay). In the seeding experiment, the developed acid adsorption–alkaline elution gave high sensitivity of rotavirus detection (125 PFU/g of oyster). From August 2005 to February 2006, 120 oyster samples ( Crassostrea belcheri) were collected from local markets and oyster farms, concentrated, and tested for naturally occurring rotaviruses. Four oyster samples were group A rotavirus-positive. Based on phylogenetic analysis of rotavirus DNA sequences in those positive samples, the oyster samples contained the sequences associated with human rotavirus G9 (two samples), G3 (one sample), and G1 (one sample). The present study demonstrates the successful application of developed virus concentration method and RT-nested PCR for the detection of rotaviruses in naturally contaminated oyster samples. The method might be used as a tool for evaluating the presence of enteric viruses in shellfish for monitoring and control of public health.

Assessment of human enteric viruses in shellfish from the northern Adriatic sea

Incidence and circulation of different strains of hepatitis A and Norovirus in shellfish were studied on 235 samples ( Tapes philippinarum, Mytilus galloprovincialis, Ostrea spp. and Chlamys spp.) obtained from different sites, representing the shellfish production areas of the northern Adriatic sea. Shellfish were harvested in the period of one year and, after depuration, were examined for bacterial ( Escherichia coli and Salmonella) and viral (HAV and NoV) contamination. Viral contamination was present on average in 22% of samples: specifically, 6% of samples tested positive for HAV, 14% for NoV and 2% for both viruses. None of the samples revealed the presence of Salmonella, and in most of them (93%) the number of E. coli was below the European legislation limit of 230 MPN/100 g. T. philippinarum was the species most often contaminated, as well as being the only species in which the legal limit for E. coli was, in some cases, exceeded. Both HAV and NoV contamination were detected throughout the year; NoV detection was slightly more frequent during winter months, but positive samples were also present in summer. The sequencing of the PCR products showed the circulation of only one HAV genotype (IA) and four different NoV genotypes (Hawaii, Melksham, Lordsdale and GGIIb) with a prevalence of the GGIIb genotype in the second period of the monitoring.

Detection of human enteric viruses in shellfish collected in Tunisia

The aim of this study was to detect the main pathogenic human RNA enteric viruses able to persist in the environment such as astrovirus, enterovirus, norovirus and hepatitis A virus (HAV) in shellfish collected from two locations in northern Tunisia. Viruses were eluted from digestive tissues and concentrated by polyethylene glycol precipitation before nucleic acid extraction and purification. After checking for inhibitors, all viruses were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by hybridization. Overall, 83% of the samples were found positive for at least one virus. Astrovirus was detected in 61% of the samples, norovirus in 35% and HAV in 26%. Surprisingly, only one sample was found positive for enterovirus. The mean number of positive samples found in this study is in accordance with the data found in the literature, indicating that no real difference exists in this respect among countries studied. A notable exception is HAV, which reflects the epidemiological status of the population. This study highlights the interest to analyse shellfish samples from different production areas. These data will be helpful to understand virus circulation and to improve shellfish safety. The results, which confirm contamination, necessitate the development of appropriate studies and monitoring in all shellfish-producing countries.

Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage

Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.

Detection of enteric viruses in shellfish from the Norwegian coast.

Common blue mussels (Mytilus edulis), horse mussels (Modiolus modiolus), and flat oysters (Ostrea edulis) obtained from various harvesting and commercial production sites along the Norwegian coast were screened for the presence of norovirus by a real-time reverse transcription (RT)-nested PCR assay and for possible indicators of fecal contamination, i.e., for F-specific RNA bacteriophages (F-RNA phages) by plaque assay and for human adenoviruses and human circoviruses by nested PCR assay. The aims were to obtain relevant information for assessing the risk of transmission of enteric viruses by shellfish and to investigate the potential of various indicator viruses in routine screening. Noroviruses were detected in 6.8% of the samples, and the indicators were detected in 23.8% (F-RNA phages), 18.6% (adenoviruses), and 8.0% (circoviruses) of the samples. A seasonal variation was observed, with the exception of circoviruses, with more positive samples in the winter. A positive correlation was found between F-RNA phages and noroviruses. However, F-RNA phages were present in only 43% of the norovirus-positive samples. The results show that mussels from the Norwegian coast can constitute a risk of infection with enteric viruses and that routine testing of samples may be justified. Advantages and disadvantages of various options for screening are discussed.

Detection and Occurrence of Enteric Viruses in Shellfish: A Review

During feeding, bivalve mollusks (oysters, mussels and clams) can accumulate pathogenic human enteric viruses when present in sewage-polluted seawater. It has been well established that infectious hepatitis virus is transmitted by consumption of raw or inadequately cooked shellfish. But because of the lack of epidemiologic techniques, transmission of other enteric viruses by shellfish has not been established. Other enteric viruses, such as polio, echo, coxsackie and reo, have been detected in shellfish. Enteroviruses have been detected in shellfish taken from both “open” and “closed” areas, based on bacteriological standards used at present in the United States. Field and laboratory studies have indicated that enteric viruses can survive for long periods in seawater and in shellfish. Recent advances in methodology have led to development of more rapid and less expensive methods for detection of a greater number of enteric viruses in shellfish.

Coliphages as indicators of enteric viruses in shellfish and shellfish raising estuarine waters

The practicality of a coliphage indicator system for human enterovirus in polluted waters was studied. Parallel examinations of sewage effluents, shellfish and shellfish growing waters for coliphage and enteric virus indicated a wide dissemination of coliphage throughout the estuary, generally occurring in the absence of detectable enteric virus activity. A majority of the enteric virus isolations were observed in samples yielding no coliphage activity. Under controlled conditions, oysters were observed to accumulate more coliphage than enteric virus. Replication of coliphage in the estuary during the summer months was shown to occur when proper host cell was present. Two major coliphage types were observed in field samples based on their reactivity with different Escherichia coli strains. A shift in dominant coliphage type was seen to occur during the study. Survival times of coliphage and enteric virus in estuary waters along with retention values in oysters were shown to be similar with a slight advantage shown by coliphage. Inability to correlate accurately coliphage and enteric virus occurrence in field samples along with the potential for the presence of more than one dominant coliphage type indicated the serious shortcomings of the coliphage indicator system as a method of enteric virus detection, leaving its overall use in the polluted environment subject to grave doubt.