Viruses In Shellfish Research Articles

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.

Heat Inactivation of Hepatitis A Virus in Shellfish Using Steam.

Shellfish are an important cause of foodborne viral illness. Consumer-friendly cooking recommendations for shellfish could improve food safety and decrease the risk for infection from contaminated products. Thermal inactivation parameters were established for hepatitis A virus (HAV) in mussels and validated with cooking experiments. Steaming for only 2-5min was not sufficient to inactivate HAV in mussels in all layers of a steamer. Steaming mussels for 6min was sufficient to inactivate HAV in all layers. These cooking guidelines produce shellfish with a reduced risk for foodborne virus transmission.

RNA extraction method for the PCR detection of hepatitis A virus in shellfish

Viruses are the leading cause of foodborne illness associated with the consumption of raw or slightly-cooked contaminated shellfish. This study evaluated the E.Z.N.A. Mollusc RNA extraction and purification kit for the detection of HAV in shellfish. The E.Z.N.A. method, based on the cationic detergent, cetyltrimethyl ammonium bromide, in conjunction with a selective RNA binding silica matrix, efficiently isolated viral RNA with a detection limit of 1 TCID 50/ml by hemi-nested PCR. This method proved to be faster and less expensive than PEG precipitation-based procedures. It is also technically undemanding, requiring no extensive processing steps or excess manipulation, minimizing RNA degradation and ensuring the absence of PCR inhibitors. The E.Z.N.A. method applied to HAV screening of shellfish samples from the Apulian region, revealed a high level of contamination. These results confirm that conventional faecal indicators are unreliable for demonstrating the presence or absence of viruses.

Viruses in Shellfish

Viruses in Shellfish

International Standardisation of a Method for Detection of Human Pathogenic Viruses in Molluscan Shellfish

The viruses primarily associated with shellfish-borne illness are norovirus, causing gastroenteritis and hepatitis A virus (HAV). Recent years have seen a proliferation of publications on methods for detection of these viruses in shellfish using polymerase chain reaction (PCR). However, currently no standard harmonised procedures have been published. Standardisation is necessary before virus methods can be considered for adoption within a regulatory framework. A European standardisation working group is developing a two-part (quantitative and qualitative) standard method for virus detection in foodstuffs, including shellfish, which has the potential to be incorporated into EU legislation as a reference method. This article describes the development of the standard method and outlines the key methodology principles adopted, the controls and other quality assurance measures supporting the method and future necessary developments in the area.

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.

Duplex Real Time PCR for the detection of hepatitis A virus in shellfish using Feline Calicivirus as a process control

The consumption of bivalve shellfish is a common cause of foodborne outbreaks of viral origin. The evaluation of the sanitary quality of these products, however, is still based on bacterial indicators of fecal contamination (Reg. (EC) No. 2073/2005 and No.1441/2007) even if it is known that they are not reliable indicators of viral contamination. In this study a duplex Real Time PCR method for quantitative detection of hepatitis A (HAV) in shellfish was developed. Feline Calicivirus (FCV) was used as a control for assessing the effectiveness of the concentration and extraction process and the ability to eliminate PCR inhibitors present in the food matrix. The specific primers and probes for detection of HAV and FCV, chosen respectively from the 5′-UTR region and in the ORF1 region, were labeled with two different dyes and detected simultaneously. The method was applied on spiked and non-spiked shellfish from a local market. The amplification of HAV in the presence of FCV showed good linearity ( R 2 = 0.994) and the sensitivity limit of the reaction was at least 5 × 10 2 TCID 50 g −1 of an hepatopancreas extract.

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.

Evaluation of an extracting method for the detection of Hepatitis A virus in shellfish by SYBR-Green real-time RT-PCR

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In the present study, we evaluated a rapid and simple extraction method to concentrate and purify enteric viruses from shellfish tissues for their detection by real-time RT-PCR. This procedure consists of an alkaline elution with a glycine buffer, solids removal by slow speed centrifugation, purification by chloroform extraction and virus concentration by ultracentrifugation. The efficiency of this method to recover Hepatitis A virus (HAV) from oysters seeded with this virus, was assessed by real-time RT-PCR and conventional RT-nested PCR after extracting viral RNA by a commercial isolation kit. Real-time RT-PCR yielded higher detection sensitivity than the obtained by conventional RT-nested PCR. Besides the improvements in detection sensitivity, the real-time RT-PCR, by quantifying HAV RNA, allowed to check the overall extraction procedure and the recovery efficiency after each processing step. After the last phase, i.e. virus concentration by ultracentrifugation, the RNA purity was high but the estimated HAV recovery efficiency was however low, probably due to virus losses and the presence of RT-PCR inhibitors in sample concentrates. In contrast, the HAV recovery percentage was higher after the virus elution step while the RNA purity was lower. Real-time RT-PCR detection could allow to eliminate some purification and concentration steps that are required for conventional RT-nested PCR detection. The overall procedure for detecting HAV could be then simplify avoiding virus losses during manipulation.

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.

Inactivation of a calicivirus and enterovirus in shellfish by high pressure

Abstract This study demonstrates the potential of high pressure (HP) processing to reduce viral contamination in shellfish. Bovine enterovirus, which is structurally similar to hepatitis A virus, was more pressure-resistant than feline calicivirus, a surrogate for norovirus. Both viruses were more pressure-resistant when treated in “naturally” contaminated mussels and oysters, compared to seawater and culture medium, suggesting that the medium can have a significant protective effect against HP treatment. Treatment at pressures of 250 MPa showed only a limited inactivation of either virus in shellfish, suggesting that relatively mild HP treatments (approximately 260 MPa) currently used for the commercial processing of oysters, principally to assist the shucking process, may be insufficient to ensure the safety of shellfish for human consumption, particularly in relation to human pathogenic viruses. Industrial relevance High pressure (HP) processing is increasingly being used in the commercial processing of oysters, principally to assist the opening or shucking of oysters. Little is known about the effect of HP treatment on pathogens in shellfish, particularly human enteric viruses, which are the predominant cause of shellfish-borne disease. This article demonstrates the inactivation of surrogate animal viruses, as potential models for noroviruses and hepatitis A virus, by HP processing and compares the efficacy of inactivation when viruses are treated in culture medium, seawater and shellfish. The potential of HP processing to reduce viral contamination in shellfish is discussed in light of these findings.

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.

High pressure-induced inactivation of Qβ coliphage and c2 phage in oysters and in culture media

High pressure (HP) treatment inactivates bacteria in shellfish, but its effects on viruses in shellfish have not yet been determined, although viral illness is frequently associated with shellfish consumption. The aim of this study was to investigate the baroresistance of two bacteriophage viruses, Qβ coliphage and c2 phage, in oysters and in culture media. High numbers (≥ 10 7 ml − 1 or g − 1 ) of both phages were obtained in culture media and in oysters. Samples were HP treated at 200–800 MPa at 20 °C for up to 30 min. Little or no inactivation of either phage was observed in oysters or in culture media after treatment at ≤ 400 MPa. High levels of inactivation of both phages in oysters and in culture medium were observed following treatment at 500–700 MPa. Titres of both phages were reduced to non-detectable levels (up to 8 log inactivation) in oysters and in GM17 broth (for c2 phage) after treatment at 800 MPa. The level of Qβ coliphage in tryptone soya broth with yeast extract (10 10 PFU ml − 1 ) was reduced by ∼ 7 log units following treatment of 800 MPa. Levels of inactivation of both phages in oysters were similar to those in culture media. Increasing the duration of treatment at 550 or 600 MPa increased the level of inactivation of both phages in oysters. HP treatment may effectively inactivate phage in shellfish but HP-induced inactivation of human enteric viruses in oysters needs to be studied directly, to more accurately assess the ability of this technology to inactivate these viruses.

Artificial neural network prediction of viruses in shellfish.

A database was probed with artificial neural network (ANN) and multivariate logistic regression (MLR) models to investigate the efficacy of predicting PCR-identified human adenovirus (ADV), Norwalk-like virus (NLV), and enterovirus (EV) presence or absence in shellfish harvested from diverse countries in Europe (Spain, Sweden, Greece, and the United Kingdom). The relative importance of numerical and heuristic input variables to the ANN model for each country and for the combined data was analyzed with a newly defined relative strength effect, which illuminated the importance of bacteriophages as potential viral indicators. The results of this analysis showed that ANN models predicted all types of viral presence and absence in shellfish with better precision than MLR models for a multicountry database. For overall presence/absence classification accuracy, ANN modeling had a performance rate of 95.9%, 98.9%, and 95.7% versus 60.5%, 75.0%, and 64.6% for the MLR for ADV, NLV, and EV, respectively. The selectivity (prediction of viral negatives) was greater than the sensitivity (prediction of viral positives) for both models and with all virus types, with the ANN model performing with greater sensitivity than the MLR. ANN models were able to illuminate site-specific relationships between microbial indicators chosen as model inputs and human virus presence. A validation study on ADV demonstrated that the MLR and ANN models differed in sensitivity and selectivity, with the ANN model correctly identifying ADV presence with greater precision.

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.