Virus-contaminated oysters: a three-month monitoring of oysters imported to Switzerland

Molluscan shellfish are known to be carriers of viral and bacterial pathogens. The consumption of raw oysters has been repeatedly linked to outbreaks of viral gastroenteritis and hepatitis A. Switzerland imports over 300 tons of oysters per year, 95% of which originate in France. To assess the level of viral contamination, a 3-month monitoring study was conducted. Therefore, the sensitivities of several previously described methods for virus concentration were compared, and one protocol was finally chosen by using dissected digestive tissues. Eighty-seven samples consisting of five oysters each were analyzed for Norwalk-like viruses (NLVs), enteroviruses, and hepatitis A viruses from November 2001 to February 2002. The oysters were exported by 31 French, three Dutch, and two Irish suppliers. Eight oyster samples from six French suppliers were positive for NLVs, and four samples from four French suppliers were positive for enteroviruses; two of the latter samples were positive for both viral agents. No hepatitis A viruses were detected. The sequences of NLV and enterovirus amplicons showed a great variety of strains, especially for the NLVs (strains similar to Bristol, Hawaii, Mexico, and Melksham agent). The data obtained indicated that imported oysters might be a source of NLV infection in Switzerland. However, further studies are needed to determine the quantitative significance of the risk factor within the overall epidemiology of NLVs.     

Detection of human adenoviruses and enteroviruses in Korean oysters using cell culture, integrated cell culture-PCR, and direct PCR

Oysters are known to be carriers of food-born diseases, but research on viruses in Korean oysters is scarce despite its importance for public health. We therefore tested oysters cultivated in Goheung, Seosan, Chungmu, and Tongyeong, for viral contamination using cell culture and integrated cell culture PCR (ICC-PCR) with Buffalo green monkey kidney (BGMK) and human lung epithelial (A549) cells. Additional screens via PCR, amplifying viral nucleic acids extracted from oysters supplemented our analysis. Our methods found 23.6%, 50.9%, and 89.1% of all oysters to be positive for adenoviruses when cell culture, ICC-PCR, and direct PCR, respectively, was used to conduct the screen. The same methodology identified enteroviruses in 5.45%, 30.9%, and 10.9% of all cases. Most of the detected enteroviruses (81.3%) were similar to poliovirus type 1; the remainder resembled coxsackievirus type A1. A homology search with the adenoviral sequences revealed similarities to adenovirus subgenera C (type 2, 5, and 6), D (type 44), and F (enteric type 40 and 41). Adenovirus-positive samples were more abundant in A549 cells (47.3%) than in BGMK cells (18.2%), while the reverse was true for enteroviruses (21.8% vs. 14.5%). Our data demonstrate that Korean oysters are heavily contaminated with enteric viruses, which is readily detectable via ICC-PCR using a combination of A549 and BGMK cells.     

Development of a Simple Method for Concentrating Enteroviruses from Oysters

The development of a simple method for concentrating enteroviruses from oysters is described. In this method viruses in homogenized oyster tissues are efficiently absorbed to oyster solids at pH 5.5 and low salt concentration. After low-speed centrifugation, the supernatant is discarded and viruses are eluted from the sedimented oyster solids by resuspending them in pH 3.5 glycine-buffered saline. The solids are then removed by low-speed centrifugation, and the virus-containing supernatant is filtered through a 0.2-micronm porosity filter to remove bacteria and other small particulates without removing viruses. The virus-containing filtrate is then concentrated to a volume of a few milliliters by ultrafiltration, and the concentrate obtained is inoculated directly into cell cultures for virus assay. When tested with pools of oysters experimentally contaminated with small amounts of different enteroviruses, virus recovery efficiency averaged 63%.     

Polyelectrolyte Flocculation as an Aid to Recovery of Enteroviruses from Oysters

A simple and rapid method for recovering enteroviruses from oysters is described. A polycation sewage flocculant promoted cohesion of oyster solids and thereby aided separation of these from the viruses. Recovery of 80 to 100% of experimentally inoculated virus was achieved, and the suspension or extract obtained could be inoculated directly into tissue cultures or concentrated first for greater sensitivity.     

Determination of enteroviruses, hepatitis A virus, bacteriophages and Escherichia coli in Adriatic Sea mussels

The aim of the present study was to evaluate the incidence of enteric viruses in mussels and to verify the possibility of using phages as indirect indicators of mussel viral contamination. Mussels (36 samples) collected from three different areas of the Adriatic Sea were analysed to determine the following parameters: Escherichia coli, somatic coliphage (T6 phage), F-Plus (MS2 phage), B40-8 (phage of Bacteroides fragilis), enteroviruses and hepatitis A virus. Most of the results of the bacteriological analysis (most probable number (MPN) ml-1) were in accordance with the bacteriological limits established by European law, with the exception of seven samples. The bacteriophage analyses were always negative for F-Plus and B40-8, with the exception of a few samples, whereas the somatic coliphages were generally between 0 and 20 MPN g-1, with the exception of two samples (110 MPN g-1). The virological analysis showed five samples positive for the presence of enteroviruses and 13 for the presence of hepatitis A virus (in three samples both viruses were present). Most of these samples presented acceptable bacteriological parameters and the bacteriophages were absent or their value was generally very low. The results show that the detection of E. coli and phages does not seem to be a good indicator of viral contamination.     

Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)

Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin–magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.     

A Persistent, Productive, and Seasonally Dynamic Vibriophage Population within Pacific Oysters (Crassostrea gigas)

In an effort to understand the relationship between Vibrio and vibriophage populations, abundances of Vibrio spp. and viruses infecting Vibrio parahaemolyticus (VpVs) were monitored for a year in Pacific oysters and water collected from Ladysmith Harbor, British Columbia, Canada. Bacterial abundances were highly seasonal, whereas high titers of VpVs (0.5 × 10⁴ to 11 × 10⁴ viruses cm⁻³) occurred year round in oysters, even when V. parahaemolyticus was undetectable (<3 cells cm⁻³). Viruses were not detected (<10 ml⁻¹) in the water column. Host-range studies demonstrated that 13 VpV strains could infect 62% of the V. parahaemolyticus strains from oysters (91 pairings) and 74% of the strains from sediments (65 pairings) but only 30% of the water-column strains (91 pairings). Ten viruses also infected more than one species among V. alginolyticus, V. natriegens, and V. vulnificus. As winter approached and potential hosts disappeared, the proportion of host strains that the viruses could infect decreased by ~50% and, in the middle of winter, only 14% of the VpV community could be plated on summer host strains. Estimates of virus-induced mortality on V. parahaemolyticus indicated that other host species were required to sustain viral production during winter when the putative host species was undetectable. The present study shows that oysters are likely one of the major sources of viruses infecting V. parahaemolyticus in oysters and in the water column. Furthermore, seasonal shifts in patterns of host range provide strong evidence that the composition of the virus community changes during winter.     

Prevalence and genetic diversity of waterborne pathogenic viruses in surface waters of tropical urban catchments

Aims: To study the virological quality of surface water from highly urbanized tropical water catchment areas and to determine predominant enteric viral genotypes in surface water. Methods and Results: A wide range of human pathogenic viruses in urban surface waters was screened by nested PCR assays after concentration by ultrafiltration. Among the 84 water samples collected, at least one virus was detected in 70 (83·3%) of these samples. Noroviruses were determined to be the most prevalent enteric viruses detected in urban surface water samples, followed by astroviruses, enteroviruses, adenoviruses and hepatitis A viruses. The molecular characterization of environmental viral isolates suggested co‐circulation of multiple genotypes of both noroviruses GI and GII, astroviruses and enteroviruses in urban surface waters. Conclusions: Human enteric viruses with great genetic diversity were detected in surface waters, indicating the presence of human origin of faecal contamination in highly urbanized water catchment areas. Significance and Impact of the Study: The present study identifies and characterizes potential viral hazards of source waters for drinking water supply and recreational activities. This will enable scientific decisions to be made regarding the selection and prioritization of human pathogenic viruses to be included in the future risk assessment and treatment evaluation for water and wastewater.     

Bioaccumulation Efficiency,Tissue Distribution,and Environmental Occurrence of Hepatitis E Virus in Bivalve Shellfish from France

Hepatitis E virus (HEV), an enteric pathogen of both humans and animals, is excreted by infected individuals and is therefore present in wastewaters and coastal waters. As bivalve molluscan shellfish are known to concentrate viral particles during the process of filter feeding, they may accumulate this virus. The bioaccumulation efficiencies of oysters (Crassostrea gigas), flat oysters (Ostrea edulis), mussels (Mytilus edulis), and clams (Ruditapes philippinarum) were compared at different time points during the year. Tissue distribution analysis showed that most of the viruses were concentrated in the digestive tissues of the four species. Mussels and clams were found to be more sensitive to sporadic contamination events, as demonstrated by rapid bioaccumulation in less than 1 h compared to species of oysters. For oysters, concentrations increased during the 24-h bioaccumulation period. Additionally, to evaluate environmental occurrence of HEV in shellfish, an environmental investigation was undertaken at sites potentially impacted by pigs, wild boars, and human waste. Of the 286 samples collected, none were contaminated with hepatitis E virus, despite evidence that this virus is circulating in some French areas. It is possible that the number of hepatitis E viral particles discharged into the environment is too low to detect or that the virus may have a very short period of persistence in pig manure and human waste.     

New tools for the study and direct surveillance of viral pathogens in water

Half a century ago scientists attempted the detection of poliovirus in water. Since then other enteric viruses responsible for gastroenteritis and hepatitis have replaced enteroviruses as the main target for detection. However, most viral outbreaks are restricted to norovirus and hepatitis A virus, making them the main targets in water. The inclusion of virus analysis in regulatory standards for viruses in water samples must overcome several shortcomings such as the technical difficulties and high costs of virus monitoring, the lack of harmonised and standardised assays and the challenge posed by the ever-changing nature of viruses. However, new tools are nowadays available for the study and direct surveillance of viral pathogens in water that may contribute to fulfil these requirements.     

Assessment of adenovirus,hepatitis A virus and rotavirus presence in environmental samples in Florianopolis,South Brazil

Aims: To assess the presence of human adenovirus (HAdV), hepatitis A (HAV) virus and rotavirus A (RV‐A) in environmental samples from the Southern region of Brazil and to provide viral contamination data for further epidemiological studies and governmental actions. Methods and Results: Water samples from various sources (seawater, lagoon brackish water, urban wastewater, drinking water sources‐with and without chlorination and water derived from a polluted creek) and oysters of two growing areas were analysed by enzymatic amplification (nested PCR and RT‐PCR), quantification of HAdV genome (qPCR) and viral viability assay by integrated cell culture‐PCR (ICC‐PCR). From June 2007 to May 2008 in a total of 84 water samples, 54 (64·2%) were positive for HAdV, 16 (19%) for RV‐A and 7 (8·3%) for HAV. Viability assays showed nonpositive samples for HAV; though, infectious viruses were confirmed for RV‐A (12·5%) and HAdV (88·8%). Oyster samples by PCR were positive for HAdV (87·5%) and RV‐A (8·3%), but none for HAV. Quantitative PCR in oysters showed means loads in genomic copies (gc) of 9·1 × 10⁴ gc g^?1 (oyster farm south) and 1·5 × 10⁵ gc g^?1 (oyster farm north) and in waters ranging from 2·16 × 10⁶ (lagoon water) to 1·33 × 10⁷ gc l^?1 (untreated drinking water). Conclusions: This study has shown a widespread distribution of the analysed viruses in this particular region with high loads of HAdV in the environment which suggests the relevance of evaluating these viruses as positive indicators of viral contamination of water. Significance and Impact of the Study: The environmental approach in this study provides data concerning the prevalence, viability and quantification of enteric viruses in environmental waters and oysters in the South region of Brazil and has indicated that their presence might pose a risk to population in contact with the environmental samples searched.