Shellfish contamination by infectious agents is classically monitored based on detection of Escherichia coli
in shellfish tissues (European regulation 91/492/EC) or fecal coliforms in growing waters (United States National Shellfish Sanitation Program). However, shellfish meeting regulation criteria have been implicated in outbreaks, and depuration, efficient at eliminating some bacteria, does not eliminate viruses (7
). The observed differences in clearance of bioaccumulated bacteria and viruses raise questions about potential interactions between oysters and viral human pathogens. This is of particular interest for NoVs, the pathogens most frequently involved in shellfish-borne outbreaks (4
). The finding that NoV-specific ligands exist in shellfish led to the hypothesis that expression of these ligands may influence bioaccumulation and behavior of these viruses in oysters (23
). The ligand for Norwalk virus, the prototype GI strain, was characterized as an A-like carbohydrate structure indistinguishable from human blood group A antigen and whose expression is restricted to the digestive tissues of these animals and shows a clear seasonal variation (23
). For the Houston GII.4 strain, we recently demonstrated that the interaction in digestive tissues involved both a sialic acid in α2,3 linkage and an A-like carbohydrate ligand and that the virus binds to gills and mantle tissue involving the sialic acid-containing ligand exclusively (30
). To evaluate the impact of these ligands on NoV bioaccumulation in oysters, three representative strains of NoV GI and GII were compared in terms of efficiency of bioaccumulation, tissue distribution, and seasonal influence. Selection of the tissues analyzed was based not only on the VLPs' binding ability but also on oyster physiology. For feeding activity, oysters pump water over their gills. Suspended particles are captured and passed on to the alimentary tract, with some sorting of particles occurring prior to ingestion to help regulate what is presented to the digestive tract. The organs involved in the ingestion and digestion of food and the elimination of feces include the mouth, a short esophagus, stomach, a crystalline-style sac, digestive diverticula, midgut, rectum, and anus. (All of these tissues were dissected and called “digestive tissues” [DT] in this study.) With the exception of a short section of the rectum, the entire alimentary canal lies within the visceral mass and is completely immobilized by the surrounding connective tissue (included here in mantle tissues). Food is moved from the mouth toward the anus by the strong ciliary activity from epithelial cells that line the alimentary tract. The digestive gland surrounds the stomach entirely and also part of the intestine. It comprises a series of branched ducts that open into the stomach, and the duct branches serially to terminate in blind-ending tubules, the location of the digestion activity (14
). Based on these physiological parameters, we chose to apply a quantitative approach to three groups of tissues: i.e., gills, digestive tissues, and mantle.
Environmental conditions have an impact on oyster growth, respiration, and nutrient assimilation (8
). As these aspects were not considered here, to avoid as much as possible variability due to environmental conditions and to follow the seasonal cycle of oysters, all experiments were performed with the same batch of oysters kept in a clean area during the 6 months of the study. The seawater was collected from the same area, and the aquarium temperature was adapted to the in situ
measured temperature. The bioaccumulation experiments (the three strains at three concentrations and the control batch) were conducted on the same day for each season, and oysters were dissected at the same time, by different members of the laboratory, to avoid any delay and differences within assays. Similarly, all tissues were then extracted by organs rather than by strains or level of contamination, and rRT-PCRs were performed in a single experiment, including all negative controls and the standard curve. These precautions were important to avoid artifactual experimental variations and to allow safe comparisons of the four experiments.
The first striking observation was that oysters concentrated the three strains with very different efficiencies and tissue distributions. The GI.1 strain was previously shown to bind specifically through an A-like carbohydrate structure to DT but not to other tissues (23
). We observed here that it was readily bioaccumulated in DT, with less than 1% of the virus detected in other tissues after 1 h and a 1,000-fold difference between gills/mantle and DT after 24 h, consistent with the lack of a ligand in gills and mantle. The high concentration of GI.1 recovered in DT is also consistent with earlier observations (1
). The efficiency of this DT-specific bioaccumulation paralleled the season-dependent expression level of the carbohydrate ligand, strongly arguing in favor of its involvement in the bioaccumulation process. Moreover, we previously observed that GI.1 VLPs bioaccumulated in a manner dependent on this carbohydrate recognition since mutant VLPs that had lost the carbohydrate ligand-binding property were less well accumulated (30
). We also previously observed that in the environment, the ratio between genome copies in oysters and in water was much higher (50 times) for GI strains than for GIII strains that have no carbohydrate ligand in oyster tissues (56
). Collectively, the previously reported observations and those presented here represent compelling evidence for a major role of the TD-specific carbohydrate ligand in the highly efficient GI.1 strain bioaccumulation in oysters. In contrast, the GII.4 strain was poorly bioaccumulated, regardless of the month considered, and showed a different distribution within the shellfish body. Even though a preferential accumulation in DT occurred after 24 h, after 1 h, a large percentage of virus was detected in gills and mantle, consistent with the binding to sialic acid in the α2,3 linkage previously detected by ELISA and histochemistry (30
). Bioaccumulation of GII.4 strains in gills or labial palps of Pacific oysters (Crassostrea gigas
) or in gills but not in DT of Crassostrea ariakensis
) was reported, suggesting that the behaviors of various NoV GII.4 strains may be similar in distinct oyster species, even if the results obtained here are to be considered for the GII.4 Houston strain. Over the last 15 years, strains of the GII.4 genotype became predominant across human populations (up to 80 to 90% of clinical cases) and have been responsible for several large outbreaks (3
). Despite this high prevalence in human infections and thus large amounts of GII.4 particles discharged in sewage (46
), strains of this cluster are not predominant in oyster-related outbreaks (11
). Different factors such as viral load in human feces, resistance to sewage treatment, and adsorption to different particles may influence the behavior of viral particles (12
). However, our observation of very poor accumulation by oysters compared to other NoV strains is concordant with this epidemiological observation.
What is the reason for the weak DT-specific bioaccumulation of GII.4? Assays of water in the bioaccumulation tanks at the end of the last two experiments showed negligible numbers of virus genome copies leftover for all three strains compared to the inocula (data not shown), suggesting that the three strains have almost entirely been captured by the oysters. Although the low GII.4 bioaccumulation might be explained by a lower stability compared to that of other strains, as previously suggested (9
), control experiments showed no decrease in genome copy numbers or capsid immune reactivities in seawater during the short period of time considered (24 h). However, it should be noted that detection of RNA by RT-PCR may not correlate with infectivity. Furthermore, the very low bioaccumulation of GII.4 compared to GI.1 was already clearly visible after 1 h. It is therefore unlikely that the inefficient bioaccumulation of the GII.4 strain can be explained by a lower stability in seawater in such short periods. The binding of the GII.4 strain to gills (and the mantle) may prevent the passage of viral particles to the mouth and thus to DT. Even after 1 day and irrespective of the season or concentrations, GII.4 virus persists on the gills (and the mantle) in accordance with the binding with VLPs detected by ELISA (30
). Because we were unable to detect residual virus in the seawater after a 24-h bioaccumulation period (as observed also for the two other strains tested), we hypothesize that binding to gills and mantle through a sialic acid-containing ligand prevents the passage into the digestive tract and is followed by rapid destruction of the virus by unknown mechanisms that need to be further analyzed and identified.
GII.3 NoVs have an ELISA binding pattern to oyster tissues similar to that observed for the GII.4 strain, with VLPs binding to DT, gills, and mantle. However, the bioaccumulation efficiency was much higher than that observed for the GII.4 strain. After 1 h, as observed for the GII.4 strain, NoV GII.3 was detected in gills and mantle but also in DT. After 24 h, gills and mantle tissues displayed concentrations 1,000-fold lower than in DT, suggesting that after being transiently retained in the gills, probably due to binding to sialic acid, they are either destroyed, as observed for the GII.4 strain, or they are released to enter the mouth, as observed for the GI.1 strain. The release from the gills or mantle might occur if the GII.3 strain has a lower binding affinity for the sialic acid-containing ligand, an aspect that will require further investigation.
GI NoVs represent about 30% of NoV-contaminated shellfish (field or market studies) (5
), and this genogroup is also frequently detected in shellfish outbreaks (18
). Similarly, NoV GII.3 strains are frequently reported in shellfish-related outbreaks (11
). These data fit with our observation that even after 1 h, mimicking an accidental contamination, there is already considerable uptake of GI.1 and GII.3 viruses in oysters. The efficiency of oysters at bioaccumulating GI strains such as Norwalk virus, particularly during the winter months (January through March) when oyster consumption is highest in France, may explain why this genogroup of NoVs are so often implicated in shellfish-related outbreaks despite their relatively low frequency of detection in the community. Virus contamination of oysters can occur in the absence of a specific ligand, as observed through a field study with GIII NoV strains (56
). However, it is far less efficient than when a digestive-tissue-specific ligand is present, as in the case of the GI.1 strain.
From a public health perspective, identification of a correlation between ligands in shellfish and bioaccumulation efficiency may help to predict periods of high risk, guide the development of testing protocols that will help to increase the sanitary quality of shellfish put on the market, or even lead to the selection of oyster species that may be less sensitive to NoV contamination.