The role of bivalve mollusks in the transmission of Noroviruses is not fully understood. The nature of accumulation of NoVs in these animals was thought in the past to be passive. However, considering that these viruses can persist for a long time in shellfish, an active mechanism of virus concentration was suggested (Le Guyader et al., 2006). Recent studies permitted to elucidate the mechanism of transmission of these viruses by shellfish bivalve mollusks.
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It has been hypothesized that these animals, while feeding, can concentrate viruses by mechanisms such as mechanical entrapment, direct chemical bonding, Van der Waals bonding, H+ ion bonding and other ionic bondings (Tian et al., 2007).
A number of environmental and biological factors can influence NoV binding to shellfish tissues. For example, NoV accumulation in oysters may depend on factors such as water temperature, mucus production, glycogen content, or gonadal development (Le Guyader et al., 2006b).
It is well documented that noroviruses can bind to human gastrointestinal cells through involvement of histo-blood group carbohydrates such as human ABH and Lewis carbohydrates (Tian et al., 2007; Le Guyader et al., 2006a). Therefore, Le Guyader et al. (2006a) examined the possibility of a similar binding to oyster tissues of Norwalk virus and recombinant VLPs. The authors analyzed accumulation of NoV genogroup I in Pacific oysters after 12 and 24 hours. Both VLPs and native virions bound to oyster digestive tissues, namely to the midgut, main and secondary ducts of the digestive diverticula and to tubules. No binding to connective tissue was observed.
Using immunohistochemistry, authors determined that this attachment to oyster digestive tissues was carbohydrate-dependent like in the case of human epithelial cells. This was confirmed by testing the ability of saliva of different ABO and secretor phenotypes to block the binding of VLPs to shellfish tissues. Type A saliva secretor completely blocked binding of VLPs, type O saliva secretor strongly reduced binding, while type B or nonsecretor saliva did not block binding of VLPs to shellfish tissues. Authors suggested that attachment of VLPs to oyster tissue involved carbohydrate binding sites overlapping those that attach to human digestive cells, in the viral capsid P2 domain (Le Guyader et al., 2006a).
Genogroup I and II strains of norovirus show various binding patterns with different carbohydrate structures of the histo-blood group family, suggesting the coevolution of these viruses with their host, or carrier vector. Since Norwalk virus binds to oyster tissues using the same binding site as in the case of human cells, this could mean that a coevolution mechanism occurred and viruses adapted to oysters, their intermediate hosts, in order to reach humans, their definitive hosts (Le Guyader et al., 2006a).
However, specific binding of noroviruses to also other bivalve mollusk species could occur. In fact, Tian et al. (2007) demonstrated that, similarly as in the case of oysters, also mussels and clams contain type A-like HBGAs, although in case of this species binding of MAbs to type A HBGAs was significantly lower compared to species such as Pacific or American oysters. Authors demonstrated that manila clams contain also type O-like HBGAs, like oysters, which are absent in mussels (Tian et al., 2007).
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Comelli et al. (2008) analyzed the binding affinity of NoV GI.3b and GII.4 genotypes to blue mussel digestive tissue by performing a bioaccumulation experiment. Authors could not detect the GI.3b strain with the tested methods, and they suggested that this NoV strain cannot be efficiently bioaccumulated in mussels, considering also that several other authors could detect several NoV genotypes belonging to both NoV genogroups I and II in mussels, but no one detected GI.3b strain (Comelli et al., 2008).
Maalouf et al. (2011) analyzed 1 hour and 24 hours bioaccumulation of three different NoV genotypes (GI.1, GII.3 and GII.4) in oysters, during the cold period of the year (October, November, January and March). Authors used three different virus concentrations in the experiment and digestive tissue, gills and mantle was analyzed.
NoV GI.1 was efficiently bioaccumulated at all three doses, and the bioaccumulation was dose- dependent, hence it showed the highest quantities of virus in shellfish digestive tissues for the highest virus concentration in the tank. Increase in bioaccumulation was observed during the month of January compared to October and November experiments. In January, after 1 hour of bioaccumulation as much as 41% of seeded virus in the water was found in the digestive tissues, compared to only 1% found during other months. After 24 hours, oysters accumulated 88% of seeded virus, compared to 1,2 – 27% in other months. Gills and mantle presented about 100 times inferior concentrations which were stable between 1 hour and 24 hours test.
NoV GII.3 was also efficiently bioaccumulated in oysters, although definitely worse compared to genogroup I strain. Unlike in the case of NoV GI.1, no significant variation in bioaccumulation was observed during different months. After 1 hour, only up to 0,5% of virus inoculum was accumulated in oyster digestive tissues, and after 24 hours up to 4% of virus could be detected.
NoV GII.4 showed very poor bioaccumulation in oysters. Even at higher doses, less than 0,01% of the seeded virus was concentrated in digestive tissues, and, unlike in the case of NoV GI.1 genotype, bioaccumulation was not dose-dependent, and the poorest results were observed in January. Unlike for the two other strains which were concentrated more efficiently in digestive tissue, NoV GII.4 was accumulated similarly in digestive tissue, gills and mantle.
Maalouf et al. (2010) analyzed the tissue distribution and seasonal variation of oyster ligands specific to norovirus GI.1 and GII.4 strains through a developed ELISA assay, immunohistochemistry and bioaccumulation experiments. Binding of VLPs to digestive tissues, gills and mantle was examined. ELISA results confirmed that NoV GI.1 VLPs bind strongly to digestive tissues, but not to gills and mantle, whereas NoV GII.4 bound strongly not only to digestive tissues, but also to gills and mantle.
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Bioaccumulation experiments with VLPs confirmed that GI.1 VLPs bound very efficiently only to oyster digestive tissues. NoV GII.4 VLPs were not found in oysters, even if seawater was seeded with very high concentrations, because they lost their structural integrality when got in contact with seawater.
Seasonal variations in binding to oyster tissues were confirmed for NoV GI.1 VLPs, with an increased binding during winter and spring months (from January to May) and lower binding from June to December.
Contrarily, variations encountered for GII.4 VLP binding to oysters were far less evident compared to GI.1 VLPs, although still the binding activity was higher during winter months.
McLeod et al. (2009) analyzed the distribution of norovirus in Pacific oysters after 48 hours of bioaccumulation with GII.4 strain. Authors detected the virus in digestive tract and also in gills and labial palps, albeit in minor concentrations.