Scientific Opinion on Norovirus (NoV) in oysters: methods, limits and control options : Norovirus in oysters

Background & Aims: Norwalk Virus (NV) is a member of the Caliciviridae family, which causes acute epidemic gastroenteritis in humans of all ages and its cellular receptors have not yet been characterized. Another calicivirus, Rabbit Hemorrhagic Disease Virus, attaches to H type 2 histo-blood group oligosaccharide present on rabbit epithelial cells. Our aim was to test if, by analogy, recombinant NV-like particles (rNV VLPs) use carbohydrates present on human gastroduodenal epithelial cells as ligands. Methods: Attachment of rNV VLPs was tested on tissue sections of the gastroduodenal junction and on saliva from individuals of known ABO, Lewis, and secretor phenotypes. It was also tested on human Caco-2 cells and on animal cell lines transfected with glycosyltransferases complementary DNA (cDNA). Competition experiments were performed with synthetic oligosaccharides and anticarbohydrate antibodies. Internalization was monitored by confocal microscopy. Results: Attachment of rNV VLPs to surface epithelial cells of the gastroduodenal junction as well as to saliva was detected, yet only from secretor donors. It was abolished by α1,2fucosidase treatment, and by competition with the H types 1 and 3 trisaccharides or with anti-H type 1 and anti-H types¾ antibodies. Transfection of CHO and TS/A cells with an α1,2fucosyltransferase cDNA allowed attachment of VLPs. These transfectants as well as differentiated Caco-2 cells expressing H type 1 structures internalized the bound particles. Conclusions: rNV VLPs use H type 1 and/or H types¾ as ligands on gastroduodenal epithelial cells of secretor individuals. GASTROENTEROLOGY 2002;122:1967-1977

A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5' noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness.

The epidemiological data clearly demonstrates that filter feeding bivalve shellfish can, and do, act as efficient vehicles for the transmission of enteric viruses transmitted by the faecal-oral route. This identified hazard has been documented as a cause for concern by various international agencies and has a long history. Disease outbreaks can occur on an epidemic scale as graphically illustrated by an outbreak of Hepatitis A in Shanghai, China in 1988 involving about 300,000 cases. Improvement of harvesting area water quality offers the most sustainable route to improvement in the virological quality of bivalve shellfish sold live. However there is growing awareness, and concern, that current regulatory standards based on faecal coliform monitoring do not fully protect the shellfish consumer from viral infection. New viral test methods based on PCR, and the development of alternative more reliable faecal pollution indicators, offer new approaches for the further development of public health controls. However, further work is required to build a scientific consensus and to understand the implications of their introduction into legislation.