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Developing Methods for the Isolation and Detection of Viruses in Shellfish, Particularly Noroviruses

Institutions
University Hospital Southampton
Start date
2000
End date
2003
Objective
This research project aims to develop a simple, rapid and inexpensive method to detect Norovirus contamination of shellfish and other foods.

The most common cause of outbreaks of viral gastroenteritis are Noroviruses (NV), also known as Norwalk-like viruses (NLV), small round structured viruses (SRSV) and winter vomiting disease. Foodborne virus outbreaks have been associated with the consumption of raw oysters or lightly cooked shellfish or the contamination of food by infected food handlers.

Electron microscopy remains the only catch-all method of identifying viruses causing gastroenteritis, but it is not sufficiently sensitive to detect viruses in food. Molecular techniques such as Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) are able to detect very low levels of virus particles but these are expensive, require specialised equipment, highly skilled staff and may not be suitable for routine screening of food samples.

The aim of this project is to develop a cheap and simple colorimetric test (ELISA) for the detection of human noroviruses as an alternative approach to electron microscopy. Such an assay could be used in a routine diagnostic food laboratory and would have to be specific and sensitive enough to detect low numbers of viruses found in contaminated shellfish.

More information
Monoclonal antibodies with desired reactivities will be generated against existing Norovirus recombinants (Southampton virus, Lordsdale virus, Babbacombe virus and Jena virus).

The selection of the most appropriate monoclonal antibodies will initially be based on ELISA for detection of caliciviruses in clinical samples. The optimal combination of antibodies will then be applied to detection of caliciviruses in shellfish and other foodstuffs.

Work during the first half of the project will involve two rounds of monoclonal antibody production using recombinant antigens. Production of novel recombinant viral capsid and candidate viral antigens such as VPg will proceed in parallel with antibody production.

The second half of the project will involve further rounds of monoclonal antibody production including immunisation with alternative viral antigens such as the VPg protein. Monoclonal antibodies generated during the first phase of this project will be screened for reactivity to genetic variants detected during routine surveillance of outbreaks due to enteric caliciviruses.

The final stage of the project will involve 'fine tuning' the Enzyme Linked Immunosorbent Assay (ELISA) test to ensure reliability, sensitivity and the ability to discriminate non-pathogenic animal viruses from the human viruses. It will be necessary to adapt the ELISA for facilitating antigen capture from large volumes of tissue homogenates, food and water. One approach would be the use of antibody-coated magnetic particles as a means to concentrate viral antigen from dilute samples. Validation of antigen capture ELISA will be performed using local bivalve molluscs seeded with differing concentrations of non-infectious virus-like particles. The RT-PCR procedure will be compared with traditional viral RNA extraction using oyster tissue seeded with NVs from ELISA positive clinical samples.

The final report, "Developing Methods for the Isolation and Detection of Viruses in Shellfish, Particularly Noroviruses" is available at Foodbase, an open access repository of the FSA.

Find more about this project and other FSA food safety-related projects at the Food Standards Agency Research webpage.

Funding Source
Food Standards Agency
Project number
B04003
Categories
Bacterial Pathogens
Commodities
Seafood