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Developing methods for the isolation and detection of viruses in shellfish, particularly SRSVs
Project Code: B04003
Molecular Microbiology and Infection, Southampton General Hospital
Lambden, P ; Clarke, I
Nucleic acid amplification tests, especially quantitative RT – PCR, that are used to detect very low levels of contamination by viruses are expensive and require both high tech equipment and highly skilled staff. Such tests are only appropriate in research centres and are not for routine screening of food samples. Other cheaper approaches such as looking for surrogate markers of sewage contamination e.g. enteric bacteria and phages are also inappropriate especially in the case of shellfish as the tests do not discriminate between human and animal contamination. This is important because shellfish are filter feeders and may well concentrate animal enteric viruses that are of no threat to humans. The Food Standards Agency funded this project with the objective to develop a cheap and simple colourimetric test (enzyme linked assay) for the detection of human noroviruses. Such an assay could be used in a routine diagnostic food laboratory and would have to be specific and sensitive enough to detect the low numbers of viruses found in contaminated shellfish. Prior to this study there were no such methods for the diagnosis of norovirus contamination in shellfish.
The approach in this project was firstly to produce the antigens for immunisation and then to prepare monoclonal antibodies for development of the diagnostic tests. The project contained seven key objectives:
1. Expression of viral antigens
In the absence of a cell culture system for the noroviruses the only way to produce virus capsid antigen (at the outset of this project) was by expression of the protein in insect cells using recombinant baculoviruses. This was the first key objective and was achieved for genogroup 1 and genogroup 2 human noroviruses. The capsid antigen from some of the strains selected assembled to form virus – like particles (VLPs).
Later in the project we discovered how to obtain expression of the capsid protein in E.coli using GST fusion vectors. This proved necessary to provide an alternative source of capsid antigen for screening hybridomas. In addition, we expressed the viral VPg protein in E.coli using pRSET vectors. We also expressed the capsid protein of a sapovirus in insect cells but this did not assemble to form VLPs and therefore was not used for antibody production.
2. Antibody production
Mice were immunised using a collection of recombinant antigens (VLPs) described above. Fusion of spleen cells with mouse myeloma cells generated hybridomas which were screened for monoclonal antibody production using recombinant GST fusion proteins expressing norovirus capsid antigens. The purpose of this approach was to generate broadly cross – reacting monoclonal antibodies that could be used in a ‘catch – all’ ELISA. A series of hybridomas were selected that produced monoclonal antibodies specific for noroviruses.
3. Outbreak surveillance
Throughout the project clinical samples from outbreaks were screened by ELISA and RT-PCR. ELISA negative samples that were RT-PCR positive were ‘grouped’, either genogroup 1 or genogroup 2 by sequence analysis of PCR amplicons. Lordsdale virus (genogroup 2) was the most prevalent strain throughout the duration of the project.
4.Collection of clinical samples
A collection of stool samples representing the major genetic variants has been assembled from the outbreak monitoring during the study. Monoclonal antibodies have been tested for their ability to react with or discriminate isolates in the clinical samples.
5. Development of antigen trap ELISA
A method for the detection of noroviruses by ELISA has been successfully developed using polyclonal antiserum antigen trap in combination with monoclonal detector antibodies. The ELISA was optimised using our panel of clinical samples collected during the project.
6. Development of an enhanced ELISA
The sensitivity of the standard ELISA that uses enzyme reactions to allow development of colourimetric indicators has been increased 10 to 50 fold by the use of europium conjugates for DELFIA assay.
7. Application of ELISA/DELFIA to shellfish
The shellfish tissue (hepatopancreas) was dissected out and homogenised. Tissue preparation was optimised (in consultation with CEFAS, Weymouth) and analysed for the presence of noroviruses by RT-PCR, ELISA and DELFIA. Samples were seeded with noroviruses across a series of dilutions and the DELFIA assay was capable of detecting noroviruses at a level expected in natural contamination of shellfish. Shellfish samples were screened by DELFIA and some were positive for noroviruses, these results were confirmed by RT-PCR.
Key findings and technical evaluation
The project has given 4 conclusions:
- Monitoring of clinical samples indicates that the predominant ‘strain’ of norovirus circulating in the community is the genogroup 2 ‘Lordsdale virus’ (LV).
- ELISAs have been developed that detect LV and a number of other noroviruses.
- Use of the DELFIA/ELISA gives an assay that is sensitive enough to apply to shellfish samples for the detection of norovirus contamination.
- More research investment is required to develop cross reacting monoclonal antibodies capable of detecting a range of noroviruses.
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