| Popis: |
The working hypothesis tested in this thesis that there was no strain variation between louping-ill virus isolates was found wanting. Diversity of antigenic subtypes and biotypes among louping-ill virus isolates was detected and the identified types appeared to be confined to distinct geographical location. A panel of 21 mouose monoclonal antibodies raised against the standard isolate of louping-ill virus (li.31) were selected on the basis of reaction to the virus in indirect immunofluorescence (IIF) tests. The monoclonal antibodies, further characterised on their ability to neutralise virus, inhibit haemagglutination, influence the course of infection in mice and their reactivity in an ELISA, fell into five groups based on their established characterisitics. Five monoclonal antibodies neutralized the virus. Only one antibody also possessed haemagglutination-inhibition (HAI) activity, and two protected mice very significantly against subcutaneous challenge with 2 x 104 plaque-forming units of louping-ill virus, where as for non-neutralizing antibodies enhances the viral pathogenicity as determined by the number and time when mice became affected. A two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed and a combination of 170 monoclonal antibody pairs were evaluated for detection of soluble viral antigens in this system. Competitive assay with seven selected monoclonal antibodies showed that epitopes defined by five antibodies were either close together on the virus particle or over lapped whereas the epitopes defined by the other two antibodies appear to be spatially distant from the other epitopes. The application of monoclonal antibodies in functional assays (neutralization and HAI) as well as in binding assays (IIF and ELISA) detected antigenic variation among 21 louping-ill virus isolates from 4 countries in the British Isles and 3 countries in mainland Europe. According to their reactivity in these tests isolates could be placed in five distinct groups three of which are divisible into two subgroups. Isolates from the same geographical location tended to be of the same group as did isolates from the same location from different species isolated over a 50 year period. Further characterisation of the isolates indicated that additional differences could be detected on the basis of plaque morphology and pathogenicity for mice which correspond to the groupings identified with the monoclonal antibodies. |
