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Title: Development of a diagnostic serological assay for ovine herpesvirus-2
Author: Fraser, Sarah Jane
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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The aim of this project was to develop a serological assay based on recombinant antigens derived from the OvHV-2 genome and validation of the ELISA test using clinical serum samples. Eight OvHV-2 genes were selected for expression in three bacterial systems. Four genes could in at least one system. Three genes were expressed in an in vitro transcription-translation system (Ov8, OV8.5 and orf65). Protein purification, using epitope-tag affinity purification and conventional chromatography, was unsuccessful. Due to the lack of recombinant antigen expression, crude antigen preparations from cultured AIHV-1 and OvHV-2 infected cells were analysed. Antigens suitable for the detection of MCF virus specific serum antibodies could not be extracted from an OvHV-2 infected cell line but AIHV-1 WC11 antigen could be used to differentiate MCF-positive and –negative sera. An ELISA based on this crude antigen was established, designated WC11-ELISA. Initial validation revealed over 93% concordance between WC11-ELISA and commercial ELISA, while a comparison with serological assays, a PCR test and final veterinary evaluations, showed good agreement between tests. The WC11-ELISA showed 57% agreement to PCR while commercial CI-ELISA showed 66% agreement for the same samples. This indicates the value of the ELISA as an economical, rapid and specific serological assay for the detection of antibodies against MCFV. Potentially antigenic components of the ELISA lysates were analysed by western blotting in comparison with other bovine herpesviruses, and fractionation by gel filtration showed clear antigenic bands in the eluent. Proteomic analysis of antigenic bands identified proteins encoding viral thymidine kinase (orf21), a virus capsid protein (orf25) and a viral antigen (orf54) which encodes a dUTPase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available