Due to the lack of recombinant antigen expression, crude antigen preparations from cultured A1HV-1 and OvFlV-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 A1F1V-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.

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 EL1SA test using clinical serum samples. Eight OvHV-2 genes were selected for expression in three bacterial systems. Four genes could be expressed in at least one system, but only a fragment of the Ov8 protein and ORF65 were found to be recognised by antiMCF sera. Additionally Ov2.5, expressed in mammalian cells, was not recognised by anti-MCF sera. 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 so that these polypeptides could not be evaluated as recombinant EL1SA antigens, requiring further work on large scale expression and purification.

Malignant catarrhal fever (MCF) is a sporadic but fatal lymphoproliferative viral disease of cattle, deer and other ruminants. The causative agents are highly-cellassociated herpesviruses of the subfamily gammaherpesvirinae including Alcelaphine herpesvirus-1 (A1HV-1) and Ovine herpesvirus-2 (OvHV-2). The identification of MCF-affected animals is based on the detection of antibodies to A1HV-1 in a variety of assays including indirect-immunofluorescence tests (IFAT) and a competitive-inhibition enzyme-linked immunosorbent assay (CI-ELISA), although histopathological examination remains the most recognised diagnostic procedure. PCR assays are also available for the detection of MCFV DNA.

ction of antibodies against MCFV. Potentially antigenic components of the ELISA lysate 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 (ORF5), ORF52 (homologue to tegument protein HSV UL49) and a viral antigen (ORF54) which encodes a dUTPase.

The simplicity of the WC11-ELISA and its lack of cross-reactivity make this assay a reliable, accessible and low cost tool for routine MCFV diagnosis and epidemiological studies.