Abstract: This study focused on developing an antibody-detecting ELISA capable of distinguishing BEFV infected and vaccinated animals. Based on the identified specific reactivity between the truncated BEFV G_NS protein and sera from BEFV infected cattle, the full-length G_NS recombinant protein was expressed in prokaryotic system, purified and used for developing an antibody-detecting ELISA to distinguish BEFV infected and vaccinated animals under the optimized reaction conditions. The G_NS was expressed in Escherichia coli and deposited mainly in inclusion bodies. The purified recombinant protein had a molecular weight of 73 ku. Western blot demonstrated that sera from BEFV infected cattle but not sera from BEF vaccine immunized cattle reacted strongly with the purified G_NS protein. The optimal concentrations of plate-coating antigens and the dilution of sera were 0.50 μg·mL^-1 and 1:20, the HRP conjugated secondary antibody was determined to be 1:4 000, and the S/P ratio=0.206 9 was selected as the negative/positive cut-off value. Acceptable specificity and repeatability were confirmed by no cross reactivity with positive sera of BVDV, FMDV and IBRV as well as coefficient of variation less than 10% in intra-batch and inter-bath. The DIVA (differentiation of infected from vaccinated animals) ELISA was developed and can be used for distinguish BEFV infected and vaccinated cattle.

Key words: bovine ephemeral fever, bovine ephemeral fever virus, BEF inactivated vaccine, G_NS protein, differential diagnostic ELISA