Development and validation of a DIVA ELISA for differentiating BEFV infected from vaccinated animals

Inactivated vaccine is considered safe and used for prevention of bovine ephemeral fever in several endemic countries. To differentiate between BEFV-infected and vaccinated animals, we developed an ELISA capable of detecting infection-related antibodies against BEFV. Recombinant proteins, including N, P, M, L, G_NS, α2, β and γ, were expressed in E. coli and screened by Western blotting and ELISA. The results showed G_NS, α2 and β specifically reacted with sera from BEFV infected cattle but not sera from vaccinated cattle. A DIVA ELISA based on a C-terminal truncated form of G_NS was developed, with 100% sensitivity and 98.0% specificity at a sample to positive-control optical density ratio (S/P) threshold of 0.18. Specificity analysis showed that the assay has no cross-reactivity with antisera of other common bovine viruses. Anti-G_NS antibody appears at 3-4 days post infection (dpi) and persists up to 240-300 dpi in the experimentally infected cattle. Sero-epidemiological survey using sera collected from vaccinated cattle in an endemic area in Jiangsu Province revealed sero-positive rate of 2.36% (6/254), indicating that the DIVA ELISA could be used as a reliable diagnostic tool for differentiating BEFV infected from vaccinated animals.