Development of an ORF154-DIVA ELISA for serological differentiation of LSDV-infected and vaccinated animals

Mass vaccination to achieve disease-free status requires a serological test to differentiate infected and vaccinated animals (DIVA) towards the end of the control program. The lumpy skin disease (LSD) vaccines currently in use (licensed) are not DIVA-compatible. India recently approved a new live-attenuated LSD vaccine derived from the local LSD virus (LSDV) strain (Ranchi). Unlike field LSDV strains, the Ranchi strain has a distinct 801-nucleotide deletion in its inverted terminal repeat (ITR) region, affecting ORF003/ORF154. In this study, we successfully cloned and expressed LSDV ORF154 into pET28a and purified the His-tagged protein using Ni-NTA affinity chromatography. SDS-PAGE and Western blot analyses confirmed the presence of a ∼28 kDa protein, consistent with the predicted molecular weight. An optimized antigen concentration of 500 ng/well and serum dilution of 1:50, and at a positivity cut-off of 22.63 %, the assay showed high sensitivity (95.77 %) and specificity (95.42 %), effectively distinguishing infected from vaccinated cattle. These findings demonstrate the potential of an ORF154-based ELISA as a reliable serological diagnostic tool for LSDV surveillance and disease control programs, making the Ranchi strain-based LSDV vaccine the first DIVA-compatible vaccine.