Development of an optimized E0-based indirect ELISA for serological detection of bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV), a globally prevalent immunosuppressive pathogen, continues to challenge the cattle industry, prompting the search for more effective diagnostic and immunogenic targets. The envelope glycoprotein E2, once the central focus of vaccine and diagnostic research, is now considered suboptimal because of its pronounced sequence variability, which limits cross-subtype efficacy. In contrast, the E0 protein, a more conserved outer membrane glycoprotein, has emerged as a promising alternative owing to its immunogenicity and cross-subtype stability. In this study, the E0 gene of BVDV 1b (XZ02) was engineered by removing its membrane anchor region and incorporating heterologous signal peptides (SPs) to enhance secretion efficiency in an HEK293 suspension cell system. Among the evaluated SPs, the tissue plasminogen activator signal peptide yielded the highest level of secreted E0 protein (up to 2.4 mg/mL after purification). Based on the optimized recombinant E0 antigen, an indirect ELISA was developed to detect BVDV-specific antibodies. The assay demonstrated high sensitivity, specificity, and reproducibility, with a diagnostic agreement rate of 96.53 % (κ = 0.9110), when compared with the commercial IDEXX BVDV Total Antibody kit. These results support the potential of the engineered E0 protein as a reliable, scalable antigen for the serological diagnosis of BVDV.