Shuhui Qi ¹ Jing Wang ^2,3 Ting Le ² Chao Sun ² Jitao Chang ² Zhigang Jiang ^2* Xin Yin ^2* Quanhai Pang ^1*

• ¹ College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, China, Taigu, China
• ² Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Harbin, China
• ³ Molecular Biology, Teaching and Research Center, University of Liège, Gembloux, Belgium

Bovine viral diarrhea virus (BVDV), a positive-sense single-stranded RNA virus, causes significant economic losses in the farming industry. Current diagnostic methods for BVDV vary in sensitivity and specificity, underscoring the need for more rapid and accurate detection approaches. Here, we developed a novel competitive ELISA (cELISA) to detect antibodies against the BVDV E2 protein. Three monoclonal antibodies (mAbs)-3E6, 2D5, and 5B9 were generated by immunizing the mice with purified BVDV E2 protein expressed in Expi293F cells. Among these, mAb 3E6 exhibited superior competitive binding abilities to the E2 protein, enabling effective differentiation between BVDV positive and negative sera. Notably, mAb 3E6 demonstrated pan-genotypic recognition of various BVDV strains, including BVDV-1a, -1b, -1c, -1m, -1p, -1v, and -2a, without cross-reactivity to classical swine fever virus (CSFV). Employing AlphaFold 3 for computational modeling revealed domain B of the E2 protein as the primary region for mAb 3E6 binding. Based on these findings, we then established a cELISA using mAb 3E6 and the recombinant E2 protein. Receiver-operating characteristic (ROC) analysis demonstrated excellent diagnostic performance, with a sensitivity of 99.26% and a specificity of 98.99%. Further specificity testing revealed that there were no crossreactivity of mAb 3E6 with antisera from BCoV, BHV-1, BRV, AKAV, LSDV, BLV, and CSFV infected or immunized animals, validating the assay's specificity for detecting BVDV antibodies. Consistency was observed between results from the BVDV E2 cELISA and traditional virus neutralization test (VNT) across antisera from BVDV-1 and BVDV-2 immunized animals, demonstrating high sensitivity for monitoring antibody dynamics. In performance evaluations, the established cELISA showed high concordance with VNT when evaluating 160 vaccinated sera and 190 clinical samples. In summary, the BVDV E2 cELISA, based on the mAb 3E6 targeting domain B of the BVDV E2 protein, presents a reliable and effective serological diagnostic tool for the detection of both BVDV-1 and BVDV-2 antibodies. This method holds significant potential for applications in clinical diagnosis and evaluation of vaccine efficacy.