Vaccination against Newcastle disease (ND) is the most effective means of controlling the disease, and these vaccines are commercialized only after their safety and effectiveness have been verified through tests that comply with Korean Standards of National Lot Release for Veterinary Biologics. This study investigated whether a relatively convenient and safe serological test can be used in place of the challenge test using highly virulent ND virus. Hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA) were considered positive of log2 2 or more and cutoff value of 200 or more, respectively, in both live and inactivated vaccines. However, when the antibody levels of the live and inactivated vaccines induced using the Ulster 2C, KBNP-C4152R2L, and K148/08 strains were compared, the antibody titers for inactivated vaccines were significantly higher than those for live vaccines in both the HI assay and ELISA. A strong positive correlation was observed between HI and ELISA antibody titers. The live vaccines corresponded to a survival rates of ≥ 80% and the inactivated vaccines corresponded to 100% survival rates. This study confirmed that standard efficacy tests can serve as serological tests, and can replace the challenge test and that the vaccine approval process can be improved.

Conventional Japanese encephalitis virus (JEV) hemagglutination inhibition (HI) antigens are contingent on sucking mouse brain (SMB), and present ethical and standardization challenges. In this study, we established an alternative cell culture platform using LFBK cells to produce JEV genotype I (GI) and III (GIII) antigens. Viral replication and hemagglutination (HA) activity were evaluated in six cell lines. Although raw supernatants exhibited limited HA activity, 100-fold polyethylene glycol (PEG) 8000 concentration and binary ethylenimine (BEI) inactivation of LFBK cultures successfully resulted in high-titer antigens for the K95 (GI, 256 HAU), K87 (GIII, 64 HAU), and Nakayama (GIII, 1024 HAU) strains. Diagnostic validation with swine sera showed 100% concordance between the cell-derived Nakayama antigen and commercial SMB standard. Notably, cell-concentrated GI antigens (K95 and K87) demonstrated significantly higher sensitivity (p < 0.05) for several positive samples. These results indicate that the LFBK cell platform can effectively replace traditional SMB methods and provide an ethically sustainable and highly sensitive tool for genotype-specific JEV serosurveillance.