Development of Cell Culture-Derived Hemagglutination Antigens for Japanese Encephalitis Virus Genotypes 1 and 3

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.