Development of a novel antigen for hemagglutination inhibition of Japanese encephalitis virus genotype 5

Japanese encephalitis virus (JEV) is a zoonotic arbovirus transmitted by mosquitoes and poses a significant public health concern. JEV is classified into 5 genotypes (G1–G5). Since 2010, JEV genotype 5 (G5) has been predominant in the Republic of Korea; however, a specific antigen for hemagglutination (HA) inhibition (HI) assays has been lacking. This study aimed to develop a novel antigen to enable genotype-specific HI antibody detection for JEV G5. The Sangju strain of JEV G5 was serially passaged 30 times in Vero and C6/36 cells, resulting in Sangju-30, which exhibited 2 envelope protein mutations and an HA titer of 8, increasing to 512 following polyethylene glycol 8000 concentration. The antigen demonstrated a strong correlation (R = 0.95, p < 0.05, 95% confidence interval, 6.3–31.9) with HI antibody levels in pig sera. These findings suggest that the Sangju-30 antigen is a viable alternative for JEV G5 serological monitoring, enhancing diagnostic accuracy and offering a replacement for traditional murine-derived antigen production.