Cross-Reactivity of 15 JEV Antigen-Directed Rabbit Antisera with Three Clinically Important JEV Serogroup Members:...

Japanese encephalitis virus (JEV), the prototype member of the JEV serogroup within the genus Orthoflavivirus (family Flaviviridae), is closely related to West Nile virus (WNV), Murray Valley encephalitis virus (MVEV), and St. Louis encephalitis virus (SLEV). To characterize antigenic cross-reactivity within this group, we evaluated 15 region-specific rabbit antisera, previously generated against nearly the entire JEV protein-coding region, by immunoblotting whole-cell lysates from BHK-21 cells infected with WNV, MVEV, or SLEV, with JEV included as a reference. Six antisera (α-E^N-term, α-NS2B, α-NS3^N-term, α-NS3^C-term, α-NS5^N-term, and α-NS5^C-term) robustly recognized homologous proteins across all three viruses. The remaining nine antisera displayed lineage-restricted or virus-specific reactivity: (a) α-C cross-reacted strongly with WNV and MVEV but weakly with SLEV; (b) α-M, α-NS1, α-NS4A, and α-NS4B^C-term cross-reacted with WNV and MVEV but not SLEV; (c) α-E^C-term and α-NS1' cross-reacted only with WNV; (d) α-Pr cross-reacted exclusively with MVEV; and (e) α-NS4B^N-term showed no detectable cross-reactivity under the experimental conditions used. Notably, α-NS1 and α-NS1' detected heat-labile multimers of NS1 and NS1'. These serological patterns mirror the established phylogeny of the JEV serogroup, with JEV clustering most closely with MVEV, followed by WNV and then SLEV. Together, these findings provide a comprehensive cross-reactivity map of JEV antigen-directed antisera and establish a practical framework for dissecting antigenic relationships among JEV serogroup members. These results enhance our understanding of orthoflavivirus antigenic evolution and support the future development of improved diagnostics, broad-acting vaccines, and experimental reagents for emerging and re-emerging encephalitic orthoflaviviruses.