Diagnostic Challenges of West Nile Virus Serology in Japanese Encephalitis-Endemic Regions

Background: In South Korea, where Japanese encephalitis virus (JEV) is endemic, autochthonous human West Nile virus (WNV) infections have not been confirmed.Interpreting WNV serological results is difficult owing to cross-reactive antibodies.This study examined the relationship between WNV enzyme-linked immunosorbent assay (ELISA) reactivity and neutralization in paired serum samples from patients with confirmed JEV.Methods: Thirty-one pairs of acute and convalescent serum samples from laboratoryconfirmed JEV patients from 2021 to 2023 were analyzed.JEV-and WNV-specific IgM and IgG responses were measured by ELISA.Changes in immune status ratio (ISR) values and inter-virus correlations were assessed.Neutralization activity was evaluated using plaque reduction neutralization test (PRNT) to compare temporal changes and titers of JEV and WNV.Concordance between WNV ELISA and PRNT results was analyzed by cross-tabulation, regression, and nonparametric tests.Results: ELISA indices showed significantly higher ISR values in the convalescent sera compared with those in the acute sera.JEV and WNV ISR values positively correlated within the same antibody class.In contrast, PRNT analysis showed that JEV neutralizing titers increased during convalescence across all tested JEV genotypes, whereas WNV log 2 (PRNT 50 ) values remained generally low and increased only modestly from the acute to the convalescent phase.Across all genotypes, JEV log 2 (PRNT 50 ) values consistently exceeded WNV values, with regression slopes < 1, indicating a persistent functional gap between the two viruses.Cross-tabulation of WNV ELISA and PRNT results showed substantial discordance: a subset of ELISA-positive sera lacked detectable WNV neutralization, whereas some ELISA-negative sera showed measurable neutralizing activity.Conclusion: JEV infection induced cross-reactive WNV ELISA responses, raising median WNV ISR from 4.5 to 14.1 for IgM and from 1.2 to 3.3 for IgG.WNV neutralizing activity remained relatively low, with median WNV log 2 (PRNT 50 ) values increasing only from 0.0 to 6.3.The discrepancy between ELISA and PRNT results suggests that WNV ELISA detects cross-binding antibodies but does not reliably indicate neutralization, highlighting the need for confirmatory tests, such as PRNT, in JEV-endemic areas.