Fig 1. Differential neutralization capacity and antibody binding properties of immune sera against ECSA and Asian CHIKV.

(A) Sera collected from ECSA and Asian CHIKV outbreaks have differential neutralizing capacity against MY/08/065 (ECSA) and MY/06/37348 (Asian) isolates of CHIKV. Results are expressed as a percentage of virus control. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA with the Bonferroni multiple comparisons test. Data are presented as means ± SEM from 23 (ECSA) and 40 (Asian) individual serum samples. (B) Neutralization titers (NT₅₀) of DTT-treated sera were determined by non-linear regression fitting. ***P<0.001, Wilcoxon matched-pairs signed rank test. (C) ECSA and Asian sera were cross-screened against both CHIKV isolates (10⁵ pfu, treated with 1% Triton X-100) in ELISA at different serum dilutions. Data are presented as means ± SEM from 23 (ECSA) and 40 (Asian) individual serum samples. **P<0.01, ***P<0.001, two-way ANOVA with the Bonferroni multiple comparisons test. (D) Seroneutralization was performed against different strains of CHIKV, ICRES1 (ECSA) and CAR (Asian), which were rescued from icDNA CHIKV, at a serum dilution of 1:800. **P<0.01, ***P<0.0001, Wilcoxon matched-pairs signed rank test. (E) Immunoblotting was performed under non-reducing and reducing conditions against rE2 and CHIKV from MY/08/065 and MY/06/37348. Mouse anti-His was used as a control and pooled sera were diluted at 1:1000. (F) Immunoblotting was performed under non-reducing and reducing conditions against ICRES1 and CAR. Mouse anti-E2 was used as a control and pooled sera were diluted at 1:1000. (G) Antibody titers of CHIKV immune sera (IgG) were quantified by end-point titer ELISA using whole virus antigen and recombinant E2 (rE2) derived from MY/08/065. Middle line, median; plus sign, mean; upper and lower boundaries of the box, inter-quartile range; whiskers, range of values.