Viral load and immune responses after reinfection.

(A) Experimental design of the primary infection and reinfection experiment. (B) Serum anti-E2 IgG was assessed by ELISA at 0, 6, and 16 days after reinfection. (C) Splenocytes were isolated at 2, 6, and 16 days after reinfection and stimulated with CHIKV-derived capsid, E2 and E1 peptides for 4.5 h, and the frequencies of IFN-γ- and TNF-α-producing CD8+ T cells were assessed by FACS. (D) Expression of viral positive strand RNA in feet was assessed by qRT–PCR using nsP1-specific primers and probes. Dashed lines in D, average value of PBS-inoculated mice. n = 4–5 per group. All data are presented as the mean ± SD. *: p< 0.05; **: p< 0.01; ***: p< 0.001; statistical relevance was determined by one-way ANOVA.