FIG 2.

Chikungunya virus is less sensitive to serum complement factors but is readily neutralized by anti-CHIKV antibody in seropositive donor sera. Complement-dependent neutralization of CHIKV and CHPV was assessed by incubating equal amounts of PFU of the virus with MEM or a 2-fold dilution of NHS or HI-NHS for 1 h at 37°C followed by plaque assay. (A) Effect of NHS/HI-NHS on CHIKV. A reduction in the number of plaques can be observed with both the NHS- and HI-NHS-treated samples compared to the CHIKV-alone control. No significant differences were observed within the NHS- and HI-NHS-treated groups at every dilution tested except at the highest serum concentration of 1:5. The data represent the mean ± SEM of all data points obtained from 7 individual donor serum samples. The symbols in the figure indicate significance as follows: #, P ≤ 0.0001; ^, P ≤ 0.01; *, P ≤ 0.05; ns, nonsignificance. (B) Comparison of susceptibility of CHIKV and CHPV to NHS. Chandipura virus was highly sensitive to NHS compared to CHIKV, with neutralization observed in all serum concentrations tested. Heat-inactivated serum (1:10) was not capable of limiting both CHIKV and CHPV. The data are the mean ± SEM of three independent experiments performed in duplicate. ***, P ≤ 0.0001; **, P ≤ 0.01; ns, nonsignificance. (C) Effect of HI sera from both seropositive and seronegative donors on CHIKV. Equal PFU of CHIKV were incubated with HI-sera from seropositive/seronegative donors, and the remaining viral infectivity was determined using a plaque assay. Heat-resistant factors in seropositive donors and not seronegative serum neutralized CHIKV. The data represent the mean ± SEM of 6 independent experiments. ***, P ≤ 0.001. (D) ELISA to detect anti-CHIKV antibodies in seropositive donors. Purified CHIKV adsorbed to ELISA plates was incubated with 2-fold serial dilutions of HI-donor sera, and the bound anti-CHIKV antibody was estimated colorimetrically at 405 nm. Binding was observed only with the serum from the seropositive donor and positive control and not from the seronegative serum. Serum from a seronegative donor (D8) and immune sera from rabbits immunized with UV-inactivated CHIKV (P) served as the negative and positive controls, respectively. The data are the mean ± SEM of 3 independent experiments.