FIG 6.

A recombinant MR766 ZIKV with an S368R point mutation escapes neutralization by AC10 in vitro. Serial passaging of MAb AC10 led to the identification of critical residue S368, which lies in the lateral ridge epitope of domain III of the ZIKV envelope protein. Site-directed mutagenesis was performed to generate recombinant MR766 ZIKV with the S368R point mutation. (A) Rescue titer after 72 h posttransfection demonstrates a reduction in viral titer. The dotted line represents the limit of detection. (B) Multicycle growth curves were performed to compare viral fitness between wild-type (WT) and S368R viruses. Supernatants were collected at the indicated time points, and titers were determined by plaque assays. Data points represent the means for two biological replicates, and error bars represent standard errors of the means (SEM). (C) To confirm that the S368R mutation is sufficient for escape from neutralization by MAb AC10, a plaque reduction neutralization test was performed with equivalent amounts of wild-type or S368R MR766 ZIKV. The assay was performed in duplicate, and error bars represent SEM. (D and E) Antibodies GA3 and AA1, which induce escape mutations in different sites, were tested by the same plaque reduction neutralization test. The assay was performed in duplicate, and error bars represent SEM. *, P < 0.05.