FIG 4.

PF-06409577 inhibits replication of WNV. (A) Lack of virucidal effect of PF-06409577. WNV (∼4 × 10⁸ PFU) was treated with EGCG (5 μg/ml), PF-06409577 (50 or 100 μg/ml), or drug vehicle (control) for 1 h at 37°C in culture medium. Then the infectivity in each sample was determined by plaque assay. EGCG was included in the experiments as a positive-control compound with virucidal activity (n = 2 or 3). (B) PF-06409577 inhibits WNV multiplication when added after virus infection. Vero cells were infected with WNV (MOI of 1 PFU/cell) and treated with PF-06409577 (50 μg/ml) only 1 h before virus inoculation (drug preinfection), 1 h after virus inoculation (drug p.i.), 1 h before virus inoculation, and during infection (drug pre- and p.i.), or left untreated (control). Virus yield in supernatant was determined at 24 h p.i. by plaque assay (n = 3 or 4). (C) Amount of cell-associated viral RNA in cell cultures infected with WNV (MOI of 1 PFU/cell) and treated with PF-06409577 (50 μg/ml) from 1 h p.i. Viral RNA was determined by quantitative RT-PCR at 24 h p.i. (n = 5). (D) Accumulation of dsRNA intermediates in WNV-infected cells. Vero cells were infected with WNV (MOI of 1 PFU/cell) and treated with PF-06409577 (50 μg/ml). Cells were fixed and processed for immunofluorescence using a monoclonal antibody to detect dsRNA at 24 h p.i. Uninfected cells are shown as a control of the specificity of the antibody against dsRNA. Nuclei are shown in blue. Bar, 10 μm. (E) Quantification of dsRNA fluorescence in WNV-infected cells treated or not (control) with PF-06409577 as described in panel D (n = 28 to 32). AU, arbitrary units. (F) Reduction of WNV infection in BHK-21 cells treated with PF-06409577. Cells were infected with WNV (MOI of 1 PFU/cell), and drug was added 1 h after virus inoculation and maintained during the assay. Virus yield in supernatant was determined by plaque assay at 24 h p.i. (n = 3). (G) Evaluation of the cytotoxicity of PF-06409577 on BHK-21 cells by determination of ATP content at 24 h posttreatment (n = 3 or 4). Data are expressed as mean ± SD. Points represent independent biological replicates or individual cells analyzed. Two-tailed Student's t test P values between each condition and control were Bonferroni-corrected in the case of multiple comparisons shown in panels A, B, F, and G. Welch's correction was applied in the case of different variances in panel C. *, P < 0.05; **, P < 0.01; and ***, P < 0.005.