Figure 2

In vitro characterization of influenza vRNA transcription/replication inhibitor candidates.

(A) Cytotoxicity of the identified compounds. 293 cells were cultured with the indicated compound (10 μM each: FP, favipiravir) or DMSO (Ctrl) for 48 h. Cell viability was measured by using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Error bars indicate standard deviations of triplicate experiments. (B) Effects of identified compounds on influenza virus transcription/replication in a minigenome assay. 293 cells were transfected with plasmids for the expression of a virus-like RNA encoding the firefly luciferase gene together with expression plasmids for PB2, PB1, PA, NP and Renilla luciferase, which served as an internal control. Simultaneously, the indicated compound (10 μM each: FP, favipiravir) or DMSO (Ctrl) was added to the cells. Twenty-four hours later, luciferase activity was measured by use of the Dual-Luciferase® Reporter Assay System (Promega). Error bars indicate standard deviations of triplicate experiments. Statistical significance was assessed by use of the Student's t-test: *, P < 0.05. (C) Effects of identified compounds on influenza virus replication. 293 cells were infected with A/WSN/33 (H1N1) or A/California/04/09 (H1N1) at a multiplicity of infection of 0.001. One hour later, the indicated compound (10 μM each: FP, favipiravir) or DMSO (Ctrl) was added to the cells. Virus titers in the culture supernatants at 48 h post-infection were determined by plaque assays in MDCK cells. Error bars indicate standard deviations of triplicate experiments. Statistical significance was assessed by use of the Student's t-test: *, P < 0.05.