FIG 2.

Assessment of NPP3C and 6A1HI broad-spectrum antiviral activity. (A) Huh7.5 cells were infected with ZIKV H/PF/2013 at an MOI of 0.1. Two hours postinfection, cells were treated with DMSO, NITD008 (5 μM), NPP3C (50 μM) or 6A1HI (50 μM). Two days postinfection, supernatants were subjected to plaque assays and intracellular viral replication was assessed by quantification of intracellular viral RNA by RT-qPCR and detection of NS5 viral protein expression by western blotting. ****, P-value ≤ 0.0001; **, P-value ≤ 0.01; *, P-value ≤ 0.05; ns, nonsignificant (n = 3). (B) Huh7.5 cells were infected with ZIKV-R2A (MOI = 0.001), treated at 4 h postinfection with NPP3C or 6A1HI at different concentrations. Two days postinfection, viral replication was measured by measuring luminescence and EC₅₀ values (± SEM) were determined based on three independent experiments. Displayed data are relative to the DMSO treatment. (C) Huh7.5 cells were infected with WNV NY99 at an MOI of 0.1. Two hours postinfection, cells were treated as in A. Two days postinfection, extracellular infectious titers were determined by plaque assays. ***, P-value ≤ 0.001; *, P-value ≤ 0.05 (n = 3). (D) Huh7.5 cells were electroporated with JFH-1-Luc RNA and 2 h posttransfection, cells were treated as in A. Two days later, viral replication was measured by quantifying firefly luciferase activity. Treatment with 1 μM the HCV NS3 protease inhibitor BILN2061 was used as positive control. (E) Huh7.5 cells were infected with SARS-CoV-2 at an MOI of 0.1. Two hours postinfection, cells were treated as in A. Remdesivir (1 μM) was used as positive control. 2 days postinfection, extracellular infectious titers were determined by plaque assays. (F) DBT cells were infected with MHV-A59 (MOI = 0.0001). One hour postinfection, the viral inoculum was removed, and cells were treated as in A. One day postinfection, supernatants were collected to measure infectious viral titers by plaque assays. Results shown in D–F are representative of two independent experiments.