Fig. 2. The cellular toxicity and antiviral activity of compounds 9 and 12 against PRRSV GD-XH in MARC-145 cell cultures. (A) Cytotoxicity of 9 and 12 was examined in MARC-145 cells using MTT assay at 48 h post compound treatment and results are shown as relative cell viability of the viable cells in the absence of the compound (set as 100%). (B and C) Antiviral activity of compounds 9 (Com-9) and 12 (Com-12) against PRRSV infection in MARC-145 cells was examined using immunofluorescence assay (IFA) at 48 hpi. Cells grown in 96-well plates were infected with PRRSV GD-XH (0.05 MOI) for 2 h at 37 °C and then cultured in fresh medium containing various concentrations of 9 or 12. IFA for the N protein of PRRSV was performed at 48 hpi using Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (red). Nuclei were counterstained using 4,6-diamidino-2-phenylindole (DAPI) (blue). The EC₅₀ value (concentration of compound required to protect 50% cells from infection) was determined based on IFA as described in the Methods. (B) Representative IFA images from one of the three independent experiments. Scale bar: 250 μm. (C) Dose–response curves for 9 and 12 from the three independent IFA experiments.