Fig. 2.
In vitro antiviral activity of AM580. a Multi-cycle MERS-CoV growth assay in the presence or absence of AM580. Huh7 cells were infected with MERS-CoV (0.01 MOI). Viral titers in cell culture supernatants were quantified by plaque assay at different time points. Differences between DMSO (open circle) and AM580 (20 µM, black square) groups were analyzed by Student’s t-test. b Western blot showed reduced MERS-CoV NP production after AM580 treatment. Huh7 cells were infected with 1 MOI MERS-CoV. c Upper panel: MERS-CoV-NP-positive cells quantitated by flow cytometry. Lower panel: immunofluorescence staining of MERS-CoV-NP antigen (green) and cell nucleus (blue). Scale bar:10 µm. d AM580 reduced MERS-CoV replication in cell culture supernatants of A549 (0.1 MOI), Calu-3 (0.1 MOI), and Vero (0.01 MOI) cells at 24 hpi. AM580 reduced MERS-CoV replication in cell lysates of monocyte-derived macrophage cells (MDM, 1 MOI) and THP-1 (0.1 MOI) at 24 hpi. Differences between DMSO and AM580 groups were analyzed by Student’s t-test. e AM580 inhibited MERS-CoV replication in human primary small airway epithelial cells (HSAEC) that were infected by 1 MOI MERS-CoV and treated with (red square) or without AM580 (black triangle). Supernatant and cell lysate were collected at the indicated time points and titrated by plaque assay and RT-qPCR assay, respectively. f, g AM580 showed anti-MERS-CoV activity in human intestinal organoid (intestinoid). f One-way ANOVA was used for comparison of the AM580 treated with the DMSO treated. g Representative images of intestinoids, after immunofluorescence staining for MERS-CoV NP (green), DAPI, and Phalloidin (purple), were 3D-imaged with a confocal microscope. Scale bar: 20 µm. h AM580 showed broad-spectrum antiviral effects against six different viruses as indicated. Plaque reduction assays were performed to evaluate antiviral activity of AM580 in MERS-CoV (Huh7 cells, magenta triangle) and SARS-CoV (Huh7 cells, magenta rectangle), ZIKV (Vero cells, black triangle), H1N1 virus (MDCK cells, black diamond), EV-A71 (RD cells, blue dot). TCID50 assays were used for AdV5 titration (HEp-2 cells, blue triangle). Shown are the PFU or TCID50 of indicated concentrations relative to controls in the absence of compound (%). The experiments were performed in triplicate and replicated twice. The results are shown as mean ± s.d. ***p < 0.001, **p < 0.01, *p < 0.05