(B) Antiviral effect of CHLA against multiple viruses. (C) Antiviral effect of PUG against multiple viruses. Results are plotted against values for the DMSO control treatment of virus infections and the data shown are means ± the standard errors of the mean (SEM) from three independent experiments. See text for details. Table 2 Cytotoxicity and antiviral activity of CHLA and PUG against different virus infections a Virus Cell

type Compounds CC50(μM)b Antiviral effect         EC50(μM)c SId HCMV HEL CHLA 306.32 ± 7.00 25.50 ± 1.51 12.01     PUG 299.32 ± 9.14 16.76 ± 0.88 17.86 HCV Huh-7.5 CHLA 237.61 ± 4.53 12.16 ± 2.56 19.54     PUG 222.61 ± 3.41 16.72 ± 2.55 13.31 DENV-2 Vero CHLA 159.63 ± 7.46 13.11 ± 0.72 12.18     PUG 151.44 ± 9.31 7.86 ± 0.40 19.27 MV CHO-SLAM CHLA 351.83 ± 4.54 34.42 ± 4.35 10.22     PUG 283.76 ± 11.54 25.49 ± 2.94 11.13 RSV HEp-2 CHLA 244.17 ± 17.40 0.38 ± 0.05 642.55     PUG 264.83 ± 23.72 0.54 ± 0.04 490.43 VSV A549 CHLA 316.87 ± 9.01 XAV939 61.28 ± 5.50 5.17     PUG 318.84 ± 4.99 36.98 ± 4.59 8.62 ADV-5 Repotrectinib A549 CHLA 316.87 ± 9.01 198.14 ± 14.07 1.60     PUG 318.84 ± 4.99 196.67 ± 20.05 1.62 a Values shown are means obtained from three independent experiments with each treatment performed in triplicate. b Cytotoxic effects were evaluated by XTT assay to determine the concentration of 50% cellular cytotoxicity (CC50) of the tested compounds. c Antiviral

effects were evaluated by infection analysis to determine the effective concentration that achieved 50% inhibition (EC50) against the specific virus CBL0137 mouse examined. d SI, selectivity index. SI = CC50/EC50. For assessing the antiviral activities of the tannins on the panel of viruses, HEL (1 × 105 cells/well), Vero (2 × 105 cells/well), HEp-2 (1.5 × 105 cells/well), and A549 (2 – 3 × 105 cells/well) cells were seeded in 12-well plates and co-treated with the respective viral inoculum (Figure 2A) and increasing concentration of test compounds for 1 – 2 h. The inoculum and drug mixtures were removed from the wells that were subsequently washed with PBS

twice and then overlaid with 2% FBS medium containing either Carnitine dehydrogenase methylcellulose (Sigma; HCMV: 0.6%; DENV-2: 0.75%; RSV and VSV: 1%) or SeaPlaque agarose (Lonza, Basel, Switzerland; ADV-5: 1%). After further incubation for 24 h – 10 days depending on the specific virus, wells containing ADV-5, HCMV, and VSV infections were analyzed by standard plaque assays, and wells containing DENV-2 and RSV infections were analyzed by immunohistochemical staining as described above. Viral infection (%) and the 50% effective concentration (EC50) of test compounds against different viral infections were calculated as previously described [33]. For evaluating the antiviral activities of the tannins on MV-EGFP infection, CHO-SLAM cells (2 × 104 cells/well) were seeded in 96-well plates and viral inoculum and increasing concentration of the test compounds were co-added onto the cell monolayer for 1.5 h.