*3.1. Brevilin A Shows a Broad-Spectrum Antiviral Activity against IAV*

In our previous work, brevilin A showed potent antiviral activity against PR8 virus assessed by cytopathogenic effect (CPE) reduction assay and the cell viability assay [20]. To further verify its anti-IAV activity, brevilin A was tested in a plaque reduction assay using several IAV strains including A/PR/8/34 H1N1, A/FM/1/47 H1N1, A/Hong Kong/498/97 H3N2, and A/chicken/Guangdong/1996 H9N2 viruses. Ribavirin served as a positive control. The concentration for 50% of maximal effect (EC50) of brevilin A obtained with PR8 for viral plaque formation was calculated to be 2.96 ± 1.10 μM. This result concurs with the EC50 of 1.75 ± 0.59 μM that we evaluated in previous work. Comparable to PR8, the EC50 values of brevilin A obtained with H1N1 (FM1), H3N2, and H9N2 were 1.60 ± 1.14, 3.28 ± 1.09, and 2.07 ± 1.12 μM, respectively (Table 1). While the EC50 of ribavirin obtained with these four IAV strains were between 7.05 to 10.76 μM. These results indicate that brevilin A exhibits better anti-IAV activity than ribavirin, and the effects of both are not IAV type/subtype specific. In order to test whether brevilin A possesses antiviral activity against other RNA viruses, the effect of brevilin A on respiratory syncytial virus (RSV) was evaluated by a CPE reduction assay. However, brevilin A did not show inhibitory effect on RSV at a noncytotoxic concentration.



*<sup>a</sup>* Effective concentration required for reducing virus-induced plaque number by 50%. *<sup>b</sup>* Selectivity index, CC50/EC50.

#### *3.2. Brevilin A Inhibits Progeny Virus Production in Various Virus-To-Cell Ratios*

To examine to what extent the anti-IAV activities of brevilin A is affected by virus-to-cell ratio, the cells were infected with PR8 at a MOI (MOI, defined as the ratio of input infectious viral particles per target cell) of 0.001 or 1 in the presence of either brevilin A (8 μM) or vehicle control (DMSO). Virus titers in the supernatants at the indicated time points were quantified by plaque assays. As shown in Figure 2A, after infection with virus at a MOI of 0.001, the amount of progeny virus in the supernatants increased over the incubation time and peaked at 48 hpi in vehicle control, while treatment with brevilin A could significantly reduce the production of infectious virus from cells at 24 or 48 hpi. Even when cells were infected with virus at a higher MOI (MOI = 1), treatment of brevilin A also significantly decreased virus production by about 10-fold at 8 and 12 hpi (Figure 2B). These findings imply that the treatment of brevilin A strongly suppresses the replication of IAV, of note, the inhibitory activity of brevilin A is still rather effective against a relatively higher dose of input virus.

Additionally, we also analyzed the impact of brevilin A on replication of H1N1 (FM1), H3N2, or H9N2 in MDCK cells over multiple replication cycles. As shown in Figure 2C–E, compared to the vehicle, virus titers at each time point were markedly reduced by treatment with brevilin A.

**Figure 2.** The inhibitory effect of brevilin A on the growth curves of various influenza A viruses (IAV) strains. Madin–Darby canine kidney (MDCK cells) were infected with influenza A/PR/8/34 H1N1 virus at a MOI of 0.001 (**A**) or 1 (**B**), or A/FM/1/47 H1N1 virus (**C**), A/Hong Kong/498/97 H3N2 virus (**D**), or A/chicken/Guangdong/1996 H9N2 virus (**E**) at a MOI of 0.001. Cells were then treated with 8 μM of brevilin A or vehicle. At the indicated time points after infection, virus titers in the supernatants were determined by a plaque assay. The data represent means ± SD. \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001 are considered statistically significant, compared to vehicle.

#### *3.3. Brevilin A Is E*ff*ective at the Viral Genome Replication and Translation Stage*

The life cycle of influenza virus is around 8–10 h and is divided into three steps: virus entry (0–2 h), viral genome replication and translation (2–8 h), and progeny virion release (8–10 h) [23]. To investigate the stage of viral cycle where brevilin A exhibits its activity against PR8 virus, we next performed time of addition experiments. MDCK cells were infected with PR8 virus, and brevilin A was added or removed at the indicated time points. The expression levels of influenza M2 protein in infected cells were measured at four time-intervals, 0–2, 2–4, 4–8 and 8–10 hpi. We found that the M2 level at the interval 4–8 hpi was reduced around 70%, compared to the vehicle. In contrast, no antiviral activity was detected for the remaining three intervals (0–2, 2–4, 8–10 hpi) (Figure 3A). These data indicate that brevilin A is effective at the viral genome replication and translation stage. No inhibitory effect was observed at virus entry stage or progeny virion assembly/release stage.

We then performed two other experiments to examine the mode of action of brevilin A. First, brevilin A was added to the IAV infected cells at 0, 4, 6, or 8 hpi, the supernatant was collected at 24 hpi, and the virus titers were determined by plaque assay. The virus titers were decreased when brevilin A was added at 0–4 hpi, compared to vehicle. However, similar titers were observed in the treatment and vehicle control when the compound was added 6 h after infection (Figure 3B). Moreover, the results obtained with an immunofluorescent assay showed that compared to the vehicle control, the expression of viral protein M2 was markedly reduced when brevilin A was added at 0 to 4 hpi, and addition of brevilin A at 6 hpi still had a minor impact on M2 expression (Figure 3C). These data suggest that brevilin A blocks the intermediate stage (s) of the influenza virus life cycle between approximately 4 to 6 hpi.

**Figure 3.** Influence of different treatment conditions of brevilin A on IAV replication. (**A**) MDCK cells were infected with PR8 at a MOI of 1, brevilin A or vehicle was present at four time-intervals, 0–2, 2–4, 4–8, and 8–10 hpi. At 12 hpi, cell lysates were analyzed by Western blot assay; (**B**) MDCK cells were infected with PR8 at a MOI of 0.1, then treated with brevilin A (8 μM) at 0, 2, 4, or 6 hpi. The virus titers in the supernatant were determined by plaque assay at 24 hpi; (**C**) MDCK cells infected with PR8 (MOI = 1) were treated with brevilin A at the indicated times. The M2 protein expression was determined by immunofluorescence assay at 12 hpi. The scale bar in the images is 100 μm. The data represent means ± SD. \*, *p* < 0.05 is considered statistically significant compared to vehicle.
