This study aimed to investigate in vitro the anti-influenza B/Lee/40 virus effect of sakuranetin and mode of its action. The sakuranetin exhibited potent antiviral activity against influenza B/Lee/40 virus, reducing the formation of a visible cytopathic effect, with a 50% inhibitory concentration (IC ) of 7.21 μg/ml and no cytotoxicity at a concentration of 100 μg/ml, and the derived therapeutic index (TI) was >13.87. Oseltamivir showed weak anti-influenza B/Lee/40 virus activity with IC of 80.74 μg/ml, 50% cytotoxicity concentration of >100 μg/ml, and TI of >1.24. Sakuranetin also showed effective inhibitory effects when added at the viral attachment, entry, and postentry steps. Moreover, sakuranetin effectively inactivated influenza B/Lee/40 virus infection in dose- and temperature-dependent manners. Sakuranetin indicated an inhibitory effect in viral RNA synthesis in the presence of 100 μg/ml of sakuranetin. Overall, this research revealed that sakuranetin could inhibit influenza B/Lee/40 virus replication and that sakuranetin may be involved in the virus attachment, entry, and postentry. Therefore, sakuranetin is a good candidate for a chemopreventive agent for influenza virus-related diseases.

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