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This study probed antiviral activity of<jats:italic>I. indigotica<\/jats:italic>extract and its marker compounds against Japanese encephalitis virus (JEV).<jats:italic>I. indigotica<\/jats:italic>methanol extract, indigo, and indirubin proved less cytotoxic than other components, showing inhibitory effect (concentration-dependent) on JEV replication<jats:italic>in vitro<\/jats:italic>. Time-of-addition experiments proved the extract, indigo, and indirubin with potent antiviral effect by pretreatment (before infection) or simultaneous treatment (during infection), but not posttreatment (after entry). Antiviral action of these agents showed correlation with blocking virus attachment and exhibited potent virucidal activity. In particular, indirubin had strong protective ability in a mouse model with lethal JEV challenge. 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