LAUSR

&NA; Cardenolides are plant‐derived toxic substances. Their cytotoxicity and the underlying mechanistic signaling axes have been extensively documented, but only a few anti‐viral activities of cardenolides and the associated signaling pathways have been reported. Previously, we reported that a variety of cardenolides impart anti‐transmissible gastroenteritis coronavirus (TGEV) activity in swine testicular (ST) cells, through targeting of the cell membrane sodium/potassium pump, Na+/K+‐ATPase. Herein, we further explore the potential signaling cascades associated with this anti‐TGEV activity in ST cells. Ouabain, a representative cardenolide, was found to potently diminish TGEV titers and inhibit the TGEV‐induced production of IL‐6 in a dose dependent manner, with 50% inhibitory concentrations of 37 nM and 23 nM respectively. By pharmacological inhibition and gene silencing, we demonstrated that PI3K_PDK1_RSK2 signaling was induced in TGEV‐infected ST cells, and ouabain imparted a degree of anti‐TGEV activity via further augmentation of this existing PI3K_PDK1 axis signaling, in a manner dependent upon its association with the Na+/K+‐ATPase. Finally, inhibition of PI3K by LY294002 or PDK1 by BX795 antagonized the anti‐viral activity of ouabain and restored the TGEV virus titer and yields. This finding is the first report of a PI3K_PDK1 signaling axis further induced by ouabain and implicated in the suppression of TGEV activity and replication; greatly illuminates the underlying mechanism of cardenolide toxicity; and is expected to result in one or more anti‐viral applications for the cardenolides in the future. Graphical abstract Figure. No caption available. HighlightsOuabain eliminated TGEV titers and inhibited viral replication.Ouabain diminished TGEV induced IL‐6 production.Ouabain enhanced PI3K or PDK1 activation induced by TGEV via Na+/K+‐ATPase.PI3K or PDK1 inhibition antagonized the anti‐TGEV activity of ouabain.Ouabain augmented the PI3K_PDK1 axis signaling that inhibited TGEV activity.