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.