Betanodavirus induces RIPK3/MLKL-mediated necroptosis and IL-21/adaptive immune response via superoxide stress/mTOR/ULK1-mediated autophagy signal in the context of brain damage in grouper (Epinephelus coioides).

Betanodavirus infection induces viral nervous necrosis (VNN) in fish. However, the role of macroautophagy/autophagy in the pathogenesis of VNN remains unknown. In this study, we found that RGNNV can trigger a superoxide-induced stress signal that, in turn, triggers an mTOR/ULK1-mediated autophagy signal and host cell necroptosis and immune responses in fish fin-cells (GF-1) and a species of grouper (Epinephelus coioides). The results revealed that autophagy began in the early-middle replication stage (12-36 h), as identified via RFP-LC3 phagosome formation and an immune EM approach, and was inhibited by the 3-methyladenine autophagy inhibitor, 3-MA. Then, RGNNV infection activated superoxide (O2-) signaling pathways in the mitochondria. O2- triggered autophagy and activated mTOR/ULK1-mediated signaling pathways, which were blocked by an antioxidant (diphenyleneiodonium, DPI) treatment. Furthermore, in the middle replication stage (24-48 h), the autophagy inhibitor 3-MA blocked RIPK3/MLKL-mediated necroptosis induction in GF-1 cells. Furthermore, in an in vivo analysis, we found that RGNNV could induce mTOR/ULK1-mediated autophagy signaling (as determined via Western blot analysis) and RIPK3/MLKL-mediated necroptosis signals (as determined via a qRT-PCR approach). Then, in a transcriptome analysis conducted to screen for innate and adaptive immune response gene induction, indicators of innate (i.e., IL-1β) and adaptive (i.e., IL-10 and Il-21) responses were found to have dramatically increased, where this increase was inhibited by antioxidant (DPI) treatment. Taken together, our data suggest that superoxide (ROS)-mediated stress signals can induce mTOR/ULK1-mediated autophagy activation. Then, the autophagy inhibitor 3-MA is activated to block RIPK3-mediated necroptosis signals. Finally, necroptotic cell damage is correlated with the induction of innate and adaptive responses in the damaged brains of the fish. These new insights are expected to facilitate the treatment of RGNNV infections.