Betanodavirus protein α induces cell apoptosis or secondary necrosis by a poorly understood process. In the present ork, red spotted grouper nervous necrosis virus (RGNNV) RNA 2 was cloned and transfected into tissue culture cells (GF-1) which then underwent apoptosis or post-apoptotic necrosis. In the early apoptotic stage, progressive phosphatidylserine externalization was evident at 24 h post-transfection (p.t.) by Annexin V-FLUOS staining. TUNEL assay revealed poptotic cells at 24–72 h p.t, after which post-apoptotic necrotic cells were identified by acridine orange/ethidium romide dual dye staining from 48 to 72 h p.t. Protein α induced progressive loss of mitochondrial membrane potential (MMP) which was detected in RNA2-transfected GF-1 cells at 24, 48, and 72 h p.t., which correlated with cytochrome c release, especially at 72 h p.t. To assess the effect of zfBcl-xL on cell death, RNA2-transfected cells were co-transfected with zfBcl-xL. Co-transfection of GF-1 cells prevented loss of MMP at 24 h and 48 h p.t. and blocked initiator caspase-8 and effector caspase-3 activation at 48 h p.t. We conclude that RGNNV protein α induces apoptosis followed by econdary necrotic cell death through a mitochondria-mediated death pathway and activation of caspases-8 and -3.
