Dynamics of nontypical apoptotic morphological changes visualized by greenfluorescent protein in living cells with infectious pancreatic necrosis virus infection

Morphologically, apoptotic cells are characterized by highly condensed memb rane blebbing and formation of apoptotic bodies. Recently, we reported that apoptosis precedes necrosis in a fish cell line infected with infectious p ancreatic necrosis virus (IPNV). In the present study, we tested the possib ility that nontypical apoptosis is a component of IPNV-induced fish cell de ath. A variant type of green fluorescent protein (EGFP) was expressed in a fish cell line such that EGFP served as a protein marker for visualizing dy namic apoptotic cell morphological changes and for tracing membrane integri ty changes during IPNV infection. Direct morphological changes were visuali zed by fluorescence microscopy by EGFP in living cells infected with IPNV, The nontypical apoptotic morphological change stage occurred during the pre -late stage (6 to 7 h postinfection). Nontypical apoptotic features, includ ing highly condensed membrane blebbing, occurred during the middle apoptoti c stage. At the pre-late apoptotic stage, membrane vesicles quickly formed, blebbed, and were finally pinched off from the cell membrane. At the same time, at this pre-late apoptotic stage, apoptotic cells formed unique small holes in their membranes that ranged from 0.39 to 0.78 mu m according to e xamination by scanning electron microscopy and immunoelectron microscopy. Q uantitation of the intra- and extracellular release of EGFP by CHSE-214-EGF P cells after IPNV infection was done by Western blotting and fluorometry. Membrane integrity was quickly lost during the late apoptotic stage (after 8 h postinfection), and morphologic al change and membrane integrity loss c ould be prevented and blocked by treatment with apoptosis inhibitors such a s cycloheximide, genistein, and EDTA before IPNV infection. Together, these findings show the apoptotic features at the onset of pathology in host cel ls (early and middle apoptotic stages), followed secondarily by nontypical apoptosis (pre-late apoptotic stage) and then by postapoptotic necrosis (la te apoptotic stage), of a fish cell line. Our results demonstrate that nont ypical apoptosis is a component of IPNV-induced fish cell death.