Apoptosis Precedes Necrosis of Fish Cell Line with Infectious Pancreatic Necrosis Virus Infection

AbstractThe current view of infectious pancreatic necrosis virus (IPNV) infection includes a necrotic process that relies primarily on the histological appearance of tissue after the degenerative process. We tested this view by examining the possibility that apoptosis is a component of double-stranded RNA virus (IPNV) that induces fish embryonic cell death. Four kinds of assays for apoptosis were used in analyzing IPNV-infected CHSE-214 cells: (1) assay with terminal deoxynucleotidyl transferase (TdT)-mediated end-labeling of DNA in nuclei of intact cells during virus infection, (2) assay for procoagulant activity, (3) assay for DNA ladders, and (4) electron microscopic assays for the ultrastructural changes in characteristic apoptotic cells. In all p.i. samples, both low and high m.o.i. groups contained apoptotic nuclei, according to TdT-mediated dUTP labeling of intact cells, but in control CHSE-214 cells, apoptotic nuclei were rare at all levels of incubation sampled by TdT-mediated dUTP labeling. Prenecrotic or postnecrotic cells were found to express phosphatidylserine on the surface by annexin V–FITC labeling, but normal cells did not. DNAs from both 4 h p.i. of high m.o.i. and 8 h p.i. of low m.o.i. were found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. The IPNV-infected CHSE-214 cells were analyzed with an electron microscope and showed a pattern of ultrastructural change, indicating that apoptosis appears before pathological changes of necrosis, including condensed chromatin, fragmented nuclei, nuclei with chromatin marginations, and secondary necrosis from prenecrotic cells in IPNV-infected CHSE-214 cells. Together, these findings show that apoptosis precedes any detectable necrotic change in CHSE-214 cells that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in host cells and is followed by necrotic processes