Thesis Title

Induction of Apoptosis and Necrosis By Infectious Pancreatic Necrosis Virus in Chinook Salmon Embryo Cells

Date of Graduation

Summer 1997

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Christopher Field

Subject Categories

Biology

Abstract

Viruses are associated with the induction of apoptosis or necrosis in the cells they infect. Infectious Pancreatic Necrosis Virus (IPNV) is a birnavirus that has been shown to cause severe necrosis in pancreatic, intestinal epithelial, kidney, and liver tissues in infected salmonid fish, but no research indicates the incidence of apoptasis associated with IPNV infection. Histological evidence from infected fish tissue demonstrates pyknotic nuclei within damaged tissue distinct from surrounding necrotic tissue. The focus of this investigation was to determine the incidence of apoptosis and necrosis in IPNV (strain LWVRT)-infected CHSE-214. The incidence of apoptosis and necrosis in IPNV-infected cells (m.o.i of 10) incubated for 0 hr, 4 hr, 8 hr, 16 hr, and 24 hr was determined morphologically by epifluorescence microscopy using acridine orange and ethidium bromide and biochemically by 2% agarose gel electrophoresis and Hoescht 33256 fluorometric analysis of isolated DNA. Data indicate that CHSE-214 cells are capable undergoing spontaneour apoptosis. Furthermore, IPNV (LWVRT) induces severe necrosis by 16 hr post-inoculation and mild apoptosis by 24 hr post-inoculation as indicated by epifluorescence microscopy. Apoptotic CHSE-214 cells demonstrate a 180-200 bp DNA ladder indicative of an endogenous endonuclease activity, also found in other apoptotic systems. Increases in the percent of DNA fragmentation and the appearance of a 180-200 bp DNA ladder by 24 hr indicate that cell damage occurs late in infection. This is the first known report of apoptosis in CHSE-214 cell line.

Copyright

© Timothy M Doyle

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