Thesis Title

Increased Apoptosis in the Host By Guanidine Hydrochloride Treatment of Infectious Pancreatic Necrosis Virus

Date of Graduation

Fall 1999

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Christopher Field

Subject Categories

Biology

Abstract

Apoptosis is an orchestrated type of cell death, characterized early by phosphatidylserine flipping to the outer leaflet of the membrane, followed by cell shrinkage and cell blebbing. DNA isolates from apoptotic cells typically present a laddering effect on agarose gels. Recent publications indicate the early protein products of some viruses are able to repress apoptosis within their host. Particles or empty capsids that produce inefficient or no protein products will allow host cells to undergo apoptosis. Empty capsids are mass produced by some viruses or can be created from wild type virus by nucleic acid extrusion. This process has been done with Bovine Herpes Virus (BHV) using guanidine hydrochloride (GuHC1) to extrude the DNA and leave the capsids intact. These 'created' empty capsids were used to demonstrate that attachment of the empty capsid induced apoptosis. Different strains of Reovirus, a naked double stranded RNA virus similar to Infectious Pancreatic Necrosis Virus (IPNV), have also been treated with GuHCI to show inactivation of ơ1 protein, responsible for haemagglutinin, without disrupting other capsid proteins. However, the researchers did not investigate different types of cytopathic effects. IPNV is primarily known to be a necrotic virus, not an apoptotic virus. Therefore, IPNV was treated with GuHC1 to test the effects it had on viral activity and apoptosis in Chinook Salmon Embryo cells (CHSE-214). Standard research methods of apoptotic detection were used: vital dye exclusion, fluorescent microscopy and DNA laddering in electrophoretic gels. The vital dye staining showed a significant decrease in viral activity post treatment with 4M GuHC1. Fluorescent microscopy results indicated examples of increased phosphatidylserine (PS) flip to the outside membrane in 4M GuHC1 treated virus versus wild type virus infected cells. DNA laddering in cells was observed in 4M GuHC1 treated viral samples compared to controls of positive apoptotic and positive necrotic samples. These results indicate that 4M GuHC1 treatment of IPNV will decrease viral activity and increase apoptosis in CHSE-214 cells, suggesting a viral product, which normally blocks apoptosis, is not operative.

Copyright

© Dan Sturdevant

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