Date of Award

5-2007

Level of Access

Campus-Only Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

Advisor

John T. Singer

Second Committee Member

Keith Hutchison

Third Committee Member

Carol Kim

Abstract

Infectious pancreatic necrosis (IPN) causes high mortalities in salmonid fry and has a significant economic impact on the aquaculture industry. IPN virus (IPNV) is lethal only in young fish, but once infected, survivors become life-long carriers of IPNV, serving as a reservoir for horizontal and vertical transmission. To date, there is no effective vaccine that blocks the establishment of the carrier state. We tested the ability of a genetic vaccine to prevent both infection and the formation of the carrier state in fish challenged with IPNV. A plasmid vector, pIPN, was constructed to express IPNV segment A proteins VP2, NS, and VP3 under the control of the immediate-early cytomegalovirus promoter. Transcription of segment A from pIPN in Chinook salmon embryo (CHSE) cells and in fish muscle tissue after intramuscular injection was confirmed by RT-PCR analysis of RNA isolated from pIPN-transfected CHSE cells and from the injection site of pIPN-injected fish. A modified version of pIPN, pIPNGFP, containing a gene fusion between segment A VP3 and a promoterless GFP reporter gene was constructed to demonstrate translation of segment A mRNA in both cultured fish cells and in vivo in fish muscle tissue after intramuscular injection. We demonstrated successful IPNV infection in zebrafish and used a zebrafish model to test the efficacy of pIPN. The mean IPNV titer in zebrafish challenged with 106 TCID50 of IPNV was 2.3 x 104 TCID50/ g offish tissue. IPNV was detected in fish tissues up to 8 weeks post challenge. Histological and immunohistochemical analysis confirmed a diagnosis of IPN. The immune response in zebrafish after vaccination with pIPN or challenge with IPNV was assessed by determining the level of expression of anti-viral cytokines. pIPN induced expression of inflammatory cytokines II-1 and TNF and both pIPN and IPNV induced expression of Mx and type I and type II interferons in zebrafish kidney cells. The difference in viral titer in fish vaccinated with pIPN compared to viral titers in control fish was not statistically significant. Vaccination with pIPN did not result in a rate of IPNV clearing that was statistically different from those observed with controls.

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