Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Dr. Stephanie DeWitte-Orr

Advisor Role

Master Thesis Supervisor

Abstract

Rainbow trout is the most farmed fish in Ontario, and thus is economically important to the province. Despite this, there is a lack of understanding regarding fish innate immunity, specifically with regards to interferon-stimulated genes (ISGs) and their antiviral effector functions. ISGs are the workhorses of the innate antiviral response, operating together to limit each step of virus replication. The Viral Hemorrhagic Septicemia Virus (VHSV) induced gene (Vig)-3 is a newly identified ISG within many fish species and is homologous to ISG-15 in mammals. It is a small ubiquitin-like protein inducible by type I interferon (IFN-I), and is suggested to have antiviral effects within the cell. Vig-3 has been proposed to establish an antiviral response by acting both intracellularly through covalent modification of proteins, as well as extracellularly as a signaling molecule. It is for these reasons it was investigated in Rainbow trout. To do this, Rainbow trout gonadal cells (RTG-2) were infected with two fish viruses (infectious pancreatic necrosis virus; IPNV and VHSV), as well as treated with poly I:C, and the expression of vig-3 was monitored over 24- and 48h periods at the transcript, protein, and cellular level. The transcript level of expression was analyzed via quantitative real time polymerase chain reaction (q-RT-PCR) and demonstrated that vig-3 expression was induced during treatment with VHSV, IPNV and poly I:C. Western blot analysis was used to analyze protein expression of Vig-3 during infection with the same viruses and treatment with poly I:C. It was found that during poly I:C treatment and viral infection Vig-3 protein expression was induced from 6h to 48h. It was also found that Vig-3 was able to bind to target proteins in a process known as ISGylation. Immunocytochemistry was used to determine the cellular expression of Vig-3 during viral infection with IPNV and VHSV and treatment with poly I:C. In this case it was determined that Vig-3 was upregulated at both 12h and 24h during all treatments, as well as is both localized to the cytoplasm and nucleus. These findings contribute to a better understanding of a poorly studied aspect of innate antiviral immunity in an economically valuable fish species.

Convocation Year

2019

Convocation Season

Fall

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