Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Stephanie DeWitte-Orr

Advisor Role

Supervisor

Abstract

Viral dsRNA is produced by almost all viruses sometime during their replicative cycle. These viral nucleic acids are potent inducers of both innate and adaptive immune responses, and are therefore considered important immuno-modulators. Previous studies have shown that viruses produce dsRNA when replicating in mammalian cells; however, to date no one has demonstrated viral dsRNA production in virus infected fish cells. Therefore, the goal of this study is to investigate dsRNA production by fish viruses in fish cells, verifying production and performing initial characterization of the dsRNA molecules being produced. Three different rainbow trout cell lines were used in this study: rainbow trout gill (RTgill-W1, epithelial), rainbow trout gut (RTgutGC, epithelial) and rainbow trout gonad (RTG-2, fibroblast). These cell lines were selected because innate immune responses are relatively well characterized in RTG-2; while RTgill and RTgut represent two tissues that would be first to ‘see’ a virus infection in vivo. The study also includes three different fish viruses: viral haemorrhagic septicaemia virus (VHSV), which has a negative sense single stranded RNA (-ssRNA) genome, chum salmon reovirus (CSV), which has a double stranded RNA (dsRNA) genome, and frog virus3 (FV3), which has a dsDNA genome. These viruses were selected because they have different genomes and thus different replication cycles, which is important for verifying dsRNA production is not specific to one virus genome type. dsRNA production was measured using immunofluorescence, a technique which relies on J2, a mouse anti-dsRNA antibody. Not only does immunofluorescence with J2 verify that fish viruses produce dsRNA in fish cells, but it also indicates the location of dsRNA production within the cell. An acridine orange stain was also performed to indicate the relative amount of dsRNA produced during a virus infection as well as the length of the dsRNA molecules to provide further evidence for dsRNA production by fish viruses in fish cells using an antibody-independent method. Because dsRNA is an important immuno-modulator, it has possible applications as a novel adjuvant for vaccines or as an antiviral therapy. The results from this study are important not only because it contributes to a better understanding of virus-host interactions, but characterizing viral dsRNA in fish cells could provide basic research evidence on which to build novel dsRNA-based therapies in fish.

Convocation Year

2014

Convocation Season

Fall

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