Master of Science (MSc)
Faculty of Science
Dr. Stephanie DeWitte-Orr
In Canada, annual trout production is valued at over $60M with approximately 10,000 tonnes of trout produced per year. Rainbow trout (Oncorhynchus mykiss) are one of the most commonly farmed fresh-water fish in Canada. The high-density farming practices involved in aquaculture increase rainbow trout susceptibility to aquatic viruses such as infectious pancreatic necrosis virus (IPNV). The innate antiviral immune response in rainbow trout is poorly characterized, and the functions of numerous interferon stimulated genes (ISG) in rainbow trout remain unclear. One such gene, virus-induced gene 1 (vig-1) has been poorly studied; however, the human orthologue for vig-1, viperin (also known as radical S-adenosyl methionine domain containing 2 (RSAD2)), has been widely characterized and numerous antiviral mechanisms have been attributed to it. Vig-1 is thought to be a potentially important interferon-stimulated gene since its expression is induced by various stimulants, including polyinosinic:polycytidylic acid (poly I:C) (a viral double stranded ribonucleic acid (dsRNA) mimic) and viruses. This project sought to better characterize the role of vig-1 in the innate antiviral immune response in rainbow trout cells.
This thesis explored the induction kinetics of vig-1 in two rainbow trout cell lines, rainbow trout spleen-11 (RTS-11) and rainbow trout gonad-2 (RTG-2). This was accomplished by analyzing expression of vig-1 at the transcript level using real time quantitative polymerase chain reaction (RT-qPCR) and then expression at the protein level was investigated by western blot. Both analyses were completed following treatment with a range of concentrations of high molecular weight (HMW) poly I:C, or IPNV at various incubation points to better understand the expression patterns of vig-1 at the transcript and protein levels. This work suggests that firstly, vig-1 expression peaks at 24 hours following poly I:C treatment in both RTS-11 and RTG-2 cells, with a greater fold increase observed in RTG-2 cells despite RTS-11 being an immune cell line. Secondly, vig-1 expression in RTG-2 cells was found to increase in a dose-dependent manner when treated with increasing concentrations of poly I:C. Thirdly, infection with IPNV resulted in a transient increase of vig-1 expression at both 24- and 48- hours post-infection. The first steps of identifying vig-1’s antiviral function were completed by optimizing transfection efficiencies in the rainbow trout cell line, RTG-2, and the Atlantic salmon (Salmo salar) cell line, Atlantic salmon gill-10 (ASG-10), with a green fluorescent protein (GFP) expression plasmid and a non-liposomal transfection reagent. This work found that ASG-10 cells exhibited low transfection efficiency and RTG-2 cells exhibited 0% transfection efficiency under the conditions tested, suggesting that ASG-10 cells are more effective at DNA uptake. Given the low and zero transfection efficiencies observed in this study, alternative transfection reagents or methods should be explored for these cell lines before work continues to elucidate vig-1’s antiviral function by overexpression studies. Together, this work contributes to a better understanding of rainbow trout innate immune responses.
Lum, Samantha, "The characterization of Vig-1’s expression patterns in IPNV-infected, or dsRNA-treated, rainbow trout cells" (2024). Theses and Dissertations (Comprehensive). 2631.