Master of Science (MSc)
Faculty of Science
Dr. Stephanie DeWitte-Orr
Ranavirus infections are on the rise and have been implicated in numerous species die-offs across the globe. Frog virus 3 (FV3) is the type-species of the genus, yet the immune mechanisms governing susceptibility remain poorly understood. Arguably the most important immune response to infection is the type I interferon (IFN) response. Type I IFNs trigger an “antiviral state” in host cells via the production of numerous interferon-stimulated genes (ISGs) that act to inhibit virus replication in various way, including the induction of apoptosis. Apoptosis is an important antiviral defense mechanism to limit virus replication within infected cells. This study employed the use of two rainbow trout cell lines, RTgutGC (intestinal origin) and RTG-2 (gonadal origin), previously shown to differ in susceptibility to FV3, thereby providing an excellent model to study innate anti-FV3 immune responses. Time-lapse infection videos and cell viability assays were used to quantify differences in the extent of cell death over time. RTG-2 exhibited greater cell death at a lower virus titre, compared with RTgutGC. The mechanism of cell death was investigated via DAPI staining and DNA laddering to observe nuclear condensation and intranucleosomal fragmentation, respectively, both hallmarks of apoptosis. Both cells underwent apoptosis in response to FV3. Moreover, UV-inactivated FV3 exhibited similar apoptotic cell death, suggesting that FV3-induced apoptosis is independent of productive virus replication. Likewise, poly I:C induction of IFN and ISGs inhibited virus replication, but had no effect on FV3-induced cell death. Using real-time RT-PCR IFN, ISG, and viral transcript expression was examined in both cell lines. Surprisingly FV3 elicited an equally poor IFN and ISG response in both cell lines, and was only detectable at 72h post-infection. Even when UV-inactivated, FV3 did not elicit a significant IFN response. However, viral transcript expression appears to be greater in the highly susceptible RTG-2 cell line. Further investigation into this difference in susceptibility between cell lines revealed that RTG-2 exhibited greater viral entry and cellular metabolism, which may account for the enhanced level of infection. Thus, FV3 appears to exhibit virulence factors that are independent of replication, yet the mechanisms governing susceptibility appear to be the result of intrinsic cellular features that are IFN-independent.
Lisser, Graeme Robert Jones, "USING RAINBOW TROUT CELL LINES AS A MODEL FOR UNDERSTANDING THE INNATE ANTI-FV3 IMMUNE RESPONSE" (2017). Theses and Dissertations (Comprehensive). 1899.
Video 1A: FV3 Infection Time-Lapse in RTgutGC
Video 1B - FV3 Infection Video in RTG-2 March 16.mp4 (1575 kB)
Video 1B: FV3 Infection Time-Lapse in RTG-2