Delineating the roles of cellular and innate antiviral immune parameters mediating ranavirus susceptibility using rainbow trout cell lines.

Frog virus 3 is the type species of the Ranavirus genus and the causative agent of massive mortalities of aquatic species worldwide. A critical step in limiting virus replication, particularly early in infection, is the innate immune response. Presently, little is known regarding what innate immune strategies limit FV3 at the cellular level. To this end, the present study uses two rainbow trout cell lines, RTG-2 and RTgutGC, which demonstrate susceptible and relatively resistant phenotypes to FV3 infection, to elucidate susceptibility factors to FV3. RTG-2 demonstrated a lower LD50 and significantly higher virus transcript production compared to RTgutGC. The mode of cell death appeared to be apoptosis for both cell lines; however, RTG-2 did not demonstrate fragmented nuclei typical of apoptosis in cell culture. Next, the source of RTG-2's enhanced susceptibility was pursued, in hopes of highlighting unique features of this virus-host interaction that would predispose a cell to susceptibility. The type I interferon (IFN) response is the keystone mechanism used by the innate immune system to limit virus replication. FV3 induced very low to no levels of IFNs and interferon stimulated genes (ISGs) in either cell line, nor did inducing IFNs prior to infection inhibit virus-induced cell death. A dsRNA-induced antiviral state did reduce virus replication however. UV-inactivated FV3 was also able to kill RTG-2; thus, susceptibility to FV3-induced cell death observed in RTG-2 was independent of virus replication or the cell's ability, or lack thereof, to produce an IFN response. Importantly, RTG-2 showed greater viral entry compared to RTgutGC, suggesting non-innate immune factors, such as surface receptor expression or endocytic mechanism rates, may be key contributors to FV3 susceptibility. These findings contribute to our understanding of cell-level susceptibility to this environmentally important aquatic animal pathogen.