Project description:Frog virus 3 (FV3) and other ranaviruses are responsible for an increasing number of die-offs involving wild, farmed, and captive amphibians, fish, and reptiles. Studies in Lithobates pipiens (Leopard frogs) and Xenopus laevis (African clawed frog) indicate that embryos and tadpoles are more likely to succumb to FV3 infection than adult animals. The heightened susceptibility of embryos and tadpoles to ranaviral disease likely reflects the immaturity of their immune system. To ascertain which elements of the immune system respond to ranavirus infection, we explored cellular transcriptional responses following infection of fathead minnow (Pimephales promelas) cells with either wild type (wt) FV3 or a knock out (KO) mutant targeting the 18kDa immediate early gene (18K). At 8 hr post infection (p.i.), a time when late viral gene expression has begun and host protein synthesis has been inhibited, we observed marked up-regulation of multiple cellular transcripts encoding proteins affecting innate and acquired immune responses. We found 2367 target sequences expressed 4-fold or higher in wt-infected cells versus 530 target sequences that were 4-fold or more downregulated. For example, in cells infected with wt virus, interferon (IFN) transcripts were elevated compared to mock-infected cells, along with those encoding IFN regulatory factors (IRFs), IFN stimulated genes (ISGs) such as Mx and MHC class I, and other immune related gene products such as the interleukins IL-1M-NM-2, IL-8, IL-17C and IL-12. Cells infected with the 18K KO mutant (M-bM-^HM-^F18K) showed generally similar levels of induction although quantitative and qualitative differences were noted. Collectively, these results indicate that ranavirus infection induced expression of multiple cellular genes affecting both innate and acquired immunity. It is likely that induction of various immune-related genes may slow or limit viral infection through the activation of multiple components of the innate immune system and, in a whole animal, enhance the development of acquired immunity and protect from disease. To ascertain which elements of the immune system respond to ranavirus infection, we explored cellular transcriptional responses following infection of fathead minnow (Pimephales promelas) cells with either wild type (wt) FV3 or a knock out (KO) mutant targeting the 18kDa immediate early gene (18K). Replicate confluent monolayers of FHM cells grown on 35 mm dishes were mock-infected or infected with wt- or M-bM-^HM-^F18K-FV3 at a MOI of 10 PFU/cell. At 8 hr p.i., total RNA was isolated from samples RNeasy (Qiagen, Valencia, CA, USA) for microarray analysis. Five or six replicates were used per treatment.

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