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Differential transcription of fathead minnow immune-related genes following infection with frog virus 3, an emerging pathogen of ectothermic vertebrates.

ABSTRACT: Frog virus 3 (FV3) and other ranaviruses are responsible for die-offs involving wild, farmed, and captive amphibians, fish, and reptiles. To ascertain which elements of the immune system respond to infection, we explored transcriptional responses following infection of fathead minnow cells with either wild type (wt) FV3 or a knock out (KO) mutant targeting the 18 kDa immediate early gene (18K). At 8h post infection we observed marked upregulation of multiple transcripts encoding proteins affecting innate and acquired immunity. Sequences expressed 4-fold or higher in wt-infected cells included transcripts encoding interferon (IFN), IFN regulatory factors (IRFs), IFN stimulated genes (ISGs) such as Mx and MHC class I, and interleukins IL-1?, IL-8, IL-17C and IL-12. Cells infected with the 18K KO mutant (?18K) showed qualitative differences and lower levels of induction. Collectively, these results indicate that ranavirus infection induced expression of multiple cellular genes affecting both innate and acquired immunity.

SUBMITTER: Cheng K

PROVIDER: S-EPMC4046121 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Differential transcription of fathead minnow immune-related genes following infection with frog virus 3, an emerging pathogen of ectothermic vertebrates.

Cheng Kwang K Escalon B Lynn BL Robert Jacques J Chinchar V Gregory VG Garcia-Reyero Natàlia N

Virology 20140401

Frog virus 3 (FV3) and other ranaviruses are responsible for die-offs involving wild, farmed, and captive amphibians, fish, and reptiles. To ascertain which elements of the immune system respond to infection, we explored transcriptional responses following infection of fathead minnow cells with either wild type (wt) FV3 or a knock out (KO) mutant targeting the 18 kDa immediate early gene (18K). At 8h post infection we observed marked upregulation of multiple transcripts encoding proteins affecti ...[more]

PMID: 24889227

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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-1β, IL-8, IL-17C and IL-12. Cells infected with the 18K KO mutant (∆18K) 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.

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.

Project description:Omics approaches are broadly used to explore endocrine and toxicity-related pathways and functions. Nevertheless, there is still a significant gap in knowledge in terms of understanding the endocrine system and its numerous connections and intricate feedback loops, especially in non-model organisms. The fathead minnow (Pimephales promelas) is a widely used small fish model for aquatic toxicology and regulatory testing, particularly in North America. A draft genome has been published, but the amount of available genomic or transcriptomic information is still far behind that of other more broadly studied species, such as the zebrafish. Here, we used a proteogenomics approach to survey the tissue-specific proteome and transcriptome profiles in adult male fathead minnow. To do so, we generated a draft transcriptome using short and long sequencing reads from liver, testis, brain, heart, gill, head kidney, trunk kidney, and gastrointestinal tract. We identified 30,378 different putative transcripts overall, with the assembled contigs ranging in size from 264 to over 9,720 nts. Over 17,000 transcripts were >1,000 nts, suggesting a robust transcriptome that can be used to interpret RNA sequencing data in the future. We also performed RNA sequencing and proteomics analysis on four tissues, including the telencephalon, hypothalamus, liver, and gastrointestinal tract of male fish. Transcripts ranged from 0 to 600,000 copies per gene and a large portion were expressed in a tissue-specific manner. Specifically, the telencephalon and hypothalamus shared the most expressed genes, while the gastrointestinal tract and the liver were quite distinct. Using protein profiling techniques, we identified a total of 4,045 proteins in the four tissues investigated, and their tissue-specific expression pattern correlated with the transcripts at the pathway level. Similarly to the findings with the transcriptomic data, the hypothalamus and telencephalon had the highest degree of similarity in the proteins detected. The main purpose of this analysis was to generate tissue-specific omics data in order to support future aquatic ecotoxicogenomic and endocrine-related studies as well as to improve our understanding of the fathead minnow as an ecological model.

Dataset Information

Differential transcription of fathead minnow immune-related genes following infection with frog virus 3, an emerging pathogen of ectothermic vertebrates.

Publications

Differential transcription of fathead minnow immune-related genes following infection with frog virus 3, an emerging pathogen of ectothermic vertebrates.

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