| Turbot, Scophthalmus maximus, is one of important fish species cultured widely in coastal areas of northern China. Over the past decade, turbot farming industry of China has been suffered a growing damage from various viral epidemics, e.g."turbot reddish body syndrome"(TRBS), caused by a novel virus, turbot reddish body iridovirus (TRBIV). As transcriptional regulators of type I interferon (IFN) and IFN stimulated genes (ISGs) which form the first line of antiviral defence in vertebrates, interferon regulatory factors play critical diverse roles in a wide variety of cellular processes involving antiviral response, cytokine signaling, cell growth control and lymphocyte development. IRF-3 and IRF-7 are two key IRF members due to their critical roles in transcriptional regulation of IFN gene and ISGs. Up to date, the IRF-3 and IRF-7 have not been cloned in turbot.In this study, two full-length cDNAs and genomic sequences were cloned and identified in turbot, scophthalmus maximus. The cDNA of turbot IRF-3 (SmIRF-3) is 2820 bp in length, with a 242 bp 5'-UTR and a 1180 bp 3'-UTR. IRF-7 (SmIRF-7) cDNA consists of 1976 bp with a 13 bp 5'-UTR and a 646 bp 3'-UTR. The open reading frames (ORFs) of the two cDNAs translate into 466 and 436 amino acids, with the highest identity of 56.0% -81.2% and 49.0% -80.3% to fish IRF-3 and IRF-7, respectively. Each protein possesses a putative DNA-binding domain (DBD) containing a tryptophan cluster, a characteristic of all IRF family members, an IRF association domain (IAD) and a serine-rich C terminal domain (SRD). The presence of these domains along with phylogenetic analysis places SmIRF-3 and SmIRF-7 into the IRF-3 subfamily. The SmIRF-3 gene is 6077 bp long, containing a same structure of 11-exon and 10-intron as other fish IRF-3 genes, while SmIRF-7 gene is 3999 bp long, containing a structure of 10-exon and 9-intron same with other fish IRF-7 genes. Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze tissue distribution of SmIRF-3 and SmIRF-7 mRNA in healthy fish. RT-PCR analysis revealed that turbot IRF-3 was expressed constitutively in limited tissue types. The higher expression was detected in the kidney, head kidney and spleen, low expression levels in gills, stomach, intestine, liver and heart, and no expression in brain, gonad, muscle and skin. The constitutive expression of SmIRF-7 was found ubiquitously in all tested tissues, with higher levels observed in the immune relevant organs including spleen, kidney, head kidney, gills and intestine. A weak expression of SmIRF-7 was also detected in stomach, liver and muscle. These results indicates a role of SmIRF-3 and SmIRF-7 in fish immune system.In order to explore the protential roles of SmIRF-3 and SmIRF-7 in antiviral response, their transcriptional modulations by TRBIV were studied in vivo and compared with that of turbot Mx gene. The gills, head kidney, spleen and muscle were selected to conduct the study because they represent immune and non-immune organs of fish. All these genes were up-regulated in the head kidney (10-, 12- and 6-fold increase at peak level for SmIRF-3, SmIRF-7 and Mx, respectively), spleen (16- and 4.7-fold increase at peak level for SmIRF-3 and Mx, respectively), gills (3.7- and 2.8-fold increase at peak level for SmIRF-3 and Mx, respectively) and muscle (4.5- and 2.8-fold increase at peak level for SmIRF-7 and Mx, respectively) upon challenge with TRBIV. These results indicate that IRF-3 and IRF-7 have essential roles in antiviral responses to virus infection. Our findings help an understanding of the IFN system in turbot and researches on the control of turbot viral diseases using immune methods. |
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