| Japanese flouder (Paralichthys olivaceus) is one of the most economically valuable and widely cultured species in China. During the last decade, with the development of intensive indoors farming of flouder, various diseases, especially viral disease has prevailed leading to a great economic loss in the flouder farming. An understanding of immune defense mechanisms of Japanese flouder can contribute to control of the diseases of this species.Because Japanese flouder is a kind of low vertebrate, the IFN system plays a important role in its immune defense. Interferon regulatory factors (IRFs) are a family of transcription regulators with diverse roles in lots of cellular processes.IRF-3 and IRF-7 are the key IRF members, playing a pivotal role in transcriptional activation of IFN gene and IFN stimulating genes upon viruse infection. However, up to date, the IRF-3 and IRF-7 have not been cloned in Japanese flouder.In this study, full-length cDNAs encoding IRF-3 (GenBank accession number: GU017417 and GU017418 for two cDNA variants) and IRF-7(GenBank accession number:GU017419) have been cloned using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) from the head kidney tissue of Japanese flouder, Paralichthys olivaceus. The two IRF-3 variants are generated by the different polyadenylation sites. The IRF-3 cDNA contain an open reading fame (ORF) of 1404 nucleotides (bp) encoding 467 amino acid residues, with a 5'untranslated region (UTR) of 245 bp and 3'UTR of 552 bp for the long variant and 327 bp for the short one. The putative IRF-3 protein possesses a DNA-binding domain(DBD),an IRF association domain(IAD)and a serine-rich C terminal domain (SRD) found in all IRF-3 members. The IRF-7 cDNA contains an ORF of 1293 bp encoding 430 amino acid residues, with a 5'UTR of 84 bp and a 3'UTR of 655 bp. The putative IRF-7 protein also possesses domains of DBD, IAD and SRD. Phylogenetic analysis demonstrated that IRF-3 and IRF-7 have the most highest homology to those of other vertebrates, respectively. In the phylogenetic tree, they are clustered into IRF-3 and IRF-7 groups, respectively, which constitute the IRF3 subfamily.The tissue distribution patterns of IRF-3 and IRF-7 in healthy flounders were examined by RT-PCR method. High levels of IRF-3 mRNA expression were seen in the kidney, spleen and skin. A weaker expression was detected in the esophagus, stomach, intestinal, bladder, muscle and head kidney. The IRF-7 was expressed in all examined tissues wiht a high expression level in the brain, gill, gullet, esophagus, stomach, intestinal and bladder and a weaker expression in the heart, headkidney, kidney, spleen, gonad and skin.We further investigated the expression profile of IRF-3, IRF-7 and other important genes in the IFN system of flounder upon poly I:C stimulation or lymphocystic disease virus (LCDV) infection by real time PCR. The expressions of IRF-3,IRF-7, IFN-γ(GenBank accession number:AB435093) and Mx (GenBank accession number:AB110446) were all up-regulated after challenged by PolyI:C or LCDV.However, the expression of IFN-I (GenBank accession number:AB511962) couldn't be dectable in FG9307 cells in both cases. In PolyI:C-treated FG9307 cells, the expressions of both IRF-3 and IRF-7 showed an approximate 3-fold increase at the highest levels which appeared at 24 h post stimulation (p. s), and IRF-γand Mx showed 4 and 7-fold increase, respectively, at their highest levels both of which appeared at 48 h p. s.. In LCDV-infected FG9307 cells, both IRF-3 and IRF-7 reached their highest expression levels at 72 h post infection (p. i.)with 7 and 6-fold increase, respectively, whilst both IFN-γand Mx reached their highest expression levels at 96 h p. i.with about 5 and 7-fold increase, respectively. After intraperitoneal injection of Japanese flouder with LCDV, IRF-7, IFN-I, IFN-γand Mx were all found to be up-regulated in the headkidney. The IFN-I showed an approximate 48-fold increase at the highest levels appearing at 3 d p. i.The IRF-7, IFN-y and Mx showed,approximate 14,25 and 80-fold increase, respectively, at the highest levels all appearing at 5 d p.i.. Similar to human IRF-3,the flouder IRF-3 is constitutively expressed in head kidney of Japanese flouder without being affected by virus infection. These results suggest that IFN system exists in Japanese flouder and IRF-3 and IRF-7 play important roles in innate immune responsive to virus infection. Our findings contribute to an understanding of the IFN system in Japanese flouder, thus providing a basis for the breeding of antiviral fish species by tansgenetic technology and for the development of antiviral drug to cure fish disease as well. |
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