In this report, three type I IFN genes were identified in rainbow trout (rt) Oncorhynchus mykiss and are classified into two groups based on their primary protein sequences: group I containing two cysteine residues; and group II containing four cysteines residues. The group I rtIFNs were induced in fibroblasts (RTG-2 cells), macrophages (RTS-11 cells), and head kidney leukocytes when stimulated with polyinosinic:polycytidylic acid, whereas group II IFN was up-regulated in head kidney leukocytes but not in RTG-2 and RTS-11 cells. Recombinant group I rtIFNs were potent at inducing Mx expression and eliciting antiviral responses, whereas recombinant group II rtIFN was poor in these activities. That two subgroups of type I IFN exist in trout prompted a survey of the genomes of several fish species, including zebrafish, medaka, threespine stickleback and fugu, the amphibian Xenopus tropicalis, the monotreme platypus and the marsupial opossum, to gain further insight into possible IFN evolution. Analysis of the sequences confirmed that the new IFN subgroup found in trout (group II IFN) exists in other fish species but was not universally present in fish. The IFN genes in amphibians were shown for the first time to contain introns and to conserve the four cysteine structure found in all type I IFNs except IFN-βε and fish group I IFN. The data overall support the concept that different vertebrate groups have independently expanded their IFN types, with deletion of different pairs of cysteines apparent in fish group I IFN and IFN-βε of mammals.

A homologue of interleukin 18 has been identified from rainbow trout, Oncorhynchus mykiss. The trout IL-18 gene spans 3.7 kb and consists of six exons and five introns, sharing the same gene organization with its human counterpart. The putative translated protein is 199 amino acids in length with no predicted signal peptide. Analysis of the multiple sequence alignment reveals a conserved ICE cut site, resulting in a mature peptide of 162 amino acids. The trout IL-18 shares 41-45% similarity with known IL-18 molecules and contains an IL-1 family signature motif. It is constitutively expressed in a wide range of tissues including brain, gill, gut, heart, kidney, liver, muscle, skin and spleen. Transcription is not modulated by lipopolysaccharide, poly(I:C) or trout recombinant IL-1b in primary head kidney leucocyte cultures and RTS-11 cells, a macrophage cell line. However, expression is downregulated by lipopolysaccharide and rIL-1b in RTG-2 cells, a fibroblast-like cell line. An alternatively spliced form of IL-18 mRNA has also been found and translates into a 182 amino acid protein with a 17 amino acid deletion in the precursor region of the authentic form. This alternatively spliced form is also widely expressed although much lower than the authentic form. Interestingly, its expression is upregulated by lipopolysaccharide and poly(I:C), but is not affected by rIL-1b in RTG-2 cells. The present study suggests that alternative splicing may play an important role in regulating IL-18 activities in rainbow trout.Keywords: interleukin 18; alternative splicing; expression; rainbow trout.In the last few years, major advances have occurred in the discovery of fish cytokine genes. This has been attributed mainly to the enormous progress made in genome projects for the Fugu and zebrafish genome, and the large increase of fish EST (expressed sequence tag) entries in the Genebank. Interleukin (IL) 18, produced mainly by activated macrophages, is an important cytokine with multiple functions in innate and acquired immunity [18][19][20]. One of the primary biological properties is to induce interferon gamma (IFN-c) synthesis in Th1 and NK cells in synergy with 22].It promotes T and NK cell maturation, activates neutrophils and enhances Fas ligand-mediated cytotoxicity [23][24][25].IL-18 structurally belongs to the IL-1 family but has low sequence homology with IL-1a, IL-1b and the IL-1 receptor antagonist (IL-Ra). It resembles IL-1b in many ways such as possessing a similar b-trefoil structure and secretion process but has distinct biological functions [18,26]. Like IL-1b, it is synthesized as an inactive precursor of approximately 24 kDa and is stored intracellularly. Activation and secretion of IL-18 is mainly effected through specific cleavage of the precursor after D35 by caspase 1, also termed IL-1b converting enzyme (ICE), which is believed to be one of the key processes regulating IL-18 bioactivity [27,28]. Some other enzymes, including caspase 3 and neutrophil proteinase 3, also cleave the IL-18 precursor to generate...