What is known about the biological activity of fish cytokines is reviewed. Most of the functional studies performed to date have been in teleost fish, and have focused on the induced effects of cytokine recombinant proteins, or have used loss- and gain-of-function experiments in zebrafish. Such studies begin to tell us about the role of these molecules in the regulation of fish immune responses and whether they are similar or divergent to the well-characterised functions of mammalian cytokines. This knowledge will aid our ability to determine and modulate the pathways leading to protective immunity, to improve fish health in aquaculture.

IFN-γ is one of the key cytokines in defining Th1 immune responses. In this study, an IFN-γ homologue has been identified in rainbow trout Oncorhynchus mykiss, and its biological activities have been characterized. The trout IFN-γ cDNA is 1034 bp in length and translates into a 180-aa protein. The first intron of the trout IFN-γ gene contains highly polymorphic GACA minisatellites and 44-bp DNA repeats, giving rise to at least six alleles. IFN-γ is structurally conserved among vertebrates, and a signature motif has been identified. A nuclear localization sequence known to be crucial for IFN-γ biological activities is also present in the C-terminal region of the trout IFN-γ. The IFN-γ expression was induced in head kidney leukocytes by stimulation with PHA or poly(I:C) and in kidney and spleen of fish injected with poly(I:C). rIFN-γ produced in Escherichia coli significantly stimulated gene expression of IFN-γ-inducible protein 10 (γIP-10), MHC class II β-chain, and STAT1, and enhanced respiratory burst activity in macrophages. Deletion of 29-aa residues from the C terminus containing the nuclear localization sequence motif resulted in loss of activity with respect to induction of γIP-10 in RTS-11 cells. Moreover, IFN-γ-induced γIP-10 expression was completely abolished by the protein kinase C inhibitor staurosporine, and partially reduced by U0126, a specific inhibitor for ERKs. Taken together, the present study has demonstrated for the first time a functional IFN-γ homologue in a fish species, strongly suggesting a conserved Th1 immune response is most likely present in lower vertebrates.

The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

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.

The retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) comprise three homologues: RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). They activate the host interferon (IFN) system upon recognition of viral RNA pathogen-associated molecular patterns (PAMPs) in the cytoplasm. Bioinformatic analysis of the sequenced vertebrate genomes suggests that the cytosolic surveillance system is conserved in lower vertebrates, and recent functional studies have confirmed that RIG-I is important to fish antiviral immunity. In this study, we have identified MDA5 and LGP2 homologues from rainbow trout Oncorhynchus mykiss and an additional LGP2 variant with an incomplete C-terminal domain of RIG-I. Trout MDA5 and LGP2 were constitutively produced in fibroblast and macrophage cell lines and upregulated by poly(I:C), recombinant IFN, or infection by RNA viruses (viral hemorrhagic septicemia virus and salmon alphavirus) with a single-stranded positive or negative genome. Overexpression of MDA5 and LGP2 but not of the LGP2 variant resulted in significant accumulation of Mx transcripts in cultured cells, which correlated with a marked enhancement of protection against viral infection. These results demonstrate that both MDA5 and LGP2 are important RLRs in host surveillance against infection of both negative and positive viruses and that the LGP2 variant with a deletion of 54 amino acids at the C terminus acts as a negative regulator for LGP2-elicited antiviral signaling by competing for the viral RNA PAMPs. Interestingly, MDA5 expression was not affected by overexpressed LGP2 in transfected cells and vice versa, suggesting that they likely act in parallel as positive regulators for IFN production.