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Type I interferons (IFNs) are critical cytokines for the establishment of antiviral status in fish, amphibian, avian and mammal, but the knowledge on type I IFNs is rather limited in reptile. In this study, seven type I IFN genes, designed as IFN1 to IFN7, were identified from a reptile species, the Chinese soft-shelled turtle (Pelodiscus sinensis). These identified type I IFNs have relatively low protein identity, when compared with those in human and chicken; but they possess conserved cysteines, predicted multi-helix structure and N-terminal signal peptide. The Chinese soft-shelled turtle IFN1 to IFN5 have two exons and one intron, but IFN6 and IFN7 are the single-exon genes. Chinese soft-shelled turtle type I IFNs are located respectively on the two conserved reptile-bird loci, named as Locus a and Locus c, and are clustered into the four of the five reptile-bird groups (named as Groups I-V) based on phylogenetic evidence, due to the lack of IFNK in the turtle. Moreover, the Chinese soft-shelled turtle type I IFNs can be induced by soft-shelled turtle iridovirus (STIV) infection and show antiviral activity in soft-shelled turtle artery (STA) cells, except IFN6. In addition, due to the difference in genome organizations, such as the number of exons and introns of type I IFN genes from fish to mammal, the definition and evolution of 'intronless' type I IFN genes were discussed in lineages of vertebrates. Thus, the finding of type I IFNs on two different loci in P. sinensis sheds light on the evolution of type I IFN genes in vertebrates.

Toll-like receptors (TLRs), as a family of pattern recognition receptors (PRRs), possess specific pathogen-related molecular pattern (PAMP) recognition spectrum in inducing immune responses. In this study, sixteen TLRs were identified and characterized in mandarin fish (Siniperca chuatsi). All these TLRs consist of leucine-rich repeats (LRRs), a transmembrane domain and a Toll/interleukin-I receptor (TIR) domain, with the exception of TLR5S which lacks TIR domain, and they can be clustered into five branches, i.e. TLR1 subfamily, TLR3 subfamily, TLR5 subfamily, TLR7 subfamily and TLR11 subfamily in phylogenetic tree. These TLR genes were expressed in all tested tissues and had high expression levels in immune-related tissues such as head-kidney and spleen or mucosa-related tissues such as intestine and pyloric caecum. The transcripts of TLR2a, TLR2b, TLR3, TLR13a, TLR14, TLR22 and TLR23 were all significantly up-regulated after stimulation with poly(I:C); TLR1, TLR2a, TLR2b, TLR3, TLR5M, TLR5S, TLR13a and TLR13b transcripts were all significantly up-regulated after stimulation with PGN; and TLR2a, TLR2b, TLR5M, TLR5S, TLR7, TLR8, TLR9, TLR13c, TLR14 and TLR22 transcripts were all significantly up-regulated after stimulation with LPS in isolated head kidney lymphocytes of mandarin fish. The findings in this study may provide a valuable basis for functional study on TLR genes in mandarin fish.

Toll/interleukin-1 receptor (TIR) domain-containing adaptors, serve as pivotal signal transduction molecules in Toll-like receptor (TLR) signalling pathway to mediate downstream signalling cascades. In this study, four TIR-domain containing adaptors, MyD88, TRIF, MAL and SARM, were identified in mandarin fish Siniperca chuatsi, and they all contain TIR domains, of which MyD88 and SARM had high sequence homology with their vertebrate homologues. The expression analysis at mRNA level indicated that these genes were ubiquitously distributed in different tissues, being high in immune- and mucosa-related tissues such as head-kidney and intestine. The transcripts of these adaptor genes were up-regulated by poly(I:C) and LPS stimulation in isolated head-kidney lymphocytes (HKLs) of mandarin fish. Fluorescence microscopy revealed that all these molecules were localized in cytoplasm, and further investigations showed that the over-expression of MyD88, TRIF and MAL activated the NF-κB, ISRE or type Ι IFN promoters and inhibited SVCV replication, whereas their antiviral effects were significantly impaired when co-transfected with SARM. It was also confirmed by co-immunoprecipitation (Co-IP) that SARM interacts separately with MyD88, TRIF and MAL, and MAL interacts with MyD88. However, the regulatory mechanisms of these adaptors involved in signalling pathways of different TLRs should be of interest for further research.

Interferon (IFN)-stimulated genes (ISGs) exert multiple functions in immune system, and IFN-induced protein 35 (IFP35), which is a member of ISG, has been suggested to be involved in numerous cellular activities including the regulation of antiviral immunity in mammals. However, the role of IFP35 in fish innate immunity remains largely unknown. In the present study, we characterized the IFP35 gene in mandarin fish Siniperca chuatsi, which contains two conserved Nmi/IFP35 homology domains (NIDs) at C-terminus, but no leucine zipper motif, with its genomic DNA sequence consisting of eight exons and seven introns. High and constitutive mRNA level of IFP35 was observed in all examined tissues, with the highest level being observed in gills. Moreover, the IFP35 gene was significantly induced in vivo for 120 h following the infection of infectious spleen and kidney necrosis virus (ISKNV), and its mRNA and protein level was also significantly induced in vitro following the treatment of poly I:C, IFNh, IFNc, as well as IFN-γ. The subcellular localization results indicated that exogenous IFP35 protein was mainly located in cytoplasm, while endogenous IFP35 protein was transferred into, or aggregated around, the nucleus with the induction of poly I:C or IFNs. The dual luciferase activity analysis indicated that the IFP35 promoter was activated by type I and type II IFNs through ISRE site. It is considered that IFP35 in fish is involved in antiviral, as well as in IFN-induced innate immunity.

Interferons (IFNs) can induce the expression of IFN-stimulated genes (ISGs), such as myxovirus resistance (Mx) protein, to inhibit virus replication. In this study, the expression of Mx gene in mandarin fish, and the IFN-sensitive response elements (ISREs) and gamma-interferon activated sites (GASs) in the promoter of Mx gene were analyzed in relation to the stimulation of three distinct type I IFNs, IFNc, IFNd and IFNh, and two type II IFNs, IFN-γ and IFN-γ related molecule (IFN-γrel). A single Mx gene was found in mandarin fish, and its expression was highly and constitutively observed in all organs/tissues examined. The Mx gene was significantly induced in vivo for 120 h following infectious spleen and kidney necrosis virus (ISKNV) infection. Furthermore, the overexpression and recombinant of IFNh, IFNc, as well as IFN-γ can significantly induce Mx expression in MFF-1 cells at transcript and protein levels, although all the three type I IFNs and the two type II IFNs can activate the Mx promoter. In addition, ISRE1 which is the proximal one among the three predicted ISREs seems to be the important ISRE for the higher and efficient activation of the Mx promoter. However, the possible interaction between the GASs and type II IFN signalling molecules require further study.

The interleukin (IL) -1 family members play an important role in regulating inflammatory responses and their functions are mediated by a group of receptors consisting of immunoglobulin and Toll/IL-1 receptor (TIR) domains. In humans, 10 IL-1Rs are found. In this study, 5 IL-1 receptors including IL-1R3/IL-1RAcP, IL-1R8/SIGIRR, IL-1R9a/IL-1RAcPL1a, IL-1R9b/IL-1RAcPL1b and IL-1R10/IL-1RAcPL2 were identified in grass carp (Ctenopharyngodon idella). Phylogenetic analysis reveals that the IL-1R9a/IL-1RAcPL1a and IL-1R9b/IL-1RAcPL1b share significantly high sequence similarity and are believed to have been duplicated from the same gene prior to the radiation of teleosts. Further, these two receptors closely relate to the IL-1R10/IL-1RAcPL2, suggesting that they may have evolved from a common ancestor. The IL-1R3/IL-1RAcP, IL-1R9a/IL-1RAcPL1a, IL-1R9b/IL-1RAcPL1b and IL-1R10/IL-1RAcPL2 are highly expressed in the brain. Stimulation of primary spleen leucocytes by LPS and intraperitoneal injection of fish with poly (I:C) or bacterial infection results in significant increases of IL-1R3/IL-1RAcP expression. Interestingly, the IL-1R8/SIGIRR and IL-1R10/IL-1RAcPL2 showed similar expression patterns.

IFN-γ, as the sole member of mammalian type II IFN, is a multifunctional cytokine which exerts its effects through two distinct IFN-γ receptors, IFNGR1 and IFNGR2. However, in teleost fish, another IFN-γ homologous gene, namely IFN-γ related gene (IFN-γrel), has been identified. Although IFN-γ and IFN-γrel genes have been described in some fish species, many important aspects remain poorly understood in relation with their signalling and function. In the present study, IFN-γ and IFN-γrel, as well as their receptors, cytokine receptor family B (CRFB) 17, CRFB13, two of which are homologous to IFNGR1 in mammals, and CRFB6, homolomous to IFNGR2, have been characterized in mandarin fish, Siniperca chuatsi. It was revealed that the two type IFN members exhibit antiviral activity, and IFN-γ transduces downstream signalling through CRFB13 and CRFB6, while IFN-γrel interacts with CRFB17 to activate downstream signalling. Moreover, IFN-γ and IFN-γrel have been shown to exert antiviral biological activity in a STAT1-dependent manner. Intracellular domain analysis of CRFB17 and CRFB13 demonstrated that the Y386 tyrosine residue of CRFB13 is required for the activation of the IFN-γ-mediated biologic response, and the Y324 and Y370 residues in CRFB17 are required to activate IFN-γrel signalling.

NOD-like receptors (NLRs) are a family of intracellular pattern recognition receptors (PRRs) that play critical roles in innate immunity against pathogens infection. NLRC5, the largest member of NLR family, has been characterized as a regulator of innate immunity and MHC class I expression. Alternative splicing of NLRC5 is only reported in human and zebrafish. However, the function of NLRC5 isoforms in the innate immune responses remains unknown. In the present study, we report the functional characterization of zfNLRC5a and zfNLRC5d, two splicing isoforms of zebrafish NLRC5. zfNLRC5a and zfNLRC5d are generated by exon skipping, and whose alternative splicing sites exist in the region of LRRs. Fluorescence microscopy showed that zfNLRC5 isoforms were located throughout the entire cell including nuclear staining. The expression of zfNLRC5 isoform was inducible in response to bacterial and viral infections. During SVCV infection, the in vitro and in vivo studies found that zfNLRC5d overexpression increased protection against viral infection; however zfNLRC5a overexpression had no significant effect on antiviral activity. Interestingly, zfNLRC5 isoforms but not zfNLRC5 were involved in transcriptional regulation of TLRs and NF-κB signaling. Overexpression of zfNLRC5 isoforms also contributed to negative regulation of antibacterial immune response, with the decreased expression of nfkbiaa (IκBα). All together, these results firstly demonstrate the function of NLRC5 isoforms in antiviral and antibacterial immune responses both in vitro and in vivo.

Highlights • The cDNA sequences of CXCR2, CXCR3a and CXCR3b have been cloned in rainbow trout. • The linked CXCR1/CXCR2 and CXCR3a/CXCR3b loci are hypothesised to have been present in the teleostomian ancestor. • CXCR1 and CXCR2 have likely undergone gene conversion whilst CXCR3b has been lost in mammals. • Compared with mammals, ray-finned fish possess more CXCR1–R3 receptors, but fewer ligands. • Trout CXCR1–R3 are expressed in macrophages and neutrophils, with CXCR1/R2 also abundant in B-cells., The chemokine receptors CXCR1–3 bind to 11 chemokines (CXCL1–11) that are clustered on the same chromosome in mammals but are largely missing in ray-finned fish. A second CXCR1/2, and a CXCR3a and CXCR3b gene have been cloned in rainbow trout. Analysis of CXCR1–R3 genes in lobe-finned fish, ray-finned fish and tetrapod genomes revealed that the teleostomian ancestor likely possessed loci containing both CXCR1 and CXCR2, and CXCR3a and CXCR3b. Based on this synteny analysis the first trout CXCR1/2 gene was renamed CXCR1, and the new gene CXCR2. The CXCR1/R2 locus was shown to have further expanded in ray-finned fish. In relation to CXCR3, mammals appear to have lost CXCR3b and birds both CXCR3a and CXCR3b during evolution. Trout CXCR1–R3 have distinct tissue expression patterns and are differentially modulated by PAMPs, proinflammatory cytokines and infections. They are highly expressed in macrophages and neutrophils, with CXCR1 and CXCR2 also expressed in B-cells.

Type I IFNs are considered to be the core IFN species in vertebrates because of their predominant antiviral effects. But, a puzzling question remains to be answered, as to how intronless type I IFN genes in amniotes might have evolved from intron-containing type I IFN genes in fish and amphibians. In this study, intronless and intron-containing type I IFNs were found in the amphibian model, Xenopus tropicalis, with a total of sixteen and five genes, respectively. The intronless IFNs can be divided into three subgroups, and the intron-containing ones into two subgroups, implying that a retroposition event might have occurred in amphibians, resulting in the generation of intronless type I IFN genes. Two models were tentatively proposed to explain the evolution of type I IFNs in vertebrates: in model A, fish should possess the most primitive multi-exon-containing type I IFNs, and intronless type I IFN genes in amphibians are the ancestor of modern intronless type I IFNs in amniotes; in model B, intronless type I IFN genes in X. tropicalis may just represent an independent bifurcation in this species or probably in amphibians, and intronless type I IFN genes in amniotes may have arisen from another retroposition event occurred in a transition period even when reptiles were diverged from amphibians. It is considered that the model B can reflect the current knowledge on the occurrence of intronless and intron-containing type I IFN genes in vertebrate lineages. This study thus contributes to a better understanding of the origin and evolution of type I IFNs in vertebrates, and of the occurrence of intronless I IFNs in higher vertebrates.

IFN-λ (IFNL), i.e. type III IFN genes were found in a conserved gene locus in tetrapod vertebrates. But, a unique locus containing IFNL was found in avian. In turtle and crocodile, IFNL genes were distributed in these two separate loci. As revealed in phylogenetic trees, IFN-λs in these two different loci and other amniotes were grouped into two different clades. The conservation in gene presence and gene locus was also observed for the receptors of IFN-λ, IFN-λR1 and IL-10RB in tetrapods. It is further revealed that in North American green anole lizard Anolis carolinensis, a single IFNL gene was situated collinearly in the conserved locus as in other tetrapods, together with its receptors IFN-λR1 and IL-10RB also identified in this study. The IFN-λ and its receptors were expressed in all examined organs/tissues, and their expression was stimulated following the injection of polyI:polyC. The ISREs in promoter of IFN-λ in lizard were responsible to IRF3 as demonstrated using luciferase report system, and IFN-λ in lizard functioned through the receptors, IFN-λR1 and IL-10RB, as the up-regulation of ISGs was observed in ligand-receptor transfected, and also in recombinant IFN-λ stimulated, cell lines. Taken together, it is concluded that the mechanisms involved in type III IFN ligand-receptor system, and in its signalling pathway and its down-stream genes may be conserved in green anole lizard, and may even be so in tetrapods from xenopus to human.

The mitochondrial antiviral signaling protein (MAVS) plays a key role in the signal transduction of RIG-I-like receptors (RLRs)-mediated antiviral response. In the present study, zebrafish MAVS transcript variants, namely MAVS_tv1 and MAVS_tv2, were cloned from zebrafish embryos. The putative MAVS_tv1 protein (full length form) contains an N-terminal CARD domain, a central proline region, and a C-terminal transmembrane domain (TM). MAVS_tv2 is generated by a 190 bp intron fragment insertion. The putative MAVS_tv2 protein lacked TM domain due to a frame shift, with the N-terminal 303 aa residues identical to MAVS_tv1, and no sequence homology for the C-terminal 41 aa residues. Real-time PCR showed that the expression of MAVS_tv1 in ZF4 cells was higher than that of MAVS_tv2, and MAVS variants were induced by Edwardsiella tarda and SVCV infection during the early time points of infection, whereas MAVS_tv1 unchanged or MAVS_tv2 decreased at a later time point after the infection, respectively. Overexpression of MAVS_tv1 and MAVS_tv2 in fish cells conferred antiviral resistance, and activated zebrafish IFN1 and IFN3 promoters. MAVS_tv1 overexpression induced a slow (48 hpf) increased expression of IFNI, mxa, mxb, mxe and RSAD2. In contrast, MAVS tv2 overexpression increased rapidly and transiently the expression of IFN1, IFN2, IFN3, mxc and rsad2 at 6 or 24 hpf. The simultaneous overexpression of MAVS variants and RIG-I in zebrafish embryos led to an accumulative induction of IFNs and IFN-stimulated genes including IFN1, IFN4, mxc, mxe and rsad. Furthermore, MAVS_tv1 cooperated with RIG-I in the accumulation of RIG-I transcript in a positive feedback loop; MAVS_tv2 synergized with MDA5 in the accumulation of MAVS_tv2 transcript. Collectively, these data suggest the molecular mechanisms of fish MAVS variants in antiviral immunity. (C) 2014 Elsevier Ltd. All rights reserved.

Lower vertebrates have been found to possess over 200 NACHT-domain encoding genes; but, to date, very little is known about their functional activity. This article describes the sequences and expression analysis of two zebrafish NACHT-containing proteins, namely NALPL1 and NALPL2. In addition, the functions of zebrafish NALPL1 and NALPL2, which are absent for both amino-terminal effector-binding domain (EBD) and carboxy-terminal ligand-recognition domain (LRD), were investigated for the first time in fish species. The predicted NALPL1 and NALPL2 proteins consist of 651 and 847 amino acids (aa), respectively, with both molecules only containing NACHT domain, which were different from other NACHT-family members. Phylogenetic analysis showed that zebrafish NALPL1 and NALPL2 have a closer relationship with mammalian NALP subfamily than NOD subfamily. The differential expression patterns of NALPL1 and NALPL2 in development stages and organs were observed, suggesting the difference of action phase and effector organ of NALPL1 and NALPL2. When the modulation of NALPL1 and NALPL2 in pathogen infection was analyzed, it was found that the two molecules were upregulated by both bacterial and viral infection. Overexpression of NALPL1 and NALPL2 resulted in significant inhibition for intracellular Edwardsiella tarda growth. Further studies demonstrated that NALPL1 and NALPL2 also contributed to protection against viral infection. These results demonstrate that both NALPL1 and NALPL2 are important intracellular proteins in host surveillance against both bacterial and viral infection. Interestingly, the expression of downstream signaling genes was not affected by the overexpression of NALPL1 or NALPL2, but NOD1 and MDA5 were upregulated by NALPL1 or NALPL2 overexpression, suggesting that they likely act in pathogen infection through the interaction with other PRRs. (C) 2014 Elsevier Ltd. All rights reserved.

The IFN-gamma gene was identified in a turtle, the Chinese soft-shelled turtle, Pelodiscus sinensis, with its genome consisting of 4 exons and 3 introns. The deduced amino acid sequence of this gene contains a signal peptide, an IFN-gamma family signature motif (130)IQRKAVNELFPT, an NLS motif (KRKR)-K-155 and three potential N-glycosylation sites. As revealed by real-time quantitative PCR, the gene was constitutively expressed in all tested organs/tissues, with higher level observed in blood, intestine and thymus. An induced expression of IFN-gamma at mRNA level was observed in peripheral blood leucocytes (PBLs) in response to in vitro stimulation of LPS and PolyI:C. The overexpression of IFN-gamma in the Chinese soft-shelled turtle artery (STA) cell line resulted in the increase in the expression of transcriptional regulators, such as IRF1, IRF7 and STAT1, and antiviral genes, such as Mx, PKR, implying possibly the existence of a conserved signalling network and role for IFN-gamma in the turtle. Furthermore, the infection of soft-shelled turtle iridovirus (STIV) in the cell line transfected with IFN-gamma may cause the cell death as demonstrated with the elevated lactate dehydrogenase (LDH) level and cell mortality. However, the mechanism involved in the antiviral activity may require further investigation. (C) 2013 Elsevier Ltd. All rights reserved.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of innate immunity. In this study, a long-form PGRP, designated as gcPGRP6, was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP6 is composed of 464 residues with a conserved PGRP domain at the C-terminus. The gcPGRP6 gene consists of four exons and three introns, spacing approximately 2.7 kb of genomic sequence. Phylogenetic analysis demonstrated that gcPGRP6 is clustered closely with zebrafish PGLYRP6, and formed a long-type PGRP subfamily together with PGLYRP2 members identified in teleosts and mammals. Real-time PCR and Western blotting analyses revealed that gcPGRP6 is constitutively expressed in organs/tissues examined, and its expression was significantly induced in liver and intestine of grass carp in response to PGN stimulation and in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and peptidoglycan (PGN). Immunofluorescence microscopy and Western blotting analyses revealed that gcPGRP6 is effectively secreted to the exterior of CIK cells. The over-expression of gcPGRP6 in CIK cells leads to the activation of NF-kappa B and the inhibition of intracellular bacterial growth. Moreover, cell lysates from CIK cells transfected with pTurbo-gcPGRP6-GFP plasmid display the binding activity towards Lys-type PGN from Staphylococcus aureus and DAP-type PGN from Bacillus subtilis. Furthermore, proinflammatory cytokine IL-2 and intracellular PGN receptor NOD2 had a significantly increased expression in CIK cells overexpressed with gcPGRP6. It is demonstrated that the PGRP6 in grass carp has a role in binding PGN, in inhibiting the growth of intracellular bacteria, and in activating NF-kappa B, as well as in regulating innate immune genes. (C) 2013 Elsevier Ltd. All rights reserved.

In this study, interferon gamma (IFN-γ) and interferon gamma receptor (IFN-γR) genes have been identified in non-avian reptile, the North American green anole lizard (Anolis carolinensis). Like their counterparts from other jawed vertebrates, lizard IFN-γ, IFN-γR1 and IFN-γR2 show conserved features in genomic organizations, gene loci and protein sequences. The IFN-γ gene has the full cDNA sequence of 936 bp, with 522 bp open reading frame (ORF) encoding 174 amino acids, and has the genomic organization of four exons and three introns as observed in IFN-γ genes of other classes of vertebrates. The receptors, IFN-γR1 and IFN-γR2 have the ORF of 1278 and 984 bp, coding for 425 and 327 aa, respectively, with the genome organization of seven exons and six introns. In the gene loci of IFN-γ, DYRK2, IL22, IL26 and MDM1 are found with conserved synteny in vertebrates, and similar genes adjacent to IFN-γR1 and IFN-γR2 were also found. These receptors also contain conserved motifs, such as the membrane-proximal region and the C-terminal five residue motif in IFN-γR1, and intracellular conservative sequence in IFN-γR2, which have been confirmed to mediate down-stream JAK-STAT signaling pathway in mammals. IFN-γ and its receptors, IFN-γR1 and IFN-γR2 were constitutively expressed in organs/tissues examined in the lizard, and up-regulated expression of IFN-γ was observed in organs/tissues examined following the poly(I:C) stimulation, suggesting its antiviral role in lizards. The conserved features of IFN-γ and its receptors, IFN-γR1 and IFN-γR2, in gene organization and gene locus as well as in functional domain or motif may imply that the function of type II IFN system is evolutionarily conserved in the green anole lizard, as observed in other classes of vertebrates.

In this study, we have identified 421 molecules across the vertebrate spectrum and propose a unified nomenclature for CXC chemokines in fish, amphibians and reptiles based on phylogenetic analysis. Expanding on earlier studies in teleost fish, lineage specific CXC chemokines that have no apparent homologues in mammals were confirmed. Furthermore, in addition to the two subgroups of the CXCL8 homologues known in teleost fish, a third group was identified (termed CXCL8_L3), as was a further subgroup of the fish CXC genes related to CXCL11. Expression of the CXC chemokines found in rainbow trout, Oncorhynchus mykiss, was studied in response to stimulation with inflammatory and antiviral cytokines, and bacterial. Tissue distribution analysis revealed distinct expression profiles for these trout CXC chemokines. Lastly three of the trout chemokines, including two novel fish specific CXC chemokines containing three pairs of cysteines, were produced as recombinant proteins and their effect on trout leucocyte migration studied. These molecules increased the relative expression of CD4 and MCSFR in migrated cells in an in vitro chemotaxis assay.