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In the present study, the zinc finger aspartate-histidine-histidine-cysteine (DHHC)-type containing 1 (ZDHHC1) gene was identified in a commercial fish, the Chinese perch Siniperca chuatsi. The ZDHHC1 has five putative transmembrane motifs and conserved DHHC domain, showing high amino-acid identity with other teleost fish, and vertebrate ZDHHC1 loci are conserved from fish to human. In vivo expression analysis indicated that ZDHHC1 gene was constitutively transcribed in all the examined organs/tissues, and was induced following infectious spleen and kidney necrosis virus (ISKNV) infection. It is further observed that ZDHHC1 interacts with MITA and the overexpression of ZDHHC1 in cells resulted in the upregulated expression of ISGs, such as Mx, RSAD2, IRF3 and type I IFNs such as IFNh and IFNc, exhibiting its antiviral function in fish as reported in mammals.

In fish, type I IFNs are classified into three groups, i.e. Group I, Group II and Group III, which are further divided into seven subgroups according to the number of conservative cysteines, phylogenetic relationship, and probably their receptor complexes. In the present study, four type I IFNs and four cytokine receptor family B members (CRFBs) were identified in the Asian arowana, Scleropages formosus, an ancient species in the Osteoglossomorpha with commercial and conservation values. According to multiple sequence alignment and phylogenetic relationship, the four type I IFNs are named as IFNa1, IFNa2, IFNb and IFNc, with the former two belonging to Group I, and the latter two to Group II. The four receptors are named as CRFB1, CRFB2, CRFB5a and CRFB5b. The IFNs and their possible receptor genes are widely expressed in examined organs/tissues, and are induced following the stimulation of polyinosinic polycytidylic acid (polyI:C) in vivo. It was found that IFNa1, IFNa2, IFNb and IFNc use preferentially the receptor complexes, CRFB1 and CRFB5b, CRFB1 and CRFB5b, CRFB2 and CRFB5a, and CRFB2 and CRFB5b, respectively, indicating the evolutionary diversification in the interaction of type I IFNs and their receptors in this ancient fish species, S. formosus.

Type I interferons are a subset of cytokines playing central roles in host antiviral defense, and their effects depend on the interaction with the heterodimeric receptor complex. Surprisingly, two pairs of the receptor subunits, CRFB1 and CRFB5, and CRFB2 and CRFB5, have been identified in fish, but the studies about preferential receptor usage of different fish IFN subtypes are rather limited. In this study, the three receptor chains of type I IFNs named as On-CRFB1, On-CRFB2 and On-CRFB5 were identified in Nile tilapia, Oreochromis niloticus. These three genes were constitutively expressed in all tissues examined, with the highest expression level observed in muscle and liver, and were rapidly induced in liver following the stimulation of poly(I:C). Interestingly, it is possible that all three subtypes of tilapia IFNs are able to signal through two pairs of the receptor subunits, On-CRFB1 and On-CRFB5, and On-CRFB2 and On-CRFB5. More importantly, tilapia group I IFNs (On-IFNd and On-IFNh) preferentially signal through a receptor complex composed of On-CRFB1 and On-CRFB5, and group II IFNs (On-IFNc) preferentially signal through a receptor complex comprised of On-CRFB2 and On-CRFB5. The present study thus provides new insights into the receptor usage of group I and group II IFNs in fish.

Interferons are a family of class II α-helical cytokines playing vital roles in antiviral immune response, and little information is available to date regarding the interferon system of tilapia. In this study, a type I IFN gene, named On-IFNc, was identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-IFNc contains several structural features known in type I IFNs, and On-IFNc was clustered together with the known IFNc in fish into a separated clade in the phylogenetic tree. On-IFNc gene was constitutively expressed in all tissues examined, with the highest expression level observed in liver, and was rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). In addition, recombinant On-IFNc has been proven to markedly induce the expression of the antiviral effectors, Mx and viperin, the signalling components, STAT1, STAT2, and IRF9, and the transcription factors, IRF3 and IRF7, as well as the tyrosine phosphorylation of STAT1 and STAT2 in fish cells. Furthermore, recombinant On-IFNc has been proven to possess antiviral activity against ISKNV. The present study thus contributes to a better understanding of the functional properties of the type I IFN system in tilapia.

In fish, type I IFNs are classified into three groups, i.e. group one, group two and group three, and further separated into seven subgroups based on the number of conserved cysteines and phylogenetic relationships. In the present study, four type I IFNs, named as IFNϕ1, IFNϕ2, IFNϕ3, IFNϕ4, as reported in zebrafish, were identified in a cyprinid, the topmouth culter, Culter alburnus, a species introduced recently into China's aquaculture. These IFNs may be classified as IFNa, IFNc, IFNc and IFNd in a recent nomenclature, with IFNa and IFNd having two cysteines in group one, and IFNc four cysteines in group two. These IFNs, together with their possible receptors, IFNϕ1, IFNϕ2, IFNϕ3, IFNϕ4, and CRFB1, CRFB2 and CRFB5 have an open reading frame (ORF) of 540, 552, 567, 516 bp, and 1572, 1392, 1125 bp, respectively. These IFNs have high amino acid sequence identities, being 91.1-93.6% and 66.9-77.3%, with those in grass carp and zebrafish, respectively, and are expressed constitutively in organs/tissues examined in the fish. The expression of these IFNs can be further induced following poly (I:C) stimulation. However, the possible function of these IFNs and their signalling pathway are of interest for further research.

DEAD-box helicase 41 (DDX41) is a key cytosolic DNA sensor playing critical roles in the regulation of type I IFN responses, and their functions have been well-characterized in mammals. However, little information is available regarding the function of fish DDX41. In this study, a DDX41 gene, named On-DDX41, was identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-DDX41 contains several structural features known in DDX41, including conserved DEADc and HELICc domains, and a conserved sequence "Asp-Glu-Ala-Asp (D-E-A-D)". On-DDX41 gene was constitutively expressed in all tissues examined, with the highest expression level observed in liver and muscle, and was inducible after poly(I:C) stimulation. Moreover, the overexpression of On-DDX41 can elicit a strong activation of both zebrafish IFN1 and IFN3 promoter in fish cells treated with poly(dA:dT). The present study thus contributes to a better understanding of the functional properties of DDX41 in fish.

The S100 family proteins are a group of small acidic polypeptides and have diverse functions in regulating many aspects of physiological processes. They are structurally conserved and possess two EF-hands which are central for calcium-mediated functions. In this study, 14 S100 cDNA sequences were determined in zebrafish and their genomic organizations confirmed. Re-analyzing the gene synteny of the S100 loci identified two major S100 loci in Chr16 and Chr19 which share remarkable conservation with the S100 locus in human Chr1, suggesting they may have evolved from a single locus during the teleost specific whole genome duplication event. It appears that the homologues of human S100G and S100P have been lost in zebrafish. Expression analysis reveals that S100W, ICN1 and ICN2 are markedly expressed in embryos. Further, the transcripts of S100 genes are relatively abundant in mucosal tissues such as gills and gut. Intraperitoneal injection of poly(I:C) resulted in up-regulation of most S100 genes in the gut and spleen, with highest induction of S100V2 and S100Z detected. In fish challenged with spring viremia of carp virus (SVCV), expression of most S100 family genes was increased in the spleen between day 1 and 7 post infection, with consistent induction seen for the S100A1, S100A10b, S100B, S100ICN1, S100T, S100U, S100V1 and S100Z. Interestingly, intraperitoneal injection of Edwardsiella tarda down-regulated S100 expression in the gut but resulted in induction in the spleen. The results demonstrate that the S100 family genes are differentially modulated by bacterial and viral pathogens in zebrafish.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) involved in host antibacterial responses, and their functions have been characterized in most invertebrate and vertebrate animals. However, little information is available regarding the function of frog PGRPs. In this study, a short-type PGRP (termed Xl-PGRP-S) gene was identified in the African clawed frog, Xenopus laevis. The predicted protein of Xl-PGRP-S contains several structural features known in PGRPs, including a typical PGRP domain and two closely spaced conserved cysteines. Xl-PGRP-S gene was constitutively expressed in all tissues examined, with the highest expression level observed in muscle. As a typical PRR, Xl-PGRP-S is inducible after peptidoglycan (PGN) stimulation, and has an ability to bind PGN. In addition, Xl-PGRP-S has been proven to have Zn2+-dependent amidase activity and antibacterial activity against Edwardsiella tarda. The present study represents the first discovery on the function of frog PGRPs, thus contributing to a better understanding of the functional evolution of PGRPs in early tetrapods.

As an important immune regulatory molecule, interleukin (IL)-22 has been reported in several species of fish, but its soluble receptor, IL-22 binding protein (IL-22BP), discovered as a natural antagonist of IL-22 in mammals, has not been functionally characterized in fish to date. In the present study, IL-22 and IL-22BP genes were cloned in mandarin fish Siniperca chuatsi. They all exhibited a high basal expression level in mucosa-enriched tissues, implying their possible roles in mucosal immunity. The IL-22 was found to show a potent response to LPS stimulation, acting as an inducer of antimicrobial peptide (AMP) genes, such as hepcidin and Liver-expressed antimicrobial peptide-2 (LEAP-2) in intestinal cells. IL-22BP, via co-incubation with IL-22, inhibited completely the induction of downstream genes by IL-22. Through a yeast two-hybrid assay, the interaction between IL-22BP and IL-22 was confirmed, which may account for the inhibitory effect of IL-22BP. Moreover, two hot spot residues for IL-22 binding, as reported in mammalian IL-22BP, were found to be conserved both in sequence location and function in mandarin fish IL-22BP, indicating that the interaction mode between IL-22 and IL-22BP may be also conserved in fish and mammals. In conclusion, the mandarin fish IL-22 and IL-22BP are conserved in their interaction and function with their mammalian orthologues, and these findings provide basis for future research on IL-22-IL-22BP axis in fish immunity.

Interleukin (IL)-10 is an immune-regulatory cytokine with multiple functions. In the current study, IL-10 and its two receptors, IL-10R1 and IL-10R2 were identified in mandarin fish, Siniperca chuatsi. The inhibitory effect of mandarin fish IL-10 was investigated on pro-inflammatory cytokine expression and the ligand-receptor relationship. This IL-10 possesses conserved cysteine residues, predicted α-helices and a typical IL-10 family signature motif, similar to its mammalian orthologue, and IL-10R1 harbours predicted JAK1 and STAT3 binding sites in the intracellular region. The fish IL-10 and IL-10R1 exhibit high expression levels in several immune-related organs/tissues, such as spleen, trunk kidney and head kidney, and IL-10R2 possesses a constitutive expression pattern. The expression of IL-10 shows significant increase in spleen from infectious spleen and kidney necrosis virus (ISKNV) infected mandarin fish, where the two receptors also exhibit different levels of induced expression. Mandarin fish IL-10 also exhibits significant response to the stimulation of LPS, PHA and PMA, with the two receptors exhibiting an interesting decrease in expression following the treatment of PMA. The pro-inflammatory cytokines, IL-6, IL-1β, IL-8, TNF-α, show diminished up-regulation in LPS-stimulated splenocytes pre-incubated with IL-10, indicating the anti-inflammatory roles of mandarin fish IL-10. In EPC cells transfected with different combinations of receptors, IL-10 can enhance the expression of suppressor of cytokine signalling 3 (SOCS3) only when IL-10R1 and IL-10R2 are both expressed, suggesting the participation of the two receptors in signal transduction of mandarin fish IL-10. Similar results are observed with the usage of chimeric receptors, IL-10R1/CRFB1 and IL-10R2/CRFB5. Overall, mandarin fish IL-10 shares conserved ligand-receptor system and the prototypical inhibitory activities on pro-inflammatory cytokine expression with mammalian IL-10, implying the evolutionary conservation of this cytokine.

CD44 gene is a cell surface receptor which undergoes complex alternative splicing and extensive post-translational modifications. Although many studies have showed that CD44 is involved in the process of host defense, the function of piscine CD44 in antibacterial or antiviral defense response remains unclear. In the present study, we report the functional characterization of zebrafish CD44c, which is more similar to CD44b antigen isoforms rather than CD44a based on amino acid composition and phylogenetic analysis. The expression of zebrafish CD44c was inducible in response to bacterial and viral infections. During SVCV infection, the in vivo studies revealed that CD44c overexpression led to the increased virus loads and decreased survival rate. The attenuated response by zebrafish CD44c in response to SVCV infection were characterized by the impaired production of inflammatory cytokines and the impaired expressions of IFNs, IFN-stimulated genes, MHC class I and II genes. During Edwardsiella piscicida infection, the overexpression of zebrafish CD44c facilitated bacterial growth and dissemination, but did not impact on larvae survival. The detrimental role of CD44c in host defense against E. piscicida infection was supported by a decreased production of several antibacterial molecules including defbl2, defbl3, NK-lysin and RNase3. All together, these results firstly demonstrate the negative regulation of piscine CD44c in viral and bacterial infection.

Interleukin (IL)-4 and IL-13 are T helper 2 (Th2) cytokines with pleiotropic functions. IL-4 interacts with two receptors consisting of IL-4Rα/γ chain receptor (γC) and IL-4Rα/IL-13Rα1. In contrast, IL-13 binds to IL-13Rα2 but also shares the receptor complex containing IL-4Rα/IL-13Rα1. In fish, two IL-4/13 homologs have been identified but their phylogenetic relationships with IL-4 and IL-13 are ambiguous. In this study, we identified six putative IL-4/13 receptor homologs in grass carp, including γC1, γC2, IL-4Rα1, IL-13Rα1, IL-13Rα2 and a soluble form of IL-4Rα2. Comparative sequence analyses revealed that these receptors possess conserved characteristic domains and the genes encoding them share conserved gene synteny with their human counterparts. All six receptors contain a cytokine binding homology domain (CHD) and two fibronectin type Ⅲ (FNⅢ) like domains, with IL-13Rα1 and IL-13Rα2 harbouring an extra Ig-like domain preceding the CHD domain. Interestingly, grass carp IL-13Rα1 and IL-13Rα2 lack the characteristic WSXWS motif, a typical feature of mammalian type I cytokine receptors. The IL-4/13 receptor genes are differentially expressed in tissues and primary leukocytes of head kidney and can be modulated by Flavobacterium cloumnare (F. cloumnare), suggesting they are involved in immune response against F. cloumnare infection.

Edwardsiella ictaluri, a Gram-negative intracellular pathogen, is the causative agent of enteric septicemia in channel catfish, and catfish aquaculture in China suffers heavy economic losses due to E. ictaluri infection. Vaccination is an effective control measure for this disease. In this study, an attenuated E. ictaluri strain was acquired through deletion mutation of the T3SS protein eseJ

Fish retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are critical RNA sensors in cytoplasm and are involved in antiviral innate immunity. However, some species of fish lack RIG-I gene, and the function of RLR members in RIG-I-absent fish is poorly understood. In the present study, MDA5, LGP2 and MAVS genes were identified in commercially important snakehead Channa argus. But, RIG-I gene was not found in this fish, and a systematic analysis of RLRs in available genome database of fish indicated the absence of RIG-I in the Acanthomorphata, Clupeiformes and Polypteriformes, suggesting that loss events of RIG-I gene may have occurred independently three times in the evolutionary history of fish. The MDA5, LGP2 and MAVS in snakehead have conserved protein domains and genomic location based on sequence, phylogenetic and syntenic analyses. These genes are constitutively expressed in healthy fish and can be induced by polyinosinic and polycytidylic acid (poly(I:C)) stimulation in vitro. It is further revealed that the snakehead MDA5 and LGP2 have binding capacity with dsRNA, such as poly(I:C), and MDA5 can interact with MAVS, implying the antiviral function of MDA5 in the RIG-I-absent fish.

In bony fish, CD40 and CD154 are two very important costimulatory molecules involved in T and B cell cooperation in thymus-dependent antibody production. In the current study, we identified the cDNAs of CD40 and CD154 and analyzed their genomic structures in grass carp. Quantitative real-time PCR indicated that the CD40 and CD154 were mainly expressed in immune organs. After challenge with grass carp reovirus (GCRV), these two genes were up-regulated at 72 h in head kidney and spleen. Moreover, seven and five single nucleotide polymorphisms (SNPs) were identified in the CD40 and CD154 respectively. Statistical analysis indicated that three SNPs in the coding region of the CD40 were significantly associated with the resistance of grass carp against GCRV. These results indicated that CD40 and CD154 play important roles in the responses to GCRV in grass carp. The SNP markers in the CD40 associated with the resistance to GCRV may facilitate the disease-resistant breeding of grass carp.

Teleost fish are unique in having type I and type II interferons (IFNs) only, and the type I IFNs are classified into Group one and Group two based on the presence of two or four cysteines respectively, and are further classified into seven subgroups. In the present study, three distinct type I IFNs, IFNc, IFNd and IFNh, have been identified in the genome sequences of a perciform fish, the mandarin fish Siniperca chuatsi. These IFNs are induced following the stimulation of Polyinosinic polycytidylic acid (poly(I: C)) and Resiquimod (R848) either in vivo or in vitro. But, the infectious spleen and kidney necrosis virus (ISKNV) infection caused a delayed response of IFNs, which may be resulted from the viral inhibition of type I IFN production and related signalling. The three receptor subunits, cytokine receptor family B 1 (CRFB1), CRFB2 and CRFB5 are also expressed in a similar manner as observed for the IFNs, and IFNc, IFNd and IFNh use preferentially the receptor complex, CRFB2 and CRFB5, CRFB1 and CRFB5, CRFB1 and CRFB5 respectively for their effective signalling in the induction of IFN-stimulated genes (ISGs). Moreover, the IFNs are able to induce their own expression, and also the IRF3 and IRF7 expression, leading to the amplification of IFN cascade. It is further revealed that these three IFNs are transcribed differently by IRF7 and IRF3. The composition, function, signalling and transcription of type I IFNs have been investigated in detail in a teleost fish. (C) 2018 Elsevier Ltd. All rights reserved.

The chemokine and chemokine receptor networks regulate leukocyte trafficking, inflammation, immune cell differentiation, cancer and other biological processes. Comparative immunological studies have revealed that both chemokines and their receptors have expanded greatly in a species/lineage specific way. Of the 10 human CC chemokine receptors (CCR1-10) that bind CC chemokines, orthologues only to CCR6, 7, 9 and 10 are present in teleost fish. In this study, four fish-specific CCRs, termed as CCR4La, CCR4Lc1, CCR4Lc2 and CCR11, with a close link to human CCR1-5 and 8, in terms of amino acid homology and syntenic conservation, have been identified and characterized in rainbow trout (Oncorhynchus mykiss). These CCRs were found to possess the conserved features of the G protein-linked receptor family, including an extracellular N-terminal, seven TM domains, three extracellular loops and three intracellular loops, and a cytoplasmic carboxyl tail with multiple potential serine/threonine phosphorylation sites. Four cysteine residues known to be involved in forming two disulfide bonds are present in the extracellular domains and a DRY motif is present in the second intracellular loop. Signaling mediated by these receptors might be regulated by N-glycosylation, tyrosine sulfation, S-palmitoylation, a PDZ ligand motif and di-leucine motifs. Studies of intron/exon structure revealed distinct fish-specific CCR gene organization in different fish species/lineages that might contribute to the diversification of the chemokine ligand-receptor networks in different fish lineages. Fish-specific trout CCRs are highly expressed in immune tissues/organs, such as thymus, spleen, head kidney and gills. Their expression can be induced by the pro-inflammatory cytokines, IL-1β, IL-6 and IFNγ, by the pathogen associated molecular patterns, PolyIC and peptidoglycan, and by bacterial infection. These data suggest that fish-specific CCRs are likely to have an important role in immune regulation in fish.

Intracellular NOD-like receptors (NLRs) are emerging as critical regulators of innate and adaptive immune responses. Although the NLR family member NLRC5 is functionally defined, the role of NLRC5 in regulating innate immune signaling has been controversial in mammals, and is poorly understood in teleost fish. In the present study, we report the functional characterization of zebrafish NLRC5. The cloned NLRC5 consists of 6435 bp which encodes 1746 amino acids. The N-terminal effector-binding domain of zebrafish NLRC5 is absent which is different from all other human NLRs. Fluorescence microscopy showed that zebrafish NLRC5 is located throughout the entire cell. The higher expression of zebrafish NLRC5 in embryo than in larvae was observed, suggesting the action phase of NLRC5 in zebrafish ontogenetic stages. When the modulation of NLRC5 in pathogen infection was analyzed, it was found that zebrafish NLRC5 was upregulated by both bacterial and viral infection. Overexpression of zebrafish NLRC5 resulted in significant inhibition of SVCV replication in vivo and in vitro, but failed to activate interferon (IFN) promoters and type I IFN signaling pathway. Interestingly, NLRC5 overexpression could activate mhc2dab promoter, and induce the expression of MHC class II genes. All together, these results demonstrate that zebrafish NLRC5 is involved in IFN-independent antiviral response, and also functions as a transcriptional regulator of MHC class II genes.

Interferon regulatory factors (IRFs) are transcription mediators which play vital roles in multiple biological processes, such as antiviral defense, immune response, cell growth regulation and apoptosis. A fish specific IRF, termed IRF11, has been identified in previous study through searching fish genome databases. Herein, a transcript of IRF11, EcIRF11 was cloned from orange-spotted grouper, Epinephelus coioides. The EcIRF11 cDNA sequence has 1573 bp in length, encoding a putative protein of 261 amino acids, with a high degree of similarity found between EcIRF11 and its teleost counterparts. Comparative analyses in teleost genomes revealed that IRF11 may have an ancient origin at least 450 million years ago, and the locus harbouring IRF11 might have experienced chromosomal rearrangement and/or inversion during evolution. Expression analysis revealed that the other two members, IRF1 and IRF2 also in the IRF1 subgroup (SG) as IRF11, exhibited high expression levels in early experimental infection phase in response to viral stimulation of poly I:C and to bacterial stimulation of Vibrio parahaemolyticus infections in the fish, while EcIRF11 is not transcriptionally modulated at the examined time points except in kidney at 6 h following poly I:C stimulation. Taken together, the results obtained in this study indicate that IRF11 might have been originated from the same ancestor as IRF1 and IRF2, but exhibits distinct basal and induced expression, implying its different function which needs further characterization.

Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide. Many studies have focused on the identification of protective antigens to aid in the development of novel vaccines against the disease. In this study, an immunoblotting approach was employed to identify immunogenic outer membrane proteins (OMPs) from F. columnare in two-dimensional electrophoresis (2-DE) map gels using antibacterial sera obtained from grass carp (Ctenopharyngodon idella), and anti-grass carp-recombinant Ig (rIg) monoclonal antibodies. Five unique immunogenic proteins, including the gliding motility lipoprotein GldJ (GldJ), hypothetical protein FCOL_13420 (Fco1), lipoprotein (Lip), F0F1 ATP synthase subunit beta (F0f1) and outer membrane efflux protein precursor (Omep), were characterized. Over-expression of these proteins in Escherichia coli DE3, and their immunogenicity and protective efficacy were evaluated in grass carp. The relative percent survival (RPS) of the groups immunized separately with recombinant GldJ, Lip and Omep was 72%, 64% and 68%, respectively when compared to control fish. Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of F. columnare. The results suggest that GldJ, Lip and Omep are major protective antigens and may be considered as novel candidates in the development of vaccines against columnaris disease in fish.

Peptidoglycan recognition proteins (PGRPs), which are discovered in invertebrates and vertebrates, play an important role in antibacterial immunity. However, the function of PGRPs is largely uninvestigated in reptiles. In the present study, a short-type PGRP gene, designed as C-turtle-PGRP-S, was identified in the Chinese soft-shelled turtle, Pelodiscus sinensis. The C-turtle-PGRP-S contains a highly conserved PGRP domain and has close relationship with PGRP-S orthologues in other species according to sequence and phylogenetic analyses. C-turtle-PGRP-S gene was constitutively expressed in all detected tissues and was induced by Edwardsiella tarda. Additionally, recombinant C-turtle-PGRP-S showed PGN binding activity and antibacterial function against E. tarda. Therefore, it is suggested that the function of PGRP-S is likely to be conserved in reptile vertebrates, as observed in other vertebrates, shedding light on the evolutionary conservation of PGRPs.

Interferon regulatory factors (IRFs) are a family of transcriptional factors capable of regulating the expression of distinct subsets of interferon (IFN)-stimulated genes by binding to their promoters. IRF1 was the first member identified for its ability to regulate the IFNβ gene and has now been revealed to exhibit remarkable functional diversity in the regulation of different cellular responses. In the present study, the IRF1 gene was identified and characterized in Japanese eel, Anguilla japonica (AjIRF1). The open reading frame of AjIRF1 was 804 bp in length, encoding a protein of 267 amino acids (aa) that encompasses a conserved N-terminal DNA binding domain (DBD). Sequence alignment shows the presence of six highly conserved tryptophan (W) residues in the DBD of IRF1, IRF2 and IRF11, while other IRF members have only five tryptophans. Expression analysis showed that AjIRF1 was significantly upregulated in all tested organs/tissues in response to Poly I:C stimulation or Edwardsiella tarda infection. Furthermore, the functional activity of AjIRF1 was confirmed in driving the transcription of AjIFN promoters, which depends on the highly conserved residues within DBD. Subcellular distribution analysis revealed that AjIRF1 was localized exclusively in the nucleus, which is cooperatively regulated by a bipartite NLS embedded within the DBD and a monopartite NLS located immediately downstream of the DBD. Taken together, this study presents the expression profile of AjIRF1 and defines the functional motifs required for its nuclear import and its role in activating IFN promoters, thus providing helpful information for further research on the regulatory mechanisms of teleost IRF1.

As a key molecule in innate immune signalling pathway, interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) mediates downstream signalling cascades in immune response. In the present study, an IRAK1 orthologue was characterized from rainbow trout (Oncorhynchus mykiss), with a 2115 bp open reading frame (ORF), encoding a protein of 704 amino acids (aa). Multiple alignments showed that IRAK1 contains highly conserved features among different species, with a conservative N-terminal death domain (DD) and a C-terminal conserved serine/threonine protein kinase (STKc) domain. Expression analysis indicated that IRAK1 was widely expressed in examined organs/tissues, with the highest level observed in muscle and lowest in stomach. In RTG-2 cell line, the induced expression of IRAK1 was observed following the stimulation by the fish bacterial pathogen Flavobacterium columnare. Luciferase activity assays revealed that IRAK1 induced significantly the activity of NF-κB in Human embryonic kidney 293T (HEK293T) cell line; but after co-transfected with rainbow trout IL-1 receptor-associated kinase 4 (IRAK4), the induction was significantly down-regulated when compared with the expression of IRAK1 alone. Co-immunoprecipitation (Co-IP) assays indicated that IRAK1 was associated with rainbow trout myeloid differentiation factor 88 (MyD88), IRAK4 and TNF receptor associated factor 6 (TRAF6) in transfected HEK293T cells, and may form a complex with MyD88, IRAK4 and TRAF6 during the signalling pathway.

In vertebrates, a total of eleven interferon (IFN) regulatory factors (IRFs), IRF1 to IRF11 are reported, with the conserved presence of IRF1 to IRF9 in all classes of vertebrates. However, IRF10 has been reported only in fish and birds, and IRF11 seems to be a fish specific IRF member. In this study, IRF11 in mandarin fish Siniperca chuatsi was found upregulated following virus infection, and IRF11 was localized constitutively in nucleus as revealed through immunofluorescence test. The overexpression and/or luciferase reporter assays showed that IRF11 can induce transcriptionally the ISRE activity, and the expression of type I IFNs, IFNc and IFNh, as well as the IFN-stimulated gene, Mx, thus inhibiting the Siniperca chuatsi rhabdovirus (SCRV) replication as indicated in the reduced expression of virus protein genes. It is thus suggested that IRF11 in mandarin fish and probably in other teleost fish can exert its antiviral effect through the upregulation of type I IFNs and ISGs.

TANK-binding kinase 1 (TBK1) is an essential serine/threonine-protein kinase required for Toll-like receptor (TLR)- and retinoic acid-inducible gene I (RIG-I) -mediated induction of type I IFN and host antiviral defense. In the present study, TBK1-like transcript, namely TBK1L, was cloned from zebrafish. Compared with TBK1, TBK1L contains an incomplete S_TKc domain, and lacks UBL_TBK1_like domain. Realtime PCR showed that TBK1L was constitutively produced in embryos, early larvae and ZF4 cells, and unchanged in ZF4 cells following SVCV infection. Overexpression of TBK1 but not TBK1L resulted in significant activation of zebrafish IFN1 and IFN3 promoters. Similarly, TBK1L had little impact on the antiviral state of the cells. However, the overexpression of TBK1L negatively regulated the induction of zebrafish IFN1 and/or IFN3 promoters mediated by the retinoic acid-inducible gene I-like receptors (RLRs), MAVS and TBK1. In addition, the overexpression of TBK1L in zebrafish embryos led to the decreased production of many IFN-stimulated genes induced by TBK1. Collectively, these data support that zebrafish TBK1L negatively regulates RLRs-MAVS-TBK1 pathway.

The cluster of differentiation 2 (CD2), functioning as a cell adhesion and costimulatory molecule, plays a crucial role in T-cell activation. In this paper, the CD2 gene of Nile tilapia, Oreochromis niloticus (designated as On-CD2) was cloned and its expression pattern under the stimulation of Streptococcus agalactiae was investigated. Sequence analysis showed On-CD2 protein consists of two extracellular Ig-like domains, a transmembrane region, and a long proline-rich cytoplasmic tail, which is a hallmark of CD2, and several important structural characteristics required for T-cell activation were detected in the deduced amino acid sequence of On-CD2. In healthy tilapia, the On-CD2 transcripts were mainly detected in the head kidney, spleen, blood and thymus. Moreover, there was a clear time-dependent expression pattern of On-CD2 after immunized by formalin-inactivated S. agalactiae and the expression reached the highest level at 12 h in the brain and head kidney, 48 h in the spleen, and 72 h in the thymus, respectively. This is the first report on the expression of CD2 induced by bacteria vaccination in teleosts. These findings indicated that On-CD2 may play an important role in the immune response to intracellular bacteria in Nile tilapia.

An octamer consisting of two copies of histones H2A, H2B, H3 and H4 is the nucleosome core. It is well established that histone derived antimicrobial peptides (AMPs) have anti-microbial properties in various invertebrate and vertebrate species. Different from well-known histone H2A-derived AMPs, the antimicrobial properties of the complete histone H2A are rather limited. In the present study, we report the functional characterization of the complete histone H2A from zebrafish. The expression of zebrafish histone H2A was higher in embryos than in larvae, and inducible in response to bacterial infection. Furthermore, the expression of zebrafish histone H2A was decreased by RIP2 deficiency with and/or without bacterial infection. During Edwardsiella piscicida infection, the overexpression of zebrafish histone H2A inhibited bacterial proliferation and increased the survival rate of zebrafish larvae. The overexpression of zebrafish histone H2A demonstrated an increased transcription of many antibacterial genes and MHC related genes, which was dependent on RIP2, an adaptor protein for signal propagation of the NLRs-mediated antibacterial immune response. In line with this, zebrafish histone H2A cooperated with RIP2 to induce the transcription of many antibacterial genes and MHC related genes. All together, these results firstly demonstrate the antibacterial property of the complete histone H2A against gram-negative bacteria E. piscicida in vivo and the correlation between zebrafish histone H2A and RIP2 adaptor protein on the transcriptional regulation of antibacterial genes and MHC related genes.

IRF10 gene was cloned in orange spotted grouper, Epinephelus coioides, and its expression was examined following poly(I:C) stimulation and bacterial infection. The cDNA sequence of grouper IRF10 contains an open reading frame of 1197 bp, flanked by 99 bp 5'-untranslated region and 480 bp 3'- untranslated region. Multiple alignments showed that the grouper IRF10 has a highly conserved DNA binding domain in the N terminus with characteristic motif containing five tryptophan residues. Quantitative real-time PCR analysis revealed that the expression of IRF10 was responsive to both poly(I:C) stimulation and Vibrio parahemolyticus infection, with a higher increase to poly(I:C), indicating an important role of IRF10 in host immune response during infection. A phyletic distribution of IRF members was also examined in vertebrates, and IRF10 was found in most lineages of vertebrates, not in modern primates and rodents. It is suggested that the first divergence of IRF members might have occurred before the evolutionary split of vertebrate and cephalochordates, producing ancestors of IRF (1/2/11) and IRF (4/8/9/10)[(3/7) (5/6)], and that the second and/or third divergence of IRF members occurred following the split, thus leading to the subsets of the IRF family in vertebrates.

Cyclosporine, tacrolimus and sirolimus are commonly used in renal transplant recipients to prevent rejection. Various adverse effects of these agents on the multiple organ system have been reported clinically. However, animal studies are necessary to determine and compare these effects on individual organ given the presence of multiple confounding factors and multi-pharmacy in clinical settings. In a physiologically and clinically relevant rat model of unilateral nephrectomy, the long-term impacts of commonly used immunosuppressants at doses equivalent to the therapeutic levels used for post-renal transplant patients on hepatic function and histological changes of the liver were examined. Cyclosporine induced significant hepatocellular injury, impairment of synthetic function of the liver, hyperbilirubinemia and cholestasis, and dyslipidemia accompanied by profound histological changes of hepatic structures on both light and electron microscopic examinations. On the other hand, neither tacrolimus nor sirolimus developed any hepatotoxic effects except for more remarkable dyslipidemia was observed in animals treated with sirolimus. Our study indicates that long-term administration of commonly used immunosuppressants has various impacts on biochemical parameters as well as histological alterations of the liver even at therapeutic levels. These data may therefore provide useful information for judicious selection of immunosuppressive agents based on different clinical settings.

Interferon regulatory factor (IRF) 3 and IRF7 have been identified as regulators of type I interferon (IFN) gene expression in mammals. In the present study, the two genes were cloned and characterized in the European eel, Anguilla anguilla. The full-length cDNA sequence of IRF3 and IRF7 in the European eel, named as AaIRF3 and AaIRF7 consists of 2879 and 2419 bp respectively. Multiple alignments showed that the two IRFs have a highly conserved DNA binding domain (DBD) in the N terminus, with the characteristic motif containing five tryptophan residues, which is a feature present in their mammalian homologues. But, IRF7 has only four of the five residues in other species of fish. The expression of AaIRF3 and AaIRF7 both displayed an obvious dose-dependent manner following polyinosinic:polycytidylic acid (PolyI:C) challenge. In vivo expression analysis showed that the mRNA level of AaIRF3 and AaIRF7 was significantly up-regulated in response to PolyI:C stimulation in all examined tissues/organs except in muscle, with a lower level of increase observed in response to lipopolysaccharide (LPS) challenge and Edwardsiella tarda infection, indicating that AaIRF3 and AaIRF7 may be more likely involved in antiviral immune response. In addition, some pattern recognition receptors genes related with the production of type I IFNs and those genes in response to type I IFNs were identified in the European eel genome database, indicating a relatively conserved system in the production of type I IFN and its signalling in the European eel.

Mx protein is known to play an important role in vertebrate immune response to viral infection. In this study, cDNA sequences of four Mx isoforms, designated as MxA, B, C and D were characterized in the European eel, Anguilla anguilla. These sequences contained an open reading frame of 1899, 1896, 1866, 1779 bp, flanked by 95, 53, 138, 69 bp of 5' untranslated region and 389, 241, 136, 124 bp of 3' untranslated region, respectively. A phylogenetic tree constructed with Mx peptide sequences from vertebrates revealed that MxA, C and D in the European eel formed into a clade containing zebrafish MxA and MxB and Mx proteins in other teleosts, whereas MxB in the eel was clustered together with zebrafish MxD, MxG and MxF. The transcription level of all Mx isoforms increased in a poly I:C dose-dependent manner in peripheral blood leukocytes of eels, as revealed by real-time PCR. A further experiment was conducted to reveal the temporal change in expression of these isoforms in various organs/tissues following poly I:C stimulation, and significant increase in expression was observed at various degrees in different organs or in different sampling occasions within the 12 h experimental period. In particular, MxA had the highest level of increase, while MxB had the lowest; and three isoforms, MxA, MxB and MxD had the highest increase in intestine, while the highest increase of MxC expression was observed in liver. These four isoforms of eel Mx are thus expressed differentially, and further work is certainly required to clarify the activity of promoter elements and antiviral activity of these Mx isoforms.

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from insects to mammals, recognize bacterial peptidoglycan (PGN) and function in antibacterial innate immunity. In this study, a short-form PGRP, designated as gcPGRP5 was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP5 is composed of 180 residues with a conserved PGRP domain at the C-terminus. The gcPGRP5 gene consists of four exons and three introns, spacing approximately 2.3 kb in genomic sequence. Phylogenetic analysis demonstrated that the gcPGRP5 is clustered with other PGRP-S identified in teleost fish. The gcPGRP5 is constitutively expressed in all organs/tissues examined, and its expression was significantly induced in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and PGN. Fluorescence analysis showed that gcPGRP5 is distributed in cytoplasm of CIK cells, and cell lysates from CIK cells transfected with pTurbo-gcPGRP5-GFP and ptGFP1-gcPGRP5 plasmids display the binding activity and peptidoglycan-lytic amidase activity toward Lys-PGN from Staphylococcus aureus and Dap-PGN from Bacillus subtilis. Furthermore, heat-shock protein70 (Hsp70), and MyD88, an adaptor molecule in Toll-like receptor pathway, had an increased expression in CIK cells overexpressed with gcPGRP5. It is thus indicated that gcPGRP5 exhibits amidase activity, and also possesses roles in anti-stress, and in Toll-like receptor signaling pathway. (C) 2013 Elsevier Ltd. All rights reserved.

Flavobacterium columnare, the causative agent of columnaris disease, infects freshwater fish worldwide. However, the pathogenicity of this bacterium is poorly understood due possibly to the lack of an efficient in-frame knockout technique. In order to improve electroporation efficiency, the type I restriction-modification system (R-M system) was cloned and its role in electroporation was examined in F. columnare G(4) strain. The complete sequence of type I R-M system in the bacterium, designated as Fcl, contains all three subunits of type I R-M system, named as fclM, fclS, fclR, respectively, with the identification of a hypothetical gene, fclX. Constitutive transcription of the three genes was observed in F. columnare G(4) by RT-PCR. The ORF of fclM and fclS was cloned into the plasmid pACYC184 and transformed into Escherichia coli TOP10. The resultant E. coli strain, designated as E. coli TOPmt, was transformed with the integrative plasmid pGL006 constructed for F. columnare G(4). The integrative plasmid was re-isolated from TOPmt and incubated with the lysate of F. columnare G(4). The re-isolated integrative plasmid, designated as pGL006', showed higher resistance than pGL006. With pGL006', the electroporation efficiency of the strain G(4) increased 2.6 times, while that of F. columnare G(18) was not obviously improved. Furthermore, a method to improve the electroporation efficiency of F. columnare G(4) was developed using the integrative plasmid methylated by E. coli TOPmt which contains the fclM and fclS gene of F. columnare G(4). Further analyses showed that the fcl gene cluster may be a unique type I R-M system in F. columnare G(4). It will be of significant interest to examine the composition and diversity of R-M systems in strains of F. columnare in order to set up a suitable genetic manipulation system for the bacterium.

Oncomelania hupensis is the unique intermediate host of Schistosoma japonicum. The schistosome-snail interaction is biomedically important. To identify differentially expressed transcripts in O. hupensis chronically infected with S. japonicum, suppression subtractive hybridization (SSH) was used to construct a cDNA library in each direction for transcripts that are more abundantly enriched in head-foot part of the infected O. hupensis and for those that are more abundantly enriched in the uninfected, as head-foot part contains hemocytes and hemolymph which are associated with the snail internal defense system. After differential screening, 39 transcripts were identified, including nine and 30 transcripts enriched in infected and uninfected snails, respectively. Some of the transcripts have similar homology to available sequences in current databases, including transposase, caveolin-like protein, pancreatic trypsin inhibitor-like protein, prosaposin, glutathione s-transferase (GST), and several hypothetical proteins, while most of the transcripts do not match with any sequences in available databases. The identified transcripts were involved functionally in cell growth, metabolism, signal transduction, and immune responses. Two forward library transcripts and 11 reverse library transcripts were selected for real-time PCR, and 10 of them were confirmed to be consistent with the SSH results. It is intriguing to continue functional studies for some genes such as pancreatic trypsin inhibitor; a hypothetical protein (HS576367) related to calcium ion binding; GST; and several unknown proteins (HS576353 and HS576355). These identified differentially expressed genes may be key targets for understanding the molecular mechanism of co-existence during which the snail is unable to rid itself of the schistosome in chronic infection stage. (C) 2012 Elsevier Inc. All rights reserved.

Three goose-type (g-type) lysozymes, designated as OHLysG1, OHLysG2 and OHLysG3 were identified from expressed sequence tags (ESTs) of a gastropod Oncomelania hupensis, the intermediate host of Schistosoma japonicum. The full cDNA sequences of OHLysG1, OHLysG2 and OHLysG3 consisted of 735, 909 and 808 nucleotides, with an open reading frame of 198, 214 and 249 codons containing a 21, 7 and 8 amino acid (aa) signal peptide at the N-terminus, respectively. The three g-type lysozymes shared conserved features with other g-type lysozymes, such as the substrate binding sites, the catalytic residues critical for the fundamental structure and function of g-type lysozymes. It seems possible that g-type lysozymes in molluscs shared one conserved cysteine with those in birds and mammals, and six conserved cysteines were observed for mollusc g-type lysozymes, with two unique cysteines present in the g-type lysozymes of O. hupensis. The three lysozyme genes were expressed mainly in hepatopancreas, with relatively low expression level observed in head-foot muscle and intestine. When comparing S. japonicum-infected and uninfected snails, significant increase (P < 0.05) was observed for all the three lysozymes in infected snails, with the highest increase detected in hepatopancreas, and lowest in intestine, implying their defensive role in the host-parasite, i.e. snail-trematode system. The three recombinant lysozymes expressed in Escherichia coli strain M15 showed lytic activity against Aeromonas hydrophila, Vibrio fluvialis, Aeromonas sobria and Micrococcus lysodeikticus. In conclusion, the finding of three g-type lysozymes in O. hupensis provides structural and functional evidence of multiple g-type lysozymes in gastropod, which may have evolutional implication in the snail-trematode system. (C) 2011 Elsevier Ltd. All rights reserved.

Nucleotide-binding oligomerization domain 2 (NOD2) is a cytoplasmic pattern recognition receptor (PRR), which is involved in innate antibacterial and antiviral responses. Here, two NOD2 splice variants, trNOD2a and trNOD2b, are reported in rainbow trout Oncorhynchus mykiss, that share 63% and 61% similarity with human NOD2, respectively. These two trout NOD2 splice variants were shown to be constitutively expressed in thymus, gills, skin, muscle, liver, spleen, head kidney, intestine, heart, and brain, with the expression of trout NOD2 (trNOD2) mainly contributed by trNOD2a in all the examined tissues. PolyI:C transfection up-regulated the expression of trNOD2a and trNOD2b in RTG-2 cells. The expression of trNOD2a/b was modulated by the inflammatory stimulant interferon-γ (IFN-γ) or interleukin-1β (IL-1β). Overexpression of trout NOD2 effector domains resulted in induced expression of proinflammatory cytokines including IL-1β, tumor necrosis factor-α (TNF-α), IL-6 and IL-8, the antibacterial peptide cathelicidin-2, a variety of caspases including caspase-2, -6, -7, -8, -9, and type I and type II IFN. These results suggest that fish NOD2 functions in inflammatory events, possibly via NF-κB activation, regulation of apoptosis, and triggering of antibacterial and antiviral defences.

The cDNA and genomic sequences of IgD and IgZ were characterized in grass carp Ctenopharyngodon idella in the present study, and with the identification of a BAC clone covering zeta, mu, and delta genes, the IgH locus containing these Ig genes and other V, D, J genes was also illustrated in this fish. Secretory and membrane-bound IgZ were identified, with two transmembrane exons spliced within the CH4 exon, as reported in IgM of mammals and IgZ in other teleost fish. The first and second constant domains of IgZ shows more than 90% nucleotide identity with respective domains of grass carp IgM. The IgD has a structure of delta 1-(delta 2-delta 3-delta 4)(2)-delta 5-delta 6-delta 7-TM-UTR, with the repeat of delta 2-delta 3-delta 4; but intron was not found between the two repeat, i.e. between the first delta 2-delta 3-delta 4 (delta 2.1-delta 3.1-delta 4.1) and the second delta 2-delta 3-delta 4 (delta 2.2-delta 3.2-delta 4.2), and the intron between delta 3.1 and delta 4.1 was much shorter than the intron between delta 3.2 and delta 4.2. The genomic organization of the IgH locus has a pattern of Vn-Dn-Jn-C zeta-Dn-Jn-C mu-C delta, as reported in other teleost fish. Thirteen V-H, fourteen D, and twelve J(H) genes were observed in this locus, with the similarity of three D segments and four J(H) segments being the same in the upstream of C zeta, and C mu. The transcriptional enhancer located at the mu-delta intergenic region was also analyzed and it seems possible that this enhancer is functional as verified in zebrafish and channel catfish. (C) 2010 Elsevier Ltd. All rights reserved.

The complete mitochondrial genomes of intermediate host snails for Schistosoma in China were sequenced, including the sub-species Oncomelania hupensis hupensis in two types, and O. hupensis robertsoni, intermediate hosts for S. japonicum, and Tricula hortensis, the intermediate host of S. sinensium. Four genomes have completely the same gene order as in other caenogastropods, containing 13 protein-coding genes and 22 transfer RNAs. The gene size, start codon and termination codon are mostly the same for all protein-coding genes. However, pairwise sequence alignments revealed quite different degrees of variation. The ribbed-shelled O. hupensis hupensis and the smooth-shelled but with varix O. hupensis hupensis had a lower level of genetic distance (3.1% for protein-coding genes), but the coden usages differed obviously in the mitochondrial genomes of these two types of snails, implying that their genetic difference may be larger than previously recognized. The mean genetic distance between O. hupensis hupensis and O. hupensis robertsoni was 12% for protein-coding genes, indicating a higher degree of genetic difference. In consideration of the difference in morphology and distribution, we considered that O. hupensis hupensis and O. hupensis robertsoni can be considered as separate species. The ribbed-shelled O. hupensishupensis and smooth-shelled O. hupensis robertsoni were phylogenetically clustered together within a same clade, which was then clustered with T. hortensis, confirming their close relationship. However, species or sub-species in the Oncomelania from southeastern Asian countries should be included in future study in order to resolve the phylogenetic relationship and origination of all snails in the genus.

Four new members of the SOCS family of molecules in rainbow trout (Oncorhynchus mykiss), CISH and SOCS6, 7 and 9, are described for the first time in this species. The genes had a wide tissue distribution in trout, and were detected in gills, skin, muscle, liver, spleen, head kidney, intestine and brain, with brain having the highest expression levels. Stimulation of a rainbow trout leucocyte cell line, RTS-11, (mononuclear/macrophage-like cells) with LPS or Poly 1:C had no effect on the expression of these genes, although in both cases the previously identified SOCS1-3 genes were up-regulated. Similarly, stimulation of RTS-11 or RTG-2 (fibroblasts) cells with the trout recombinant cytokines IFN-gamma or IL-1 beta had no effect on CISH or SOCS6, 7 and 9 expression. However, PMA stimulation did impact on SOCS6 and SOCS9 expression, and LPS stimulation of primary cultures or bacterial infection (Yersinia ruckeri) increased significantly CISH expression (as well as SOCS1 and SOCS2 or SOCS3 respectively). It is apparent that the type II SOCS genes (CISH, SOCS1-3) are particularly relevant to immune regulation in fish, although the intriguing expansion of the SOCS4/5 subgroup in fish requires further investigation as to their role and functional divergence. (C) 2010 Elsevier Ltd. All rights reserved.

Nuclear oligomerization domains (NODS) are cytosolic pattern recognition receptors (PRRs) to detect bacterial component. In this study, the molecular cloning and genomic characterization of grass carp NODI (gcNOD1) and grass carp NOD2 (gcNOD2) were reported. The complete open reading frame of gcNOD1 contains 2814 bp, encoding a 937-amino acid polypeptide. The gcNCD2 cDNA sequence encodes 982-amino acid polypeptide. Both gcNOD1 and gcNOD2 possess three conserved domains: carboxy terminal leucine rich repeat (LRR) domains, a central NOD, NBS or NACHT domain, and an amino terminal CARD domain (two in the case of NOD2). At the genomic level, gcNOD1 consists of 11 exons, with 10 intervening introns, spanning approximately 9 kb of genomic sequence. Whereas gcNOD2 has a length of approximately 5 kb with 9 intervening introns. Real time PCR analysis showed gcNOD1 and gcNOD2 were ubiquitously expressed in adult tissues. The highest transcript level of gcNOD1 was detected in brain, but in head kidney for gcNOD2. Grass carp reovirus significantly induced the expression of gcNOD1 and gcNOD2 in spleen (from days 1 to 6). However, expression profiles differed in time course response. Induction experiments with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyI:C revealed the differential expression and regulation of gcNOD1 and gcNOD2 in blood, head kidney, trunk kidney and spleen. All these data suggest a potential role of NOD1 and NOD2 in fish innate immune protection to bacterial and virus infections. (C) 2009 Elsevier Ltd. All rights reserved.

The ontogeny of IgM-producing cells was studied in juvenile mandarin fish Simperca chuatsi, an important fish in China's aquaculture sector. The IgM-producing cells were localised through in situ hybridisation with a probe complementary to the Ig mu-chain in lymphoid-related tissues, including head kidney, spleen, thymus, intestine and gills. In head kidney, transcripts of Ig mu were first detected at 20 days post-hatching (dph) with a few positive signals. and the number of IgM-producing cells increased obviously from 39 dph onwards. At 136 dph, a large amount of positive cells were observed in the entire organ with clusters of these cells located around the blood vessels. In spleen, IgM-producing cells were found from 26 dph onwards, followed by an increase until 67 dph: clusters of positive cells were also detected around blood vessels at 102 dph. In thymus, IgM-producing cells were first observed at 39 dph; thereafter, no obvious increase was detected until 78 dph. The positive cells in thymus were distributed mainly in the outer zone of thymus. A few IgM-producing cells were still observed in thymus of 1-year-old mandarin fish. IgM-producing cells were not detected in the intestine until 87 dph, with several discrete positively stained cells distributed in the lamina propria. IgM-producing cells, scattered mainly in primary gill filaments around blood vessels, were detected in gills from 90 dph. As in other teleosts, these results indicated that the head kidney appears to be the primary organ for IgM production in mandarin fish, and IgM-producing cells exist in all organs examined in the present study, implying their lymphoid role in fish. In addition, it is suggested that vaccination after 20 dph may be much more effective in mandarin fish. (C) 2009 Elsevier B.V. All rights reserved.

Interferons (IFNs), consisting of three major subfamilies, type I, type II (gamma) and type III (lambda) IFN, activate vertebrate antiviral defences once bound to their receptors. The three IFN subfamilies bind to different receptors, IFNAR1 and IFNAR2 for type I IFNs, IFN gamma R1 and IFN gamma R2 for type II IFN, and IL-28R1 and IL-10R2 for type III IFNs. In fish, although many types I and II IFN genes have been cloned, little is known about their receptors. In this report, two putative IFN-gamma receptor chains were identified and sequenced in rainbow trout (Oncorhynchus mykiss), and found to have many common characteristics with mammalian type II IFN receptor family members. The presented gene synteny analysis, phylogenetic tree analysis and ligand binding analysis all suggest that these molecules are the authentic IFN gamma Rs in fish. They are widely expressed in tissues, with IFN gamma R1 typically more highly expressed than IFN gamma R2. Using the trout RTG-2 cell line it was possible to show that the individual chains could be differentially modulated, with rIFN-gamma and rIL-1 beta down regulating IFN gamma R1 expression but up regulating IFN gamma R2 expression. Overexpression of the two receptor chains in RTG-2 cells revealed that the level of IFN gamma R2 transcript was crucial for responsiveness to rIFN-gamma, in terms of inducing gamma IP expression. Transfection experiments showed that the two putative receptors specifically bound to rIFN-gamma. These findings are discussed in the context of how the IFN gamma R may bind IFN-gamma in fish and the importance of the individual receptor chains to signal transduction. (c) 2009 Elsevier Ltd. All rights reserved.

Microcystins (MCs) are cyanobacterial toxins in water blooms that have received increasing attention as a public biohazard for human and animal health. Previous studies were mainly focused on the toxic effects on adult fish, rather than juvenile or larvae, and the response of fish immune system were usually neglected. This paper presents the first data of the effects of microcystin-LR (MC-LR) on transcription of several genes essential for early lymphoid development (Rag1, Rag2, Ikaros, GATA1, Lck and TCR alpha) and heat shock proteins (HSP90, HSP70, HSP60, HSP27) in zebrafish larvae. Relative changes of mRNA transcription were analyzed by real time PCR. The transcription of Rag1, Rag2, Ikaros, GATA1, Lck and TCR alpha were up-regulated when following exposure to 800 mu g/L MC-LR, which may indicate that specific lymphocytes differentiation and TCR/lg arrangement are induced to counteract the toxic effects of MC-LR. It was also interesting to note the dramatically increased transcription of HSP90. HSP70, HSP60 and HSP27, which may indicate their important roles as molecular chaperones under oxidative stress. (C) 2009 Elsevier B.V. All rights reserved.

The gene sequences of three different immunoglobulin (Ig) heavy chains, namely IgM, IgD and IgZ, were cloned from mandarin fish (Siniperca chuatsi) recently. In this study the distribution of these three kinds of Ig-producing cells in lymphoid-related tissues as head kidney, spleen, gill and intestine were investigated by using in situ hybridization, and their transcriptional changes were also analyzed by quantitative real-time PCR during 8 weeks after immunization. IgM-producing cells could be detected obviously and abundantly in all the tissues examined. A few numbers of IgD and IgZ positive cells were both detected in head kidney and spleen. IgZ positive cells could be detected in gill moderately while IgD showed negative results, otherwise no IgD or IgZ positive cells could be detected in intestine. After stimulated with bacterial pathogen Flavobacterium columnare G(4), the transcripts of these three Ig genes exhibited quite different kinetics. Significantly increased transcription of IgM gene was observed in almost all the tissues examined especially in boosted group. In contrast with IgM, seldom strong increase was examined for IgD and IgZ genes. For IgD, it seemed that the first injection could stimulate the immune response easier, since in almost all the tissues significant increase was detected at 1 or 2 weeks after injection. For IgZ, boosted injection could not enlarge the up-regulation of gene expression of first injection. This is the first case to report the transcriptional kinetics of three Ig genes in teleost after bacterin immunization. (C) 2008 Elsevier B.V. All rights reserved.