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Anthropogenic activities can lead to several devastating effects on the environment. The pollutants, which include the discharge of effluents, runoffs in the form of different lethal and sub-lethal concentrations of pesticides, heavy metals, and other contaminants, can harm exposed fauna and flora. The aquatic environment is the ultimate destination for many pollutants which negatively affect aquatic biodiversity and even can cause a species to become extinct. A pollutant can directly affect the behavior of an animal, disrupt cellular systems, and impair the immune system. This harm can be reduced and even mitigated by adopting proper approaches for the conservation of the target biota. Among aquatic organisms, cetaceans, such as the Yangtze finless porpoise, Irrawaddy dolphin, Ganges River dolphin, Amazon River dolphin, and Indus River dolphin, are at a higher risk of extinction because of lack of knowledge and research, and thus insufficient information with respect to their conservation status, management, and policies. Pneumonia is one of the leading causes of mass mortalities of cetaceans. This article reviews the limited research reported on stress and pneumonia induced by pollution, stress-induced pneumonia and immunosuppression, pneumonia-caused mass mortalities of aquatic mammals, and vaccination in wildlife with a specific focus on aquatic mammals, the role of genomics in vaccine development and vaccination, and the major challenges in vaccine development for biodiversity conservation.

Nucleotide oligomerization domain–like receptors (NLRs) and RIG-I–like receptors (RLRs) detect diverse pathogen-associated molecular patterns to activate the innate immune response. The role of mammalian NLR NOD1 in sensing bacteria is well established. Although several studies suggest NOD1 also plays a role in sensing viruses, the mechanisms behind this are still largely unknown. In this study, we report on the synergism and antagonism between NOD1 and MDA5 isoforms in teleost. In zebrafish, the overexpression of NOD1 enhances the antiviral response and mRNA abundances of key antiviral genes involved in RLR-mediated signaling, whereas the loss of NOD1 has the opposite effect. Notably, spring viremia of carp virus–infected NOD1−/− zebrafish exhibit reduced survival compared with wild-type counterparts. Mechanistically, NOD1 targets MDA5 isoforms and TRAF3 to modulate the formation of MDA5–MAVS and TRAF3–MAVS complexes. The cumulative effects of NOD1 and MDA5a (MDA5 normal form) were observed for the binding with poly(I:C) and the formation of the MDA5a–MAVS complex, which led to increased transcription of type I IFNs and ISGs. However, the antagonism between NOD1 and MDA5b (MDA5 truncated form) was clearly observed during proteasomal degradation of NOD1 by MDA5b. In humans, the interactions between NOD1–MDA5 and NOD1–TRAF3 were confirmed. Furthermore, the roles that NOD1 plays in enhancing the binding of MDA5 to MAVS and poly(I:C) are also evolutionarily conserved across species. Taken together, our findings suggest that mutual regulation between NOD1 and MDA5 isoforms may play a crucial role in the innate immune response and that NOD1 acts as a positive regulator of MDA5/MAVS normal form–mediated immune signaling in vertebrates.

Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection.

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.

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.

Aquareoviruses contain an 11-segmented double-stranded RNA genome. Previous studies indicated that NS38, a virus-encoded putative single-stranded RNA binding protein, interacts with NS80 in viral inclusion bodies (VIBs). However, the role of NS38 in aquareovirus infection remained unclear. Here, we found that NS38 interacts with inner-capsid proteins (VP1–VP4 and VP6) and the NS80-RNA complex in both transfected and infected cells. Knockdown of NS38 by siRNAs-115/219 clearly reduced viral infection, with decreased mRNA and protein yields. Moreover, NS38 can interact with host cellular eukaryotic translation initiation factor 3 subunit A (eIF3A) in transfected cells, while no association was detected between eIF3A and NS80. This study is the first to define that the NS38 is essential to viral replication. Together, our findings indicate that NS38 might function as a mediator by interacting with viral and host cellular components in VIBs during replication.

At each stage of innate immune response, there are stimulatory and inhibitory signals that modulate the strength and character of the response. RIG-I-like receptor (RLR) signaling pathway plays pivotal roles in antiviral innate immune response. Recent studies have revealed the molecular mechanisms that viral infection leads to the activation of RLRs-mediated downstream signaling cascades and the production of type I interferons (IFNs). However, antiviral immune responses must be tightly regulated in order to prevent detrimental type I IFNs production. Previous reviews have highlighted negative regulation of RLR signaling pathway, which mainly target to directly regulate RIG-I, MDA5, MAVS and TBK1 function in mammals. In this review, we summarize recent advances in our understanding of negative regulators of RLR signaling pathway in teleost, with specific focus on piscine and viral regulatory mechanisms that directly or indirectly inhibit the function of RIG-I, MDA5, LGP2, MAVS, TRAF3, TBK1, IRF3 and IRF7 both in the steady state or upon viral infection. We also further discuss important directions for future studies, especially for non-coding RNAs and post-translational modifications via fish specific TRIM proteins. The knowledge of negative regulators of RLR signaling pathway in teleost will shed new light on the critical information for potential therapeutic purposes.

No studies systematically examined the intestinal immune response for yellow stage of European eel (Anguilla anguilla) with Aeromonas hydrophila infection by time-resolved RNA-seq. Here, we examined transcriptional profiles of the intestines at three-time points following infection with A. hydrophila. Intraperitoneal injections caused mortalities within 48 h post-injection (hpi), with the survival rate 87.5% at 24 hpi and 83.9% at 48 hpi. The result from KEGG pathway enrichment analysis showed that the immune related “cytosolic DNA-sensing pathway” was significantly enriched at the first and second time points (6 hpi and 18 hpi), with the up-regulated expression of irf3, il1b, tnfaip3, cxcl8a, ap1-2, c-fos, polr3d, polr3g and polr3k both at 6 hpi and 18 hpi, but not at the third time point (36 hpi). According to the KEGG annotation, 326 immune and inflammation-related DEGs were found. The co-expression network of those 326 DEGs revealed the existence of three modules, and tlr1 was found to be in the center of the biggest module which contained massive DEGs from “signal transduction” and “transport and catabolism”. The c3 isoforms showed different expression pattern among the three time points, indicating a unique activation of complement systems at 18 hpi. Furthermore, two cathelicidins (aaCATH_1 and aaCATH_2) were highly up-regulated at the first two time points, and the bacterial growth inhibition assay revealed their antibacterial properties against A. hydrophila. Our data indicated the important roles of cytosolic DNA-sensing pathway, as well as transcripts including tlr1, c3, polr and cathelicidins in the intestine of A. anguilla in response to A. hydrophila infection. The present study will provide leads for functional studies of host-pathogen interactions.

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.

Copper has been revealed to negatively affect the hematopoietic system, which has an important function in immune pathogen defense, but little is known about the potential mechanism. In this study, copper-stressed larvae exhibited significantly increased mortality as well as reduced percentages of GFP-labeled macrophages and neutrophils after Aeromonas hydrophila (A. hydrophila) infection. However, those copper-stressed GFP-labeled macrophages and neutrophils showed more rapid responses to A. hydrophila infection. The transcriptional profiles in copper-stressed macrophages or neutrophils were unveiled by RNA-Sequencing, and KEGG pathway analysis revealed enrichment of differentially expressed genes (DEGs) in lysosome, apoptosis, oxidative phosphorylation, phagosome, etc. The copper-stressed macrophages or neutrophils were revealed to have an increase in reactive oxygen species (ROS) and mitochondria ROS (mROS)-mediated apoptosis, and a reduction in phagocytosis. Furthermore, the A. hydrophila-infected copper-stressed macrophages or neutrophils were found to be unable to maintain a consistently increased expression in immune responsive genes. This study demonstrated for the first time that copper might induce the susceptibility of fish larvae to inflammatory stimuli via triggering macrophage or neutrophil apoptosis, leading to reduced phagocytic activities and non-sustainable immune responses in immune macrophages or neutrophils.

Streptococcus agalactiae is a major aquaculture pathogen infecting various saltwater and freshwater fish. To better understand the mechanism of the immune responses to S. agalactiae in wildtype zebrafish, the transcriptomic profiles of two organs containing mucosal-associated lymphoid tissues from S. agalactiae-infected and non-infected groups were obtained using RNA-seq techniques. In the intestines, 6735 and 12908 differently expressed genes (DEGs) were identified at 24 hpi and 48 hpi, respectively. Among 66 and 116 significantly enriched pathways, 15 and 21 pathways were involved in immune system or signal transduction at 24 hpi and 48 hpi, respectively. A number of genes involved in Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, T cell receptor signaling pathway, B cell receptor signaling pathway, Antigen processing and presentation, NF-kappa B signaling pathway and PI3K-Akt signaling pathway were significantly downregulated. In the skins, 3113 and 4467 DEGs were identified at 24 hpi and 48 hpi, respectively. Among 24 and 56 significantly enriched pathways, 4 and 13 pathways were involved in immune system or signal transduction at 24 hpi and 48 hpi, respectively. More immune-related signaling pathways including Leukocyte transendothelial migration, Cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, IL-17 signaling pathway, MAPK signaling pathway, TNF signaling pathway, Complement and coagulation cascades, Hematopoietic cell lineage and Jak-STAT signaling pathway were differently enriched for upregulated DEGs at 48 hpi, which were completely different from that in the intestines. Furthmore, comparative transcriptome analysis revealed that the downregulated 1618 genes and upregulated 1622 genes existed both at 24 hpi and 48 hpi for the intestine samples. In the skins, the downregulated 672 genes and upregulated 428 genes existed both at 24 hpi and 48 hpi. Three pathways related to immune processes were significantly enriched for downregulated DEGs both in the intestines and skins collected at 24 hpi and 48 hpi, which included Antigen processing and presentation, Intestinal immune network for IgA production and Hematopoietic cell lineage. Interaction network analysis of DEGs identified the main DEGs in the sub-network of complement and coagulation cascades both in the intestines and skins. Twenty of DEGs involved in complement and coagulation cascades were further validated by Real-time quantitative PCR. Altogether, the results obtained in this study will provide insight into the immune response of zebrafish against S. agalactiae XQ-1 infection in fatal conditions, and reveal the discrepant expression pattern of complement and coagulation cascades in the intestines and skins.

Oxidative stress is a major contributor to the pathogenesis of various inflammatory diseases. Accumulating evidence has shown that oxidative stress is characterized by the overproduction of reactive oxygen species (ROS). Previous reviews have highlighted inflammatory signaling pathways, biomarkers, molecular targets, and pathogenetic functions mediated by oxidative stress in various diseases. The inflammatory signaling cascades are initiated through the recognition of host cell-derived damage associated molecular patterns (DAMPs) and microorganism-derived pathogen associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). In this review, the effects of PRRs from the Toll-like (TLRs), the retinoic acid-induced gene I (RIG-I)-like receptors (RLRs) and the NOD-like (NLRs) families, and the activation of these signaling pathways in regulating the production of ROS and/or oxidative stress are summarized. Furthermore, important directions for future studies, especially for pathogen-induced signaling pathways through oxidative stress are also reviewed. The present review will highlight potential therapeutic strategies relevant to inflammatory diseases based on the correlations between ROS regulation and PRRs-mediated signaling pathways.

Correlation studies about NOD1 and histones have not been reported. In the present study, we report the functional correlation between NOD1 and the histone H2A variant in response to Streptococcus agalactiae infection. In zebrafish, NOD1 deficiency significantly promoted S. agalactiae proliferation and decreased larval survival. Transcriptome analysis revealed that the significantly enriched pathways in NOD1−/− adult zebrafish were mainly involved in immune and metabolism. Among 719 immunity-associated DEGs at 48 hpi, 74 DEGs regulated by NOD1 deficiency were histone variants. Weighted gene co-expression network analysis identified that H2A, H2B, and H3 had significant associations with NOD1 deficiency. Above all, S. agalactiae infection could induce the expression of intracellular histone H2A, as well as NOD1 colocalized with histone H2A, both in the cytoplasm and cell nucleus in the case of S. agalactiae infection. The overexpression of H2A variants such as zfH2A-6 protected against S. agalactiae infection and could improve cell survival in NOD1-deficient cells. Furthermore, NOD1 could interact with zfH2A-6 and cooperate with zfH2A-6 to inhibit the proliferation of S. agalactiae. NOD1 also showed a synergetic effect in inducing the expression of many antibacterial genes, especially antibacterial pattern recognition receptors PGRP2, PGRP5, and PGRP6. Collectively, these results firstly highlight the roles of NOD1 deficiency in the regulation of immune-related and metabolic pathways, and the correlation between zebrafish NOD1 and histone H2A variant in the defense against S. agalactiae infection.

G protein-coupled bile acids receptor 1 (GPBAR1 or TGR5) has been widely studied as a metabolic regulator involved in bile acids synthesis, glucose metabolism and energy homeostasis. Several recent studies have shown that mammalian GPBAR1 is also involved in antiviral innate immune responses. However, the functions of piscine GPBAR1 in antibacterial or antiviral immune responses and lipid metabolism remain unclear. In the present study, we report the functional characterization of zebrafish gpbar1. Similar to mammalian GPBAR1, zebrafish gpbar1 contains similar domain composition, shows a dose-dependent activation by bile acids including INT777, LCA, DCA, CDCA and CA, and can be induced by viral infection. Compared with corresponding control groups, a significant antiviral activity against spring viremia of carp virus (SVCV) infection was observed in ZF4 cells overexpressing zebrafish gpbar1 with INT777 treatment, but not in ZF4 cells overexpressing zebrafish gpbar1 without INT777 treatment. The activation of zebrafish gpbar1 had no significant antibacterial effect against Edwardsiella piscicida infection in ZF4 cells in vitro. Transcriptome analysis revealed that zebrafish gpbar1 activation played a crucial role in activating RLR signaling pathway and inducing the production of ISGs, but not for bile acid biosynthesis and transportation. The co-occurrence analysis for antiviral-related and bile acids metabolism-related DEGs suggested a strong interaction among 2 bile acid receptors (gpbar1 and nr1h4), slco2b1 and the antiviral DEGs. The lipidomic analysis showed that zebrafish gpbar1 activation in ZF4 cells resulted a change of glycerophospholipids, but none of bile acids nor their derivatives, which were different from mammalian GPBAR1. All together, these results firstly demonstrate the conserved antiviral role of gpbar1 and its function in regulating glycerophospholipids metabolism in teleost.

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 nucleotide-binding domain and leucine-rich repeat-containing family (NLR) proteins are innate immune sensors which recognize highly conserved pathogen-associated molecular patterns (PAMPs). Mammals have small numbers of NLR proteins, whereas in some species such as in invertebrates and jawless vertebrates, NLRs have expanded into very large families. Nearly 400 NLR proteins are identified in the zebrafish genome. Members of the NLR family can be divided into two functional sub-groups based on their ability to either positively or negatively regulate host immune response or inflammatory signaling cascades. Mammalian NLRC3 has been identified as an inhibitory NLR, and serves as a negative regulator in the NF-κB-mediated inflammatory response, STING-mediated DNA sensing and PI3K-mTOR pathways. Different from mammalian NLRC3, the analysis from genomes or transcriptomes revealed that the expansions of NLRC3 existed in different species of fish. Furthermore, piscine NLRC3-like genes were confirmed to have a negative or positive regulatory function in response to different kinds of pathogen infections and in the production of proinflammatory cytokines. In this review, we summarize recent advances in our understanding of the expanding and function of NLRC3 or NLRC3-like genes in teleost fish, and give our view of important directions for future studies. The knowledge of piscine NLRC3 or expansive NLRC3-like genes-mediated biological functions in homeostasis and diseases will shed new light on the prevention and control of inflammatory and/or infectious diseases.

Both NLRC3 and NOD1 belong to regulatory NLR subfamily based on their best-characterized function. In mammals, NLRC3 was reported to function by attenuating signaling cascades initiated by other families of PRRs. In teleosts, multiple NLRC3-like genes were identified through transcriptome sequencing. However, the functions of many NLRC3-like genes, especially the fish-specific NLRC3-like genes, remain unclear. In the present study, we report the functional characterization of a novel category of NLRC3-like proteins (named as NLRC3-like 1) from the zebrafish, which consists of a fish-specific FISNA, a conserved NACHT and five C-terminal LRRs domains. The expression of zebrafish NLRC3-like 1 was inducible in response to Edwardsiella piscicida infection. During bacterial infection, the in vitro and in vivo studies revealed that zebrafish NLRC3-like 1 overexpression facilitated bacterial growth and dissemination, together with the decreased survival rate of zebrafish larvae infected with E. piscicida. The attenuated response by zebrafish NLRC3-like 1 in response to bacterial infection were characterized by the impaired expression of antibacterial genes, proinflammatory cytokines and Nox genes. Furthermore, zebrafish NLRC3-like 1 interacted with the adaptor protein RIPK2 of NODs signaling via the FISNA (Fish-specific NACHT associated domain) and NACHT domains. However, the interaction between zebrafish NLRC3-like 1 and RIPK2 inhibited the assembly of the NOD1-RIPK2 complex. Importantly, zebrafish NLRC3-like 1 inhibited NOD1-mediated antibacterial activity, NF-κB and MAPK pathways and proinflammatory cytokine production. All together, these results firstly demonstrate that zebrafish NLRC3-like 1 inhibits NOD1-RIPK2 antibacterial pathway via targeting the adaptor protein RIPK2.

RIP2 is an adaptor protein which is essential for the activation of NF-κB and NOD1- and NOD2-dependent signaling. Although NOD-RIP2 axis conservatively existed in the teleost, the function of RIP2 was only reported in zebrafish, goldfish, and rainbow trout in vitro. Very little is known about the role and mechanisms of piscine NOD-RIP2 axis in vivo. Our previous study showed the protective role of zebrafish NOD1 in larval survival through CD44a-mediated activation of PI3K-Akt signaling. In this study, we examined whether RIP2 was required for larval survival with or without pathogen infection, and determined the signaling pathways modulated by RIP2. Based on our previous report and the present study, our data demonstrated that NOD1-RIP2 axis was important for larval survival in the early ontogenesis. Similar to NOD1, RIP2 deficiency significantly affected immune system processes. The significantly enriched pathways were mainly involved in immune system, such as “Antigen processing and presentation” and “NOD-like receptor signaling pathway” and so on. Furthermore, both transcriptome analysis and qRT-PCR revealed that RIP2 was a critical regulator for expression of NLRs (NOD-like receptors) and those genes involved in MHC antigen presentation. Different from NOD1, the present study showed that NOD1, but not RIP2 deficiency significantly impaired protein levels of MAPK pathways. Although RIP2 deficiency also significantly impaired the expression of CD44a, the downstream signaling of CD44a-Lck-PI3K-Akt pathway remained unchanged. Collectively, our works highlight the similarity and discrepancy of NOD1 and RIP2 in the regulation of immune signaling pathways in the zebrafish early ontogenesis, and confirm the crucial role of RIP2 in NLRs signaling and MHC antigen presentation, but not for MAPK and PI3K/Akt pathways.

In order to get insight into the impacts of UVC/H2O2 on Microcystis aeruginosa, physiological and morphological changes as well as toxicity were detected under different UVC/H2O2 treatments. In the presence of sole UVC or H2O2, the net oxygen evolution rate decreased significantly (p < 0.05) since activity of photosystem II (PSII) was inhibited. Meanwhile, increase of intracellular reactive oxygen species (ROS), degradation of microcystin (MC) and ultrastructure destructions were observed. Under sole UVC treatment, no changes happened in the activity of photosystem I (PSI), but the degradation of D1 protein was observed. Under sole H2O2 treatment, an increase of malondialdehyde, aggregation of D1 protein and deformation of the thylakoid membrane were observed. ROS content under H2O2 treatment was about 5 times than that under UVC treatment. Combined use of UVC and H2O2, as well as 20 mJ cm(-2) UVC and 60 mu M H2O2, showed high synergetic effects. Obvious damage to membrane systems, the marked degradation of MC and inhibition of the photosystems were observed. It could be deduced that UVC worked on intracellular membrane components directly and the damaged oxygen-evolving complex, which was followed by the D1 protein degradation. H2O2 oxidised the membrane lipids via an ROS-mediated process, with thylakoid injury and the aggregation of D1 protein being the lethal mechanisms, and both PSII and PSI being the attacking targets. With regard towards the effective inactivation of M. aeruginosa and high removal of MC, UVC/H2O2 proposed a novel practical method in controlling cyanobacterial blooms. (C) 2015 Elsevier Ltd. All rights reserved.

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.

Pre-mRNA splicing is the process by which introns are removed and the protein coding elements assembled into mature mRNAs. Alternative pre-mRNA splicing provides an important source of transcriptome and proteome complexity through selectively joining different coding elements to form mRNAs, which encode proteins with similar or distinct functions. In mammals, previous studies have shown the role of alternative splicing in regulating the function of the immune system, especially in the regulation of T-cell activation and function. As lower vertebrates, teleost fish mainly rely on a large family of pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) from various invading pathogens. In this review, we summarize recent advances in our understanding of alternative splicing of piscine PRRs including peptidoglycan recognition proteins (PGRPs), nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and their downstream signaling molecules, compared to splicing in mammals. We also discuss what is known and unknown about the function of splicing isoforms in the innate immune responses against pathogens infection in mammals and teleost fish. Finally, we highlight the consequences of alternative splicing in the innate immune system and give our view of important directions for future studies.

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.

Melanoma differentiation-associated gene 5 (MDA5) is one of the three members in the retinoic acid-inducible gene I-like receptor (RLR) family, which are cytoplasmic pathogen recognition receptors recognizing intracellular viruses. In the present study, MDA5 and its spliced shorter forms, named as MDA5a and MDA5b, were identified in zebrafish. MDA5a and MDA5b can be up-regulated in cell lines following the infection of a negative ssRNA virus, the spring viraemia of carp virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, implying that the RLR may also be able to sense elements released from bacteria. The over-expression of MDA5a and MDA5b in fish cells resulted in significant induction of type I interferon promoter activity and enabled the protection of transfected cells against SVCV infection. Furthermore, the shorter spliced form, MDA5b when co-transfected with MDA5a or mitochondrial antiviral signalling protein (MAVS), induced a significantly higher level of interferon promoter activity, indicating that MDA5b may function as an enhancer in the interaction between MDA5 and MAVS.

In this study, the constant-region genes (C alpha, C beta and C gamma) that encode the T-cell antigen receptor (TCR) alpha, beta and gamma chains were cloned from mandarin fish, Siniperca chuatsi Basilewsky, an important freshwater fish species in China. The complementary DNA sequences of C alpha, C beta and C gamma were 843, 716 and 906base pairs (bp) in length and had a 465-, 289- and 360-bp 3 ' untranslated region, encoding 125, 142 and 182 amino acids, respectively. The amino-acid sequences of the constant regions of mandarin fish TCR alpha, beta and gamma chains (encoded by C alpha, C beta and C gamma, respectively) were most similar to those of their teleost counterparts, showing 60% similarity with pufferfish, 48% similarity with Atlantic salmon and 57% similarity with flounder, respectively. The phylogenetic analysis revealed that the mandarin fish C alpha, C beta and C gamma were clustered, respectively, with their vertebrate counterparts. The mandarin fish C alpha, C beta and C gamma could also be separated into four domains: immunoglobulin; connecting peptide (CP); transmembrane (TM); and cytoplasmic tail. Several conserved features in mammalian TCRs were also found in those of mandarin fish, such as a conserved cysteine residue in the CP domain of C alpha, necessary for creating an interchain disulphide bond with the TCR beta chain, and a conserved antigen receptor TM motif in C alpha and C beta. Meanwhile, transcripts of C alpha, C beta and C gamma were detectable in all examined organs, with a stronger signal observed in lymphoid organs. In addition, the temporal transcriptional changes for C alpha and C gamma were investigated, 1, 2, 3, 4, 5, 6 and 8weeks after stimulation with Flavobacterium columnare, in head kidney, spleen, blood, thymus, gill and intestine, using real-time polymerase chain reaction. The results demonstrated stimulation-dependent up-regulations in almost all tissues examined, which indicates that T cells may play important roles in preventing mandarin fish from bacterial invasion. In particular, apart from thymus, T cells were distributed mainly in gill and intestine, where striking up-regulation of C gamma was also observed. These results will facilitate functional studies of teleost TCRs and T cells.

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.

Interleukin-8 (IL-8), a CXC-type chemokine, plays a key role in acute inflammation by recruiting neutrophils in mammals. In the present study, the open reading frame (ORF) of IL -8, encoding 99 amino acids was cloned in mandarin fish, and its function in inflammation was investigated. The IL-8 contains four conserved cysteine residues, with the first two forming the CXC signature motif. The genomic sequence of mandarin fish IL -8 has four exons and three introns, a typical gene organization of the CXC chemokine. Bioactive recombinant IL-8 (rIL-8) exhibited a chemotactic effect on head kidney leukocytes in vitro , and activates the transcription of the inflammatory genes, IL -8 and IL -1β. When mandarin fish was challenged intraperitoneally with the pathogenic bacterium Flavobacterium columnare G 4 , the steady-state protein level of IL-8 was up-regulated in trunk kidney and head kidney. These results suggest that IL-8 is a functional CXC chemokine in mandarin fish, and plays a key role in the inflammatory responses towards bacterial infection.

NOD2/RIPK2 signalling plays essential role in the modulation of innate and adaptive immunity in mammals. In this study, NOD2 was functionally characterized in zebrafish (Danio rerio), and its interaction with a receptor-interaction protein, RIPK2, and RLRs such as MDA5 and RIG-I, as well as the adaptor, MAVS was revealed in fish innate immunity. The expression of NOD2 and RIPK2 in ZF4 cells has been constitutive and can be induced by the infection of Edwardsiella tarda and SVCV. The NOD2 can sense MDP in PGN from Gram-negative and -positive bacteria. It is further revealed that the NOD2 and RIPK2 can activate NF-κB and IFN promoters, inducing significantly antiviral defense against SVCV infection. As observed in the reduced bacterial burden in RIPK2 overexpressed cells, RIPK2 also has a role in inhibiting the bacterial replication. The overexpression of NOD2 in zebrafish embryos resulted in the increase of immune gene expression, especially those encoding PRRs and cytokines involved in antiviral response such as MDA5, RIG-I, and type I IFNs, etc. Luciferase reporter assays and co-immunoprecipitation assays demonstrated that zebrafish NOD2 is associated with MDA5 and RIG-I in signalling pathway. In addition, it is further demonstrated that RIPK2 and MAVS in combination with NOD2 have an enhanced role in NOD2-mediated NF-κB and type I IFN activation. It is concluded that teleost fish NOD2 can not only sense MDP for activating innate immunity as reported in mammals, but can also interact with other PRRs to form a network in antiviral innate response.

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.

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.

Multiple type I interferons (IFNs) have recently been identified in salmonids, containing two or four conserved cysteines. In this work, a novel two-cysteine containing (2C) IFN gene was identified in rainbow trout. This novel trout IFN gene (termed IFN5) formed a phylogenetic group that is distinct from the other three salmonid IFN groups sequenced to date and had a close evolutionary relationship with IFNs from advanced fish species. Our data demonstrate that two subgroups are apparent within each of the 2C and 4C type I IFNs, an evolutionary outcome possibly due to two rounds of genome duplication events that have occurred within teleosts. We have examined gene expression of the trout 2C type I IFN in cultured cells following stimulation with lipopolysaccharide, phytohaemagglutinin, polyI:C or recombinant IFN, or after transfection with polyI:C. The kinetics of gene expression was also studied after viral infection. Analysis of the regulatory elements in the IFN promoter region predicted several binding sites for key transcription factors that potentially play an important role in mediating IFN5 gene expression.

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

In Drosophila, Toll signaling cascade, which resembles the mammalian Toll-like receptor (TLR)/IL-1R signaling pathways and regulates the expression of anti-microbial peptide genes, mainly relies on peptidoglycan recognition proteins (PGRPs) for the detection of bacterial pathogens. To explore the effect of zebrafish peptidoglycan recognition protein 6 (zfPGRP6) on Toll-like receptor signaling pathway, RNA interference (siRNA) and real time quantitative PCR (RQ-PCR) methods were used to identify differentially expressed genes regulated by zfPGRP6. The target genes included TLR2, TLR3, TLR5, TLR7, TLR8, IL1R, Sterile-alpha and Armadillo motif containing protein (SARM), myeloid differentiation factor 88 (MyD88) and nuclear factor (NF)-kappa B2 (p100/p52). The results of RQ-PCR showed that RNAi-mediated Suppression of zfPGRP6 significantly down-regulated the expression of TLR2, TLR5, IL1R, SARM, MyD88 and p100/p52. The expression of beta-defensin-1 was also down-regulated in those embryos silenced by zfPGRP6. In challenge experiments to determine the anti-bacterial response to Gram-negative bacteria, RNAi knock-down of zfPGRP6 markedly increased susceptibility to Flavobacterium columnare. (C) 2008 Elsevier B.V. All rights reserved.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the TNF superfamily members, participating in many biological processes including cell proliferation and apoptotic death. In this study, a TRAIL gene was cloned from a perciform fish, the mandarin fish Siniperca chuatsi, a major cultured fish in China's aquaculture, and is named as SCTRAIL for S. chuatsi TRAIL. The full-length cDNA of SCTRAIL is 1359 bp, encoding a 283-amino-acid protein. This deduced protein contains the CYS231, a 23-mer fragment of transmembrane region, a glycosylation site and a TNF family signature, all of which are conserved among TRAIL members. SCTRAIL gene consists of six exons, with five intervening introns, spaced over approximately 9 kb of genomic sequence. Southern blotting demonstrated that the SCTRAIL gene is present as a single copy in mandarin fish genome. A 620 bp promoter region obtained by genome walking contains a number of putative transcription factor binding sites, such as Oct-1, Sp-1, NF-1, RAP-1, C/EBPaLp, NF-kappa B and AP-1. The SCTRAIL is constitutively expressed in all the analyzed tissues, as revealed by RT-PCR, which is confirmed by Western blotting analysis using polyclonal antibody against bacteria-derived recombinant SCTRAIL protein. As an apoptosis-inducing ligand, the overexpression of SCTRAIL but not the mutant SCTRAIL-C203S in HeLa cells induced changes characteristic of apoptosis, including chromatin condensation, nucleus fragmentation, DNA ladder, and increase of sub-G0/G1 cells in FACS analysis. (c) 2007 Elsevier Ltd. All rights reserved.

BACKGROUND:Interferon regulatory factor (IRF) 10 was first found in birds and is present in the genome of other tetrapods (but not humans and mice), as well as in teleost fish. The functional role of IRF10 in vertebrate immunity is relatively unknown compared to IRF1-9. The target of this research was to clone and characterize the IRF10 genes in three economically important fish species that will facilitate future evaluation of this molecule in fish innate and adaptive immunity. MOLECULAR CHARACTERIZATION OF IRF10 IN THREE FISH SPECIES:In the present study, a single IRF10 gene was cloned in grass carp Ctenopharyngodon idella and Asian swamp eel Monopterus albus, and two, named IRF10a and IRF10b, in rainbow trout Oncorhynchus mykiss. The fish IRF10 molecules share highest identities to other vertebrate IRF10s, and have a well conserved DNA binding domain, IRF-associated domain, and an 8 exon/7 intron structure with conserved intron phase. The presence of an upstream ATG or open reading frame (ORF) in the 5'-untranslated region of different fish IRF10 cDNA sequences suggests potential regulation at the translational level, and this has been verified by in vitro transcription/translation experiments of the trout IRF10a cDNA, but would still need to be validated in fish cells. EXPRESSION ANALYSIS OF IRF10 IN VIVO AND IN VITRO:Both trout IRF10 paralogues are highly expressed in thymus, blood and spleen but are relatively low in head kidney and caudal kidney. Trout IRF10b expression is significantly higher than IRF10a in integumentary tissues i.e. gills, scales, skin, intestine, adipose fin and tail fins, suggesting that IRF10b may be more important in mucosal immunity. The expression of both trout IRF10 paralogues is up-regulated by recombinant IFN-γ. The expression of the IRF10 genes is highly induced by Poly I:C in vitro and in vivo, and by viral infection, but is less responsive to peptidoglycan and bacterial infection, suggesting an important role of fish IRF10 in antiviral defense.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of almost the entire tumor necrosis factor receptor superfamily signaling pathway. In the present study, a TRAF2 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length cDNA is 3162 bp, including a 60 bp 5' untranslated region (UTR), a 1611 bp open reading frame, and a 1491 bp 3' UTR. The polyadenylation signal (AATAAA) and the mRNA instability motifs (ATTTTA, ATTTA) were followed by a poly(A) tail in the 3' UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF2 (gcTRAF2). Phylogenetic tree analysis clearly showed that gcTRAF2 is nearest to the TRAF2 gene of goldfish. The identity of gcTRAF2 with its homologs in other vertebrates ranges from 56% to 97%. It is characterized by one RING-type signature at the N-terminus, one zinc finger in the middle part, and one conserved TRAF domain consisting of a C-proximal (TRAF-C) subdomain and a N-proximal (TRAF-N) subdomain. The identity of TRAF-C among all TRAF2 homologs in vertebrates varies from 78% to 97%, whereas the identity of TRAF-N ranges from 56% to 100%. The recombinant gcTRAF2 has been expressed in Escherichia coli using pET-32a expression vector. The rabbit anti-gcTRAF2 polyclonal antibody was obtained. The expression of gcTRAF2 in different organs was examined by real-time quantitative polymerase chain reaction and Western blot analysis. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of a TRAF2 homolog molecule in fish.

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect and mammals. Using a database mining approach and RT-PCR, multiple peptidoglycan recognition protein (PGRP) like genes have been discovered in fish including zebrafish Danio rerio, Japanese pufferfish TakiFugu rubripes and spotted green pufferfish Tetraodon nigroviridis. They share the common features of those PGRPs in arthropod and mammals, by containing a conserved PGRP domain. Based on the predicted structures, the identified zebrafish PGRP homologs resemble short and long PGRP members in arthropod and mammals. The identified PGRP genes in T. nigroviridis and TakiFugu rubripes resemble the long PGRPs, and the short PGRP genes have not been found in T. nigroviridis and TakiFugu rubripes databases. Computer modelling of these molecules revealed the presence of three alpha-helices and five or six beta-strands in all fish PGRPs reported in the present study. The long PGRP in teleost fish have multiple alternatively spliced forms, and some of the identified spliced variants, e.g., tnPGRP-L3 and tnPGRP-L4 (in: Tetraodon nigroviridis), exhibited no characters present in the PGRP homologs domain. The coding regions of zfPGRP6 (zf: zebrafish), zfPGRP2-A, zfPGRP2-B and zfPGRP-L contain five exons and four introns; however, the other PGRP-like genes including zfPGRPSC1a, zfPGRPSC2, tnPGRP-L1-, tnPGRP-L2 and frPGRP-L (fr: Takifugu rubripes) contain four exons and three introns. In zebrafish, long and short PGRP genes identified are located in different chromosomes, and an unknown locus containing another long PGRP-like gene has also been found in zebrafish, demonstrating that multiple PGRP loci may be present in fish. In zebrafish, the constitutive expressions of zfPGRP-L, zfPGRP-6 and zfPGRP-SC during ontogeny from unfertilized eggs to larvae, in different organs of adult, and the inductive expression following stimulation by Flavobacterium columnare, were detected by real-time PCR, but the levels and patterns varied for different PGRP genes, implying that different short and long PGRPs may play different roles in innate immune response. (c) 2007 Elsevier Ltd. All rights reserved.

The emergence of an infectious viral disease caused by the Chinese giant salamander iridovirus (GSIV) has led to substantial economic losses. However, no more molecular information is available for the understanding of the mechanisms associated with virus–host interaction. In this study, de novo sequencing was used to obtain abundant high-quality ESTs and investigate differentially-expressed genes in the spleen of Chinese giant salamanders that were either infected or mock infected with GSIV. Comparative expression analysis indicated that 293 genes were down-regulated and 220 genes were up-regulated. Further enrichment analysis showed that the most enriched pathway is “complement and coagulation cascades”, and significantly enriched diseases include “inherited thrombophilia”, “immune system diseases”, “primary immunodeficiency”, “complement regulatory protein defects”, and “disorders of nucleotide excision repair”. Additionally, 30 678 simple sequence repeats (SSRs) from all spleen samples, 26 355 single nucleotide polymorphisms (SNPs) from the spleens of uninfected animals and 36 070 SNPs from the spleens of infected animals were detected. The large amount of variation was specific for the Chinese giant salamanders that were infected with GSIV. The results reported herein provided significant and new EST information that could contribute greatly in investigations into the molecular functions of immune genes in the Chinese giant salamander. Electronic supplementary material The online version of this article (doi:10.1186/s13567-015-0279-8) contains supplementary material, which is available to authorized users.

The gene of interferon regulatory factor-2 (IRF-2) has been cloned from the mandarin fish (Siniperca chuatsi). The IRF-2 gene has 6,418 nucleotides (nt) and contains eight exons and seven introns, encoding two mRNAs. The two IRF-2 mRNAs each contained an open reading frame of 873 nt, which both translate into the same 291 amino acids but differed in their 5' untranslated region: one mRNA was transcribed initially from the exon 1 bypassing exon 2, while the other was transcribed from the exon 2. The microsatellites (CA repeats) could be found in the carboxyl terminal region of mandarin fish IRF-2, which result in the truncated form molecules. The microsatellites' polymorphism was investigated, and eight alleles were found in 16 individuals. The microsatellites were also examined in IRF-2 of several freshwater perciform fishes. The transcription of the IRF-2 in different tissues with or without poly inosine-cytidine stimulation was analyzed by real-time PCR, and the constitutive transcription of both molecules could be detected in all the tissues examined.

TRAIL (Apo2 ligand) described as a type II transmembrane protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAIL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRF1 sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen.

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.

A tumor necrosis factor receptor-associated factor 2 binding protein (T2BP) gene was isolated from the grass carp (Ctenopharyngodon idellus) by utilizing suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The grass carp T2BP (GT2BP) gene contains an open reading frame of 579 nucleotide(s) (nt), encoding 193 amino acids, with 23 nt 5'-untranslated region and a long 3'-untranslated region of 434 nt including poly (A), 1 AUUUA motif and 4 AUUUUA motifs. No signal peptide has been detected in the predicted GT2BP, but a characteristic forkhead associated domain is present. The GT2BP mRNA shares 83% identity with the zebrafish DNA sequence, and they both have no introns in the genomic DNA. The putative transcription factor binding sites of GT2BP include two C/EBP alpha binding sites, and one c-Jun binding, one AP-1 binding, and one nuclear factor kappa B (NF kappa B) binding sites. Southern blot analysis revealed that the GT2BP was a single-copy gene. Individual difference was observed in GT2BP expression in examined organs of healthy grass carp. However, the expression of GT2BP in all examined organs in a fish with the highest copepod infection level and the significantly higher expression level in spleen and liver in infected fish may indicate its up-regulation with the parasite infection. (c) 2005 Elsevier B.V. All rights reserved.

The cDNAs and genes of two different types of leucine-rich repeat-containing proteins from grass carp (Ctenopharyngodon idellus) were cloned. Homology search revealed that the two genes, designated as GC-GARP and GC-LRG, have 37% and 32% deduced amino-acid sequence similarities with human glycoprotein A repetitions predominant precursor (GARP) and leucine-rich alpha2-glycoprotein (LRG), respectively. The cDNAs of GC-GARP and GC-LRG encoded 664 and 339 amino acid residues, respectively. GC-GARP and GC-LRG contain many distinct structural and/or functional motifs of the leucine-rich repeat (LRR) subfamily, such as multiple conserved 11-residue segments with the consensus sequence LxxLxLxxN/CxL (x can be any amino acid). The genes GC-GARP and GC-LRG consist of two exons, with 4,782 bp and 2,119 bp in total length, respectively. The first exon of each gene contains a small 5'-untranslated region and partial open reading frame. The putative promoter region of GC-GARP was found to contain transcription factor binding sites for GATA-1, IRF4, Oct-1, IRF-7, IRF-1, AP1, GATA-box and NFAT, and the promoter region of GC-LRG for MYC-MAX, MEIS1, ISRE, IK3, HOXA9 and C/EBP alpha. Phylogenetic analysis showed that GC-GARP and mammalian GARPs were clustered into one branch, while GC-LRG and mammalian LRGs were in another branch. The GC-GARP gene was only detected in head kidney, and GC-LRG in the liver, spleen and heart in the copepod (Sinergasilus major)-infected grass carp, indicating the induction of gene expression by the parasite infection. The results obtained in the present study provide insight into the structure of fish LRR genes, and further study should be carried out to understand the importance of LRR proteins in host-pathogen interactions.

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.