Functional characterization of RIP2 in large yellow croaker (Larimichthys crocea), a protein involved in the host antiviral responses via NF-κΒ, IRF3/7 related signaling.

As an adaptor protein functions essentially in the activation of NF-κΒ and MAPK signaling pathways mediated by NOD1 and NOD2, RIP2 plays important roles in the host innate immune responses. In the present study, the RIP2 ortholog termed Lc-RIP2 was identified and characterized in large yellow croaker (Larimichthys crocea). It was revealed that Lc-RIP2 is consisted of an open reading frame (ORF) of 1695 bp, encoding a protein of 564 aa, with an N-terminal kinase domain and a C-terminal caspase activation and recruitment domain (CARD). Subcellular localization assays demonstrated that Lc-RIP2 was a cytosolic protein, which was broadly distributed in the examined tissues/organs, and could be induced in response to poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulations in vivo according to qRT-PCR analysis. Notably, Lc-RIP2 overexpression in vitro was sufficient to abolish SVCV proliferation in EPC cells, and could significantly induce the activation of NF-κB, IRF3, IRF7, and IFN1 promoters. In addition, luciferase assays found that Lc-RIP2 could cooperate with Lc-MAVS, Lc-TRAF3, Lc-TRAF6, Lc-IRF3, and Lc-IRF7 in NF-κB activation, associate with Lc-TRIF, Lc-MAVS, Lc-TRAF3, Lc-IRF3, and Lc-IRF7 in IRF3 activation, enhance Lc-TRIF, Lc-MAVS, Lc-TRAF3, and Lc-TRAF6 mediated IRF7 activation, and Lc-IRF3 mediated IFN1 activation, whereas suppress NF-κB activation when co-expressed with Lc-TRIF. Co-immunoprecipitation (Co-IP) assays also demonstrated that Lc-RIP2 interacts separately with Lc-TRIF, Lc-MAVS, Lc-TRAF3, Lc-TRAF6, Lc-IRF3, and Lc-IRF7. It is thus collectively indicated that Lc-RIP2 function dominantly in the regulation of the host innate immune signaling.

TRAF5 splicing variants associate with TRAF3 and RIP1 in NF-κB and type I IFN signaling in large yellow croaker Larimichthys crocea.

As a member of the tumor necrosis factor receptor-associated factor (TRAF) family, TRAF5 acts as a crucial adaptor molecule and plays important roles in the host innate immune responses. In the present study, the typical form and a splicing variant of TRAF5, termed Lc-TRAF5_tv1 and Lc-TRAF5_tv2 were characterized in large yellow croaker (Larimichthys crocea). The putative Lc-TRAF5_tv1 protein is constituted of 577 aa, contains a RING finger domain, two zinc finger domains, a coiled-coil domain, and a MATH domain, whereas Lc-TRAF5_tv2 protein is constituted of 236 aa and only contains a RING finger domain due to a premature stop resulted from the intron retention. Subcellular localization analysis revealed that both of Lc-TRAF5_tv1 and Lc-TRAF5_tv2 were localized in the cytoplasm, with Lc-TRAF5_tv2 found to aggregate around the nucleus. It was revealed that Lc-TRAF5_tv1 mRNA was broadly expressed in examined organs/tissues and showed extremely higher level than that of Lc-TRAF5_tv2, and both of them could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulations in vivo. Interestingly, overexpression of Lc-TRAF5_tv1 and Lc-TRAF5_tv2 could significantly induce NF-κB but not IFN1 activation, whereas co-expression of them remarkably induced IFN1 activation but impaired NF-κB activation. In addition, both Lc-TRAF5_tv1 and Lc-TRAF5_tv2 were associated with TRAF3 and RIP1 in IFN1 activation, whereas only Lc-TRAF5_tv1 cooperated with TRAF3 and RIP1 in NF-κB activation. These results collectively indicated that the splicing variant together with the typical form of TRAF5 function importantly in the regulation of host immune signaling in teleosts.

Novel Findings in Teleost TRAF4, a Protein Acts as an Enhancer in TRIF and TRAF6 Mediated Antiviral and Inflammatory Signaling.

Tumor necrosis factor receptor-associated factors (TRAFs) are important adaptor molecules that play important roles in host immune regulation and inflammatory responses. Compared to other members of TRAFs, the function of TRAF4 in vertebrate immunity remains unclear, especially in teleosts. In the present study, TRAF4 ortholog was cloned and identified in large yellow croaker (Larimichthys crocea), named as Lc-TRAF4. The open reading frame (ORF) of Lc-TRAF4 is 1,413 bp and encodes a protein of 470 amino acids (aa), which is consisted of a RING finger domain, two zinc finger domains, and a MATH domain. The genome organization of Lc-TRAF4 is conserved in teleosts, amphibians, birds, and mammals, with 7 exons and 6 introns. Quantitative real-time PCR analysis revealed that Lc-TRAF4 was broadly distributed in various organs/tissues of healthy large yellow croakers and could be significantly up-regulated in the gill, intestine, spleen, head kidney, and blood under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulations. Notably, luciferase assays showed that overexpression of Lc-TRAF4 could significantly induce the activation of IRF3, IRF7, and type I IFN promoters, with the RING finger and zinc finger domains function importantly in such promoter activation. Confocal microscopy revealed that Lc-TRAF4 is located in the cytoplasm, whereas the deletion of the RING finger, zinc finger or MATH domain showed little effect on the subcellular localization of Lc-TRAF4. Interestingly, Lc-TRAF4 overexpression could significantly enhance Lc-TRIF and Lc-TRAF6 medicated IRF3 and IRF7 promoter activation. In addition, co-expression of Lc-TRAF4 with Lc-TRIF or Lc-TRAF6 could significantly induce the expression of antiviral and inflammation-related genes, including IRF3, IRF7, ISG15, ISG56, Mx, RSAD2, TNF-α, and IL-1ß compared to the only overexpression of Lc-TRAF4, Lc-TRIF or Lc-TRAF6. These results collectively imply that Lc-TRAF4 functions as an enhancer in Lc-TRIF and Lc-TRAF6 mediated antiviral and inflammatory signaling.

Molecular cloning and functional characterization of HMGB1 and HMGB2 in large yellow croaker Larimichthys crocea.

High mobility group box 1 (HMGB1) and HMGB2 have been demonstrated to be key regulators not only in DNA recombination, replication, gene transcription, but also in host inflammation and immune responses. In the present study, orthologs of HMGB1 and HMGB2 named Lc-HMGB1 and Lc-HMGB2 were characterized in large yellow croaker (Larimichthys crocea). The ORFs of Lc-HMGB1 and Lc-HMGB2 are 621 bp and 648 bp, encoding proteins of 206 aa and 215 aa, with the putative Lc-HMGB1 and Lc-HMGB2 proteins both contain two HMG domains, respectively. The genome organizations of Lc-HMGB1 and Lc-HMGB2 are both composed of four exons and three introns, which are conserved in vertebrates. Lc-HMGB1 and Lc-HMGB2 were identified as cell nucleus localized proteins, and were ubiquitously distributed in the examined organs/tissues. Additionally, Lc-HMGB1 was significantly up-regulated under LPS and PGN stimulation, whereas the stimulation of poly I:C, LPS, PGN, and Pseudomonas plecoglossicida infection could significantly induce Lc-HMGB2 expression in vivo. Notably, both Lc-HMGB1 and Lc-HMGB2 overexpression could significantly up-regulated the expression of diverse immune-related genes, including IFN1, IRF3, ISG15, ISG56, RSAD2, g-type lysozyme, and TNF-α. Moreover, overexpression of Lc-HMGB1 could also induce the expression of IRF7 and Mx. These results collectively indicate that Lc-HMGB1 and Lc-HMGB2 play important roles in host immune responses against pathogen infection.

Molecular cloning and functional characterization of RIP1 in large yellow croaker Larimichthys crocea.

Receptor interacting protein 1 (RIP1) plays important roles not only in cell-death pathways but also in host innate immune responses. In the present study, a RIP1 ortholog named Lc-RIP1 was cloned and characterized in large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of Lc-RIP1 is 2,046 bp, encoding a protein of 681 amino acids (aa), with an N-terminal kinase domain, an RHIM domain, and a C-terminal death domain. Subcellular localization analysis revealed that Lc-RIP1 was a cytosolic protein, which was broadly expressed in examined tissues/organs, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo based on qRT-PCR analysis. Notably, Lc-RIP1 could induce NF-κB, but not IRF3, IRF7 or type I IFN promoter activation. In addition, Lc-RIP1 overexpression could enhance Lc-MAVS, Lc-TRAF3, and Lc-TRAF6 mediated NF-κB promoter activation, and also Lc-TRIF and Lc-MAVS mediated IRF3 promoter activation, whereas suppress Lc-TRIF mediated NF-κB and type I IFN promoter activation, as well as Lc-TRAF3 and Lc-IRF3 mediated IRF3 promoter activation, Lc-IRF3 mediated type I IFN promoter activation and Lc-IRF7 mediated IRF7 promoter activation. These results collectively indicated that Lc-RIP1 function importantly in regulation of host innate immune signaling.

SARM suppresses TRIF, TRAF3, and IRF3/7 mediated antiviral signaling in large yellow croaker Larimichthys crocea.

As a TIR domain-containing molecular, sterile α-and armadillo motif-containing protein (SARM) acts as an adaptor in Toll-like receptor (TLR) signaling, and also plays important roles in mediating apoptosis and neuronal injury. In the present study, the ortholog of SARM, named as Lc-SARM, was cloned and identified in large yellow croaker (Larimichthys crocea). The full-length ORF of Lc-SARM consists of 2,154 bp, encoding a protein of 717 amino acids (aa), which is comprised of an N-terminal ARM domain, two SAM domains, and a C-terminal TIR domain. Confocal microscopy revealed that Lc-SARM was mainly distributed in the cytoplasm, and the mRNA expression level of Lc-SARM was broadly distributed in all the detected organs/tissues, with the highest expression level found in the brain. The expression patterns of Lc-SARM could be induced in response to poly I:C, LPS, PGN stimulations, and Pseudomonas plecoglossicida infection. Notably, although the overexpression of Lc-SARM could significantly induce NF-κB, IRF3, IRF7, and type I IFN promoter activation, whereas the co-expression of Lc-SARM with Lc-TRIF, Lc-TRAF3, Lc-IRF3, or Lc-IRF7 significantly down-regulated the induction of NF-κB, IRF3, IRF7, or type I IFN promoter activation, and suppressed the antiviral effects as well as the downstream antiviral-related genes expression compared to the only overexpression of Lc-TRIF, Lc-TRAF3, Lc-IRF3, or Lc-IRF7. Co-immunoprecipitation (Co-IP) assays also demonstrated that Lc-SARM interacts separately with Lc-TRIF, Lc-TRAF3, Lc-IRF3, and Lc-IRF7. It is thus collectively suggested that Lc-SARM functions as a negative regulator in Lc-TRIF, Lc-TRAF3, and Lc-IRF3/7 involved antiviral signaling.

MAVS splicing variants associated with TRAF3 and TRAF6 in NF-κB and IRF3 signaling pathway in large yellow croaker Larimichthys crocea.

Mitochondrial antiviral signaling protein (MAVS) acts as an essential adaptor in host RIG-I-like receptors (RLRs) mediated antiviral signaling pathway. In the present study, two MAVS transcript variants, the typical form and a splicing variant, namely Lc-MAVS_tv1 and Lc-MAVS_tv2 were characterized in large yellow croaker (Larimichthys crocea). The putative Lc-MAVS_tv1 protein contains 512 aa, with an N-terminal CARD domain, a central proline-rich region, and a C-terminal transmembrane (TM) domain, whereas Lc-MAVS_tv2 contains 302 aa and lacks the C-terminal TM domain due to a premature stop in the 102 bp intron fragment insertion. Lc-MAVS_tv1 was identified as a mitochondrion localized protein whereas Lc-MAVS_tv2 exhibited an entire cytosolic distribution. Quantitative real-time PCR revealed that Lc-MAVS_tv1 mRNA was broadly expressed in examined organs/tissues and showed extremely higher level than that of Lc-MAVS_tv2, and both of them could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo. Interestingly, overexpression of Lc-MAVS_tv2 could induce the activation of NF-κB but not IRF3, and Lc-MAVS_tv2 co-transfected with Lc-MAVS_tv1 induced a significantly higher level of NF-κB and IRF3 promoter activity. In addition, Lc-MAVS_tv2 overexpression could enhance TRAF3 and TRAF6 mediated NF-κB activation, but suppress TRAF3 and TRAF6 mediated IRF3 activation, implying that the splicing variant Lc-MAVS_tv2 may function as an important regulator in MAVS mediated signaling pathway.

Genome-wide investigation of Dmrt gene family in large yellow croaker (Larimichthys crocea).

The Dmrt (Doublesex and Mab-3 related transcription factor) gene family is a class of crucial transcription factors characterized by a conserved DM (Doublesex/Mab-3) domain. Previous researches indicate this gene family is involved in various physiological processes, especially in sex determination/differentiation and gonad development. Despite the vital roles of the Dmrt gene family in physiological processes, the comprehensive characterization and analysis of the dmrt genes in large yellow croaker (Larimichthys crocea), one of the most commercially important marine fish in China, have not been described. In this study, we performed the first genome-wide systematic analysis of L. crocea dmrt genes through the bioinformatics method. A total of seven members of the Dmrt gene family including Lcdmrt1, Lcdmrt2a, Lcdmrt2b, Lcdmrt3, Lcdmrt4, Lcdmrt5, and Lcdmrt6 were excavated based on the genome data of L. crocea. Further analysis revealed that the dmrt genes of L. crocea were distributed unevenly across four chromosomes. There were three dmrt genes (Lcdmrt1, Lcdmrt2a, and Lcdmrt3) on 3rd chromosome, one (Lcdmrt6) on 13th chromosome, one (Lcdmrt4) on 14th chromosome, two on (Lcdmrt5 and Lcdmrt2b) 17th chromosome. The gene structure analysis indicated that the number of introns of different dmrt genes of L. crocea had some differences: Lcdmrt1 had four introns, Lcdmrt2a, Lcdmrt2b, and Lcdmrt6 had two introns, Lcdmrt3, Lcdmrt4, and Lcdmrt5 had only one intron. The expression pattern analysis with published gonad transcriptome datasets and further confirmed by qRT-PCR revealed that these members of the Dmrt gene family except for Lcdmrt4 were all sexually dimorphic and preferred expressing in testis. Furthermore, the expression pattern analysis also revealed that the expression level of Lcdmrt1 and Lcdmrt6 was significantly higher than that of other members, suggesting that these two genes may play a more important role in testis. Overall, our studies provide a comprehensive insight into the Dmrt gene family members and a basis for the further study of their biological functions in L. crocea.

TRAF3 enhances TRIF-mediated signaling via NF-κB and IRF3 activation in large yellow croaker Larimichthys crocea.

As a member of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family, TRAF3 is an important regulator of NF-κB and type I interferon (IFN) activation, especially in Toll-like receptors (TLRs)- and retinoic acid inducible gene I (RIG-I)-like receptors (RLRs)-mediated signaling pathway. In the present study, a TRAF3 homologue named Lc-TRAF3 was characterized in large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of Lc-TRAF3 contains 1788 bp encoding a protein of 595 amino acids (aa). Sequence analysis indicated that Lc-TRAF3 is conserved in vertebrates, constituted with a N-terminal RING finger, two TRAF-type zinc fingers, and a C-terminal TRAF-MATH domain. The genome organization of Lc-TRAF3 is conserved in fish, with 13 exons and 12 introns, but different from that in birds or mammals, which contains 10 exons and 9 introns. Lc-TRAF3 was identified as cytosolic protein base on fluorescence microscopy analysis. Expression analysis revealed that Lc-TRAF3 was broadly distributed in examined organs/tissues, with the highest expression level in gill and weakest in brain, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo. Interestingly, overexpression Lc-TRAF3 could induce the activation of NF-κB, and Lc-TRAF3 co-transfected with Lc-TRIF induced a significantly higher level of NF-κB and IRF3 promoter activity, implying that Lc-TRAF3 may function as an enhancer in Lc-TRIF-mediated signaling pathway.

Molecular cloning and functional characterization of TRIF in large yellow croaker Larimichthys crocea.

As an adaptor in Toll-like receptor (TLR) signaling pathway, Toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon-ß (TRIF) mediates downstream signaling cascades and plays important roles in host innate immune responses. In the present study, a TRIF ortholog named Lc-TRIF was identified in large yellow croaker (Larimichthys crocea). Sequence comparison analysis revealed that Lc-TRIF has a conserved TIR domain but without TRAF6 binding motif. The genome structure of Lc-TRIF is conserved, with two exons and one intron. Syntenic comparison showed that the loci of fish TRIF was different from that in mammals or birds, and TRAM was absent in the genomes of fish, amphibians, and birds, but present in mammals and reptiles. Expression analysis revealed that Lc-TRIF was broadly expressed in examined organs/tissues, with the highest expression level in gill and weakest in brain, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation. Fluorescence microscopy results showed that Lc-TRIF exhibited a global localization throughout the entire cell including the nucleus in HEK 293T cells. Additionally, luciferase assays demonstrated that Lc-TRIF expression could significantly induce NF-κB, type I IFN, IRF3 as well as IRF7 promoter activation. These results collectively indicated that Lc-TRIF was function in host antiviral and antibacterial responses via NF-κB and IRF3/7 related signaling pathway.

Comparison of serum PCT and CRP levels in patients infected by different pathogenic microorganisms: a systematic review and meta-analysis.

To avoid the abuse and misuse of antibiotics, procalcitonin (PCT) and C-reactive protein (CRP) have been used as new approaches to identify different types of infection. Multiple databases were adopted to search relevant studies, and the articles that satisfied the inclusion criteria were included. Meta-analyses were conducted with Review Manager 5.0, and to estimate the quality of each article, risk of bias was assessed. Eight articles satisfied the inclusion criteria. The concentrations of both PCT and CRP in patients with bacterial infection were higher than those with non-bacterial infection. Both PCT and CRP levels in patients with G- bacterial infection were higher than in those with G+ bacterial infection and fungus infection. In the G+ bacterial infection group, a higher concentration of CRP was observed compared with fungus infection group, while the difference of PCT between G+ bacterial infection and fungus infection was not significant. Our study suggested that both PCT and CRP are helpful to a certain extent in detecting pneumonia caused by different types of infection.

Comparison of serum PCT and CRP levels in patients infected by different pathogenic microorganisms: a systematic review and meta-analysis

To avoid the abuse and misuse of antibiotics, procalcitonin (PCT) and C-reactive protein (CRP) have been used as new approaches to identify different types of infection. Multiple databases were adopted to search relevant studies, and the articles that satisfied the inclusion criteria were included. Meta-analyses were conducted with Review Manager 5.0, and to estimate the quality of each article, risk of bias was assessed. Eight articles satisfied the inclusion criteria. The concentrations of both PCT and CRP in patients with bacterial infection were higher than those with non-bacterial infection. Both PCT and CRP levels in patients with G− bacterial infection were higher than in those with G+ bacterial infection and fungus infection. In the G+ bacterial infection group, a higher concentration of CRP was observed compared with fungus infection group, while the difference of PCT between G+ bacterial infection and fungus infection was not significant. Our study suggested that both PCT and CRP are helpful to a certain extent in detecting pneumonia caused by different types of infection.

Zebrafish as a Model for the Study of Host-Virus Interactions.

Zebrafish (Danio rerio) has become an increasingly important model for in vivo and in vitro studies on host-pathogen interaction, offering scientists with optical accessibility and genetic tractability, and a vertebrate-type immunity that can be separated into innate and adaptive ones. Although it is shown in previous studies that few species of viruses can naturally infect zebrafish, the spring viraemia of carp virus (SVCV), a rhabdovirus that causes contagious acute hemorrhagic viraemia in a variety of cyprinid fishes, can infect zebrafish by both injection and static immersion methods in laboratory conditions. In addition, SVCV can infect zebrafish fibroblast cell line (ZF4 cells), together with the Epithelioma papulosum cyprini (EPC) cell line (EPC cells), a common cell line used widely in fish disease research. The infection and propagation of SVCV in zebrafish and especially in these cell lines can be employed conveniently in laboratory for functional assays of zebrafish genes. The zebrafish, ZF4 and EPC cell, and SVCV can serve as a simple and efficient model system in understanding host-virus interactions. In the present chapter, we provide detailed protocols for the host-virus interaction analysis based on zebrafish embryos, ZF4/EPC cells, and SVCV, including infection methods of zebrafish embryos and cell lines, analyses of immune responses by quantitative PCR (qPCR) and RNA sequencing (RNA-Seq), antiviral assays based on ZF4 and EPC cells, and the analysis of host-virus interaction using luciferase assays. These protocols should provide efficient and typical means to address host-virus interactions in a more general biological sense.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in fish: current knowledge and future perspectives.

Retinoic acid-inducible gene I (RIG-I) -like receptors (RLRs) are found conservatively present in teleost fish. All three members, RIG-I, MDA5 and LGP2, together with the downstream molecules such as MITA, TRAF3 and TBK1, have been identified in a range of fish species. However, it is unexpected that RIG-I has not been reported in fish of Acanthopterygii, and it would be important to clarify the presence and role of the RIG-I gene in a broad range of taxa in Teleostei. RLRs in fish can be induced in vivo and in vitro by viral pathogens as well as synthetic dsRNA, poly(I:C), leading to the production of type I interferons (IFNs) and the expression of IFN-stimulated genes (ISGs). Bacterial pathogens, such as Edwardsiella tarda, and their components, such as lipopolysaccharide are also found to induce the expression of RLRs, and whether such induction was mediated through the direct recognition by RLRs or through crosstalk with other pattern recognition receptors recognizing directly bacterial pathogen-associated molecular patterns awaits to be investigated. On the other hand, RLR-activated type I IFN production can be negatively regulated in fish by molecules, such as TBK-1-like protein and IRF10, which are found to negatively regulate RIG-I and MAVS-activated type I IFN production, and to block MITA or bind ISRE motifs, respectively. It is considered that the evolutionary occurrence of RLRs in fish, and their recognized ligands, especially those from their fish pathogens, as well as the mechanisms involved in the RLR signalling pathways, are of significant interest for further investigation.

Cloning and functional characterization of IRAK4 in large yellow croaker (Larimichthys crocea) that associates with MyD88 but impairs NF-κB activation.

As crucial signaling transducer in Toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling pathway, IL-1R-associated kinase 4 (IRAK4) mediates downstream signaling cascades and plays important roles in innate and adaptive immune responses. In the present study, an IRAK4 orthologue was characterized from large yellow croaker (Larimichthys crocea), named Lc-IRAK4, with a conservative N-terminal death domain and a C-terminal protein kinase domain. The genome of Lc-IRAK4 is structured into eleven exons and ten introns. Expression analysis indicated that Lc-IRAK4 was widely expressed in tested tissues, with the highest level in liver and weakest in muscle. Additionally, in the spleen, liver tissues and blood, it could be induced by poly I:C and LPS stimulation, but not be induced by Vibrio parahemolyticus infection. Fluorescence microscopy assays revealed that Lc-IRAK4 localized in the cytoplasm in HEK 293T cells. It was also determined that Lc-IRAK4 could interact with MyD88, whereas MyD88-mediated NF-κB activation was significantly impaired when co-transfected the two in HEK 293T cells. These findings collectively indicated that although Lc-IRAK4 was evolutionarily conserved in vertebrates, the exact function especially the signaling transduction mediated by IRAK4 in fish immune response was different from that in mammals, which impaired MyD88-mediated NF-κB activation.

NOD2 in zebrafish functions in antibacterial and also antiviral responses via NF-κB, and also MDA5, RIG-I and MAVS.

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.

Evolution of IFN-λ in tetrapod vertebrates and its functional characterization in green anole lizard (Anolis carolinensis).

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

Molecular and immune response characterizations of IL-6 in large yellow croaker (Larimichthys crocea).

Interleukin-6 (IL-6) is a multifunctional inflammatory cytokine which exists in multiple tissues and cell lines. In the present study, the full-length cDNA and the genomic sequence of IL-6 (LcIL-6) were cloned from large yellow croaker, Larimichthys crocea. The full-length cDNA of LcIL-6 was 1066 base pairs (bp), containing an open reading frame (ORF) of 678 bp encoding for 225 amino acids, a 5' untranslated region (UTR) of 71 bp and a 3' UTR of 317 bp. The predicted LcIL-6 protein included a 24 amino acids (aa) signal peptide and a conserved IL-6 domain. However, the polypeptide sequence identities between LcIL-6 and its counterparts in mammals and other fish are from 12% to 45%. The genome sequence of LcIL-6 gene was composed of 2126 bp, including five exons and four introns. Phylogenetic analysis revealed that LcIL-6 showed a close relationship with the IL-6 from other bony fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that LcIL-6 mRNA was expressed in most examined tissues, with the most predominant expression in stomach, followed by blood and very weak expression in other tissues. The expression levels of LcIL-6 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were investigated in spleen, head-kidney and liver. LcIL-6 transcripts were induced significantly after immune challenge, with the peak-value of 33.5 times as much as the control in the head-kidney at 3 h after LPS injection (p < 0.05). Overexpression of LcIL-6 enhanced tumor necrosis factor (TNF)-α transcripts significantly (p < 0.05) in L. crocea kidney (LCK) cells. Additionally, recombinant LcIL-6 mature peptide was obtained in the supernatant of Escherichia coli BL21 (DE3). The purified recombinant LcIL-6 fusion protein was also demonstrated to improve the transcriptional expression levels of TNF-α significantly in LCK cells (p < 0.05). However, no significant changes of Mx (myxovirus resistant protein), IL-1ß, janus kinase (JAK)2, signal transducers and activators of transcription (STAT)3 and STAT5 in LCK cells was detected after LcIL-6 overexpression or recombinant LcIL-6 protein stimulation. Our results indicated that LcIL-6 might be important in large yellow croaker immune response and improve the inflammatory response by through activation TNF-α expression.

MAVS splicing variants contribute to the induction of interferon and interferon-stimulated genes mediated by RIG-I-like receptors.

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 IFN1, 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.

Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish.

As an intracellular pattern recognition receptor (PRR), the retinoic acid-inducible gene-I (RIG-I) is responsible for the recognition of cytosolic viral nucleic acids and the production of type I interferons (IFNs). In the present study, an insertion variant of RIG-I with 38 amino acids inserted in the N-terminal CARD2 domain, as well as the typical type, named as RIG-Ia and RIG-Ib respectively were identified in zebrafish. RIG-Ia and RIG-Ib were all up-regulated following the infection of a negative ssRNA virus, the Spring Viremia of Carp Virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, indicating the RLR may have a role in the recognition of both viruses and bacteria. The over-expression of RIG-Ib in cultured fish cells resulted in significant increase in type I IFN promoter activity, and in protection against SVCV infection, whereas the over-expression of RIG-Ia had no direct effect on IFN activation nor antiviral response. Furthermore, it was revealed that both RIG-Ia and RIG-Ib were associated with the downstream molecular mitochondrial antiviral signaling protein, MAVS, and interestingly RIG-Ia when co-transfected with RIG-Ib or MAVS, induced a significantly higher level of type I IFN promoter activity and the expression level of Mx and IRF7, implying that the RIG-Ia may function as an enhancer in the RIG-Ib/MAVS-mediated signaling pathway.