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Simple Summary: TRIM family proteins form a large family of proteins that play a central role in antiviral host defense. These proteins are characterized and identified in tetrapods but the knowledge related to this family among teleost species is very limited. In this study, we analyze the evolutionary relationships, characterization, and functional annotation of common carp TRIM family. The evolutionary analysis revealed that the TRIM family among tetrapods and teleost species is conserved in sequence similarity and domain architectures. The occurrence of certain motifs across the TRIMs members implies a conserved nature and indicate a group specific function. However, the exon-intron organization is more diverse suggesting the complex alternative splicing and duplication. The functional annotation shows that most TRIM proteins are associated with ubiquitination, which can potentially mediate multicellular processes including immune response, cell cycle regulation, modulation of different substrates of important signaling pathways and post translational modifications. The study results will open up new avenues for future research and can be used to elucidate several mechanisms related to common carp development and immune response. Tripartite motifs (TRIM) is a large family of E3 ubiquitin ligases that play an important role in ubiquitylation. TRIM proteins regulate a wide range of biological processes from cellular response to viral infection and are implicated in various pathologies, from Mendelian disease to cancer. Although the TRIM family has been identified and characterized in tetrapods, but the knowledge about common carp and other teleost species is limited. The genes and proteins in the TRIM family of common carp were analyzed for evolutionary relationships, characterization, and functional annotation. Phylogenetic analysis was used to elucidate the evolutionary relationship of TRIM protein among teleost and higher vertebrate species. The results show that the TRIM orthologs of highly distant vertebrates have conserved sequences and domain architectures. The pairwise distance was calculated among teleost species of TRIMs, and the result exhibits very few mismatches at aligned position thus, indicating that the members are not distant from each other. Furthermore, TRIM family of common carp clustered into six groups on the basis of phylogenetic analysis. Additionally, the analysis revealed conserved motifs and functional domains in the subfamily members. The difference in functional domains and motifs is attributed to the evolution of these groups from different ancestors, thus validating the accuracy of clusters in the phylogenetic tree. However, the intron-exon organization is not precisely similar, which suggests duplication of genes and complex alternative splicing. The percentage of secondary structural elements is comparable for members of the same group, but the tertiary conformation is varied and dominated by coiled-coil segments required for catalytic activity. Gene ontology analysis revealed that these proteins are mainly associated with the catalytic activity of ubiquitination, immune system, zinc ion binding, positive regulation of transcription, ligase activity, and cell cycle regulation. Moreover, the biological pathway analyses identified four KEGG and 22 Reactome pathways. The predicted pathways correspond to functional domains, and gene ontology which proposes that proteins with similar structures might perform the same functions. [ABSTRACT FROM AUTHOR]

TRIF, an important adaptor downstream of Toll-like receptor signaling, plays a critical role in the innate immune response. In this study, the full-length coding sequence of TRIF from common carp (Cyprinus carpio L.) was cloned and characterized. Bioinformatics analysis showed that common carp TRIF exhibited a conserved TIR domain and had the closest relationship with grass carp TRIF. Expression analysis revealed that TRIF was constitutively expressed in the examined tissues of common carp, with the highest expression in the spleen and the lowest expression in the head kidney, and could be upregulated under Aeromonas hydrophila and poly(I:C) stimulation in vivo and under poly(I:C), LPS, PGN, flagellin, and Pam3CSK4 stimulation in vitro. Laser confocal microscopy showed that common carp TRIF colocalized with the Golgi apparatus. A luciferase reporter assay showed that carp TRIF elicited the activity of ifn-1 and nf-κb through the C-terminal domain. Additionally, crystal violet staining and qPCR assays revealed that carp TRIF inhibited the replication of SVCV in epithelioma papulosum cyprini (EPC) cells. Then, the signaling downstream of carp TRIF was investigated. Coimmunoprecipitation and Western blotting analysis demonstrated that carp TRIF interacted with TBK1 and augmented the expression of TRAF6 and phosphorylation of TBK1. Overexpression of carp TRIF significantly enhanced the expression of interferon-stimulated genes and inflammatory cytokines. Furthermore, flow cytometric (FCM) analysis suggested that carp TRIF induced apoptosis through the activation of caspase-8. In summary, our study indicated that TRIF plays an essential role in the innate immune responses of common carp against bacterial and viral infection. [ABSTRACT FROM AUTHOR]

p65 is an important subunit of the transcription factor NF-κB in the regulation of immune response. In the present study, the p65 cDNA was identified from common carp (Cyprinus carpio L.) (named Cc p65). Phylogenetic analysis revealed that Cc p65 located in the same clade as piscine p65 and exhibited closest relationship to that of Ctenopharyngodon idella. Cc p65 was constitutively expressed in all the examined tissues. Aeromonas hydrophila and poly(I:C) can induce the expression of Cc p65 in the designated tissues and the Cc p65 expression was up-regulated in HKLs following LPS and poly(I:C) stimulation. In addition, the nuclear localization signal (NLS) and C-terminal domain are the important elements of Cc p65. Immunofluorescence assay revealed that the nuclear localization signal deletion mutation of Cc p65 (Cc p65ΔNLS) failed to translocate to the nucleus even though stimulation with poly(I:C) or LPS, and the C-terminal domain deletion mutation of Cc p65 (Cc p65ΔC) did not up-regulate the luciferase activity. Furthermore, Cc p65 can induce the expression of il-1β and tnf-α. And LPS and poly(I:C) inducing the expression of il-1β and tnf-α , is dependent on the Cc p65. Taken altogether, these findings lay the foundations for future research to investigate the mechanisms underlying fish p65. • The expression of Cc p65 was up-regulated after challenged with Aeromonas hydrophila and poly(I:C). • Cc p65 activated the κB element-driven reporter. • Cc p65 induced the expression of il-1β and tnf-α. • Cc p65 was a well conserved subunit of NF-κB. [ABSTRACT FROM AUTHOR]

In the present study, interferon (IFN) regulatory factor (IRF) 9 gene (irf9) was identified and characterized in common carp Cyprinus carpio. The predicted protein sequence of Irf9 contains a DNA binding domain (DBD) that possess five tryptophans, an IRF association domain (IAD) and two nuclear localisation signals (NLS). Alignment of Irf9 of C. carpio with the corresponding Irf9 proteins of other species showed that the DBD is more highly conserved than the IAD. The putative Irf9 protein sequence of C. carpio shares higher identities with teleosts (53.8–82.3%) and lower identities with mammals (30.2–31.0%). Phylogenetic studies of the putative amino‐acid sequence of IRF9 based on the neighbour‐joining method showed that Irf9 of C. carpio has the closest relationship with the grass carp Ctenopharyngodon idella. Tissue distribution analysis showed that irf9 transcripts were detectable in all examined tissues with the highest expression in the skin and the lowest expression in the head kidney. Poly I:C and Aeromonas hydrophila stimulation up‐regulated irf9 expression in the spleen, head kidney, foregut and hindgut at different time intervals. In addition, irf9 was induced by Poly I:C and lipopolysaccharides (LPS) in vitro. These results indicate that Irf9 participates in antiviral and antibacterial immunity. Transfection of irf9 up‐regulated the expression of cytokines, including type I IFN, protein kinase R (PKR), interferon‐stimulated gene (ISG)15 and tumour necrosis factor (TNF)α in epithelioma papulosum cyprini cells (EPC) upon poly I:C and LPS stimulation. A dual‐luciferase reporter assay revealed that Irf9 has no effect on NF‐κB activation. The present study on Irf9 provides new insights into the IFN system of C. carpio and a valuable experimental platform for future studies on the immune system of fish. [ABSTRACT FROM AUTHOR]

Abstract Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are a large group of cytoplasmic pattern recognition receptors (PRRs), which play an important role in pathogen recognition and regulation of innate immune response. In fish, NLRs are divided into three distinct subfamilies: NLR-A resembling mammalian NODs, NLR-B resembling mammalian NALPs and fish-specific NLR-C. Presently, no data is available about the common carp NLR gene, and meanwhile the studies concerning fish NLR-C subfamily genes are relatively poor. In the present study, we cloned and characterized a novel NLRC gene (CcNLRC) from common carp. The full-length cDNA of CcNLRC was 3642 bp, with an ORF of 3078 bp encoding 1025 amino acids. CcNLRC appears to be unique to fish, consisting of a fish-specific NACHT associated (FISNA) domain, a NACHT domain, three LRR motifs and an extra B30.2 domain at C-terminus. Expression analysis revealed that CcNLRC was constitutively expressed in various healthy tissues, and during early developmental stages CcNLRC had two expression peaks (1 dpf and 24 dpf). In vivo stimulation with polyI:C and V. anguillarum showed significant up-regulation of CcNLRC expression in some immune-related tissues including liver, spleen, foregut, hindgut and skin. Additionally, in vitro study in common carp PBLs and HKLs stimulated with different ligands such as polyI:C, flagellin and PGN showed enhanced gene expression of CcNLRC. These results suggested that CcNLRC might play an important role in the innate immune defense of common carp against pathogen invasion. Highlights • A fish-specific NLRC gene (CcNLRC) was identified from common carp. • CcNLRC had two expression peaks during early developmental stages of common carp. • CcNLRC mRNA was up-regulated after polyI:C or V. anguillarum challenge in vivo. • Gene Expression of CcNLRC was induced by polyI:C, flagellin and PGN in vitro. [ABSTRACT FROM AUTHOR]

Abstract Toll-like receptor 8 (Tlr8) is a member of intracellular TLRs family and play a critical role in the innate immunity. In the present study, we aimed to identify tlr8 from common carp (Cyprinus carpio L.), and explored its expression profile, localization, adaptor, and signaling pathways. A novel tlr8 cDNA sequence (Cctlr8) was identified from the carp, containing a signal peptide, a LRR-N-terminal (LRR-NT), 14 leucine-rich repeats, a LRR-C-terminal (LRR-CT), a transmembrane region and a TIR domain. Phylogenetic analysis revealed that Cc Tlr8 exhibited closest relationship to that of Ctenopharyngodon idella and Danio. rerio. Subcellular localization analysis indicated that Cc Tlr8 was localized to the endoplasmic reticulum in both HeLa cells and EPC cells. Quantitative Real-Time PCR analysis demonstrated that Cctlr8 was constitutively expressed in all the examined tissues, with the highest expression observed in the spleen. After poly (I:C) injection, the expression of Cctlr8 was significantly up-regulated in all the tested tissues. In addition, the expression of Cctlr8 was up-regulated in both PBLs and HKLs following poly (I:C) stimulation. The results of immuofluorescence and coimmunoprecipitation analysis indicated that Cc Tlr8 might recruit TIRAP as the adaptor. Furthermore, Luciferase reporter assays revealed that Cc Tlr8 could activate AP-1 in 293 T cells. Taken altogether, these findings lay the foundations for future research to investigate the mechanisms underlying fish tlr8. Highlights • Cc Tlr8 was localized to the endoplasmic reticulum. • The expression of Cctlr18 was up-regulated after challenged with poly (I:C). • Cc Tlr8 recruited TIRAP as the adaptor. • Cc Tlr8 activated AP-1 in 293 T cells. [ABSTRACT FROM AUTHOR]

Toll-interacting protein (Tollip) is a mediator involved in the TLRs signaling pathway which is critical for innate immune response. In the present study, a full-length Tollip cDNA was first cloned from common carp ( CcTollip), which was 1284 bp in length, containing an open reading frame of 831 bp encoding a peptide of 276 amino acids. Multiple sequence alignment showed that the CcTollip shared the highest similarity with that of grass carp and zebrafish. Phylogenetically, the CcTollip clustered together well with their piscine family members. Quantitative real-time PCR analysis indicated that CcTollip was widely expressed in all tissues tested and showed up-regulation with challenges of Vibrio anguillarum and poly(I:C), suggesting that CcTollip was activated by V. anguillarum and poly(I:C). These data indicated that CcTollip might play an important role in immune response to bacterial and viral invasion. [ABSTRACT FROM AUTHOR]

Inflammatory Caspases are key effectors of the inflammasomes and play an important role in innate immune response. However, there are few studies on the homologs of inflammatory Caspases in bony fish. In the present study, three inflammatory Caspase genes were cloned from common carp and named Cc Caspase-A1, Cc Caspase-A2 and Cc Caspase-B. Nucleotide sequences alignment revealed that the three Caspases were very similar in structure, which contained a PYD domain in the N-terminal, and a CASc domain in the C-terminal. In the phylogenetic tree, Cc Caspase-A1 and Cc Caspase-A2 were close to the Caspase-A of grass carp, and Cc Caspase-B was close to the Dr Caspase-B of zebrafish. In healthy common carp, the expression levels of Cc Caspase-A1 and Cc Caspase-A2 were the highest in the gills, and Cc Caspase-B was the highest in the spleen. After immune stimulation with Edwardsiella tarda or Aeromonas hydrophila , the expression levels of all Cc Caspases increased significantly. The fluorescence localization assays showed that all these Cc Caspases were expressed in the cytoplasm, and were involved in the assembly of Cc NLRP1 inflammasome. These results suggest that the inflammatory Cc Caspases play a key role in immune response of common carp against bacterial infection, which may enrich the knowledge of inflammasome in fish, and provide basic data for the prevention and treatment of fish infectious diseases. • Three inflammatory Caspase genes were identified from common carp. • After bacterial stimulation, the expression of three Caspases increased. • Three Caspases were involved in the assembly of NLRP1 inflammasome. [ABSTRACT FROM AUTHOR]

Mannose receptor (MR), as a member of the C-type lectin (CLEC) family, plays an important role in the internalize pathogen-associated ligands and activate immune response. In the present study, MR was identified and characterized from Asian swamp eel (Monopterus albus) (namely Ma MR). The open reading frame of Ma MR was 4311 bp in length encoding 1437 amino acids of a ∼162.308 kDa protein, including a cysteine-rich (CR) domain, a fibronectin type II (FNII) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short cytoplasmic domain. Phylogenetic analysis indicated that Ma MR shared the highest similarity with that of Paralichthys olivaceus. The expression of Ma MR was found in all the examined tissues, with the highest expression in the spleen and kidney. After injection with Edwardsiella tarda , the transcript level of Ma MR was initially reduced and then significantly elevated in the liver, spleen, foregut and hindgut. In the isolated peripheral blood leukocytes, the expression of Ma MR was significantly induced post stimulated with LPS and LTA. Then the Ma MR-CTLD4-8 recombinant protein was purified. Bacterial agglutination and binding assay showed that r Ma MR-CTLD4-8 could bind with both Gram-positive and Gram-negative bacteria and agglutinate bacteria in the presence of calcium in vitro. Further analysis revealed that Ma MR and TLR2 coordinately induced the expression of TRAF6 and promoted the phosphorylation level of p65, leading to the expression of proinflammatory cytokines il-1β and tnf-α in EPC cells. Taken together, these results reveal that Ma MR plays an important role in the immune response of fish to pathogen infections. • Mannose receptor (MR) was identified and characterized from Monopterus albus (namely Ma MR). • Ma MR was expressed in all examined tissues and could be induced by bacteria and PAMPs. • Ma MR can agglutinate and bind Gram-positive and -negative bacteria. • Ma MR induced the expression of proinflammatory cytokine with the assistance of TLR2. [ABSTRACT FROM AUTHOR]

Common carp (Cyprinus carpio L.) is one of the most widely cultivated fish in China. Spring viraemia of carp virus (SVCV) is a highly pathogenic virus and has often caused excessive losses in carp pond fisheries. Innate immune play important roles against virus infection. To better understand the immune response of common carp against SVCV infection, transcriptome analysis was performed using the Illumina Novaseq 6000 platform. It was showed that a total of 3953 differentially expressed unigenes were identified, and the RLR signaling pathway were significantly enriched after SVCV infection. Subsequently, the role of RLR signaling pathway in SVCV infection was studied. The results showed that common carp RIG-I (Cc RIG-I) and TRIM25 (Cc TRIM25) significantly decreased the replication of SVCV by inducing the phosphorylation of TBK1, IRF3 and p65 and the expression of ifn-1 , viperin , isg15 and mx. Further studies illustrated that Cc TRIM25 could positive regulate Cc RIG-I mediated downstream signaling pathway. Finally, the mechanism of Cc TRIM25 promoting Cc RIG-I-mediated signaling was investigated. Cc TRIM25 could interact with the caspase activation and recruitment domain (CARD) of Cc RIG-I and promoted K63-linked polyubiquitination of Cc RIG-I. Altogether, the study revealed a mechanism of Cc TRIM25 regulating Cc RIG-I mediated immune response in SVCV infection. • Comparative transcriptome analysis of common carp was performed with and without SVCV-infected. • RLRs signaling pathway were significantly enriched. • Cc RIG-I and Cc TRIM25 participated in immune processes against SVCV infection. • Cc TRIM25 positively regulated Cc RIG-I mediated signal pathway through promoting its K63-linked polyubiquitination. [ABSTRACT FROM AUTHOR]

NLRP1 (NLR family pyrin domain containing 1) is the first member of NOD-like receptors (NLRs) which can form inflammasome and play critical roles in innate immunity and pathogenesis of various diseases. To date, many NLRs and inflammasome-related genes have been identified in teleost, however, the activation of NLRP1 inflammasome is only found in zebrafish, and the activator of fish NLRP1 is unclear. In the present study, the activation of Cc NLRP1 inflammasome and its function in innate immune defence of common carp was investigated. The expression of Cc NLRP1 was induced in immune-related tissues of common carp upon challenge with Edwardsiella tarda and Aeromonas hydrophila. The colocalization of Cc NLRP1 and Cc ASC, ASC oligomerization, and interaction between Cc NLRP1CARD and Cc ASC was observed in 293T, Hela and EPC cells, suggesting that the Cc NLRP1 inflammasome was activated in common carp. Furthermore, we found that MDP may be the specific ligand of Cc NLRP1, which can activate the Cc NLRP1 inflammasome. Taken together, the present study identifies a new inflammasome in common carp, and is beneficial to the control of infectious diseases in carp farming. • The Cc NLRP1 expression was induced in immune-related tissues of common carp upon challenge with E. tarda or A. hydrophila. • The results of colocalization, oligomerization and interaction suggest that Cc NLRP1 inflammasome was activated. • MDP may be the specific ligand of Cc NLRP1, which can activate the Cc NLRP1 inflammasome. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that play a critical role in innate immune responses against pathogens. In the present study, a fish-specific TLR14 was identified and characterized from Monopterus albus (named Ma TLR14), which consisted of a 2658 bp open reading frame encoding a protein of 885 amino acids. Phylogenetic analysis revealed that Ma TLR14 belong to the TLR1 subfamily and shared the highest similarity to Paralichthys olivaceus TLR14. Immunohistochemistry assay showed that Ma TLR14 mainly located in intestinal epithelial cells of hindgut. Immunofluorescence revealed that Ma TLR14 largely localized to the intracellular region and partially co-localized with cell membrane of HeLa cells. The expression levels of Ma TLR14 were upregulated in the liver, spleen, foregut and hindgut post infection with Aeromonas hydrophila. When stimulated with LPS and Flagellin, the Ma TLR14 expression was elevated in isolated peripheral blood leukocytes. Further studies showed that recombinant Ma TLR14-LRR could bind to both the gram-negative and gram-positive bacteria and cause agglutination. Subsequently, the signaling pathway of Ma TLR14 was investigated. Confocal microscopy and co-immunoprecipitation assay demonstrated that Ma TLR14 recruited MyD88 as adaptor. When overexpressed, Ma TLR14 augmented the expression of TRAF6 and phosphorylation of ERK and p65, activated NF-κB and AP-1 and elicited the expression of il-6 and tnf-α. Collectively, Ma TLR14 plays an important role in the microorganism recognition and signaling transduction. • Ma TLR14 is expressed in the intestinal epithelium of hindgut and mainly localized to internal cellular compartments. • Ma TLR14 is upregulated at mRNA expression level after bacterial and PAMPs stimulation. • Ma TLR14 can bind gram-negative bacteria as well as gram-positive bacteria through the LRR domain. • Ma TLR14 significantly induces the expression of tnf-α and il-6 via NF-κB and MAPK pathways. [ABSTRACT FROM AUTHOR]

Intelectin (ITLN) is a type of glycan-binding lectin involved in many physiological processes and some human diseases. Here we report a common carp intelectin (cITLN). Like other orthologs , cITLN also contains a conserved fibrinogen-related domain (FReD) and a unique intelectin domain, expresses in all the tissues tested with the highest level in the hindgut, and responds to bacterial challenge in the acute phase. We also expressed cITLN in Escherichia coli (E. coli) system, and the purified recombinant cITLN could neither affect the surface of bacteria nor inhibit the growth of bacteria, but it can agglutinate both gram-positive and gram-negative bacteria in a calcium-dependent manner. The cITLN's ability of agglutination of gram-positive bacteria is stronger than that of gram-negative bacteria. This is probably because recombinant cITLN could binding peptidoglycan (PGN) with a higher degree to lipopolysaccharide (LPS). Our results of cITLN provided new insight into the function of intelectin in the intestinal mucosal immunity. • One homologue of intelectin named cITLN with a FReD and a unique intelectin domain was characterized in common carp. • The cITLN is expressed in all tissues we tested and upregulated after challenge of A. hydrophila. • The cITLN can bind to both LPS and PGN. Maybe it's the early step for cITLN to exert its bacterial binding and agglutinating. [ABSTRACT FROM AUTHOR]

The common carp ( Cyprinus carpio L.) is an important farmed species worldwide. Mucosal-associated lymphoid tissues play an essential role in the fight against pathogen infection. Spring viremia of carp virus (SVCV) poses a serious threat to the common carp aquaculture industry. Understanding the molecular mechanisms driving mucosal immune responses to SVCV infection is critical. In this study, the mucosal tissues (gills, foregut and hindgut) were collected from normal and infected fishes for transcriptome analysis. A total of 932,378,600 clean reads were obtained, of which approximately 80% were successfully mapped to the common carp genome. 577, 1,054 and 1,014 differential expressed genes (DEGs) were identified in the gills, foregut and hindgut, respectively. A quantitative polymerase chain reaction assay indicated that the DEGs expression in the foregut following SVCV infection was consistent with the transcriptome results. Among them, two key genes of the retinoic acid-inducible gene I (RIG-I)-like receptor family, melanoma-differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) (i.e., Cc MDA5 and Cc LGP2), underwent further analysis. Overexpression of Cc MDA5 or Cc LGP2 increased phosphorylation of TANK-binding kinase 1 and interferon regulatory factor 3 and the expression of interferon-1 ( ifn-1 ), myxovirus resistance ( mx ), viperin and interferon-stimulated gene 15 ( isg15 ), and inhibited SVCV replication in epithelioma papulosum cyprini cells. Furthermore, Cc LGP2 significantly upregulated the Cc MDA5-induced ifn-1 mRNA expression and the activation of the ifn-1 promoter. Finally, confocal microscopy and coimmunoprecipitation experiments revealed that Cc LGP2 colocalizes and interacts with Cc MDA5 via the C-terminal regulatory domain. This study provides essential gene resources for understanding the fish immune response to SVCV infection and sheds light on the potential role of fish LGP2 in the MDA5 regulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Niu, Qi, Li, Yang and Shan.)

Background: Common carp (Cyprinus carpio L.), one of the most economically valuable commercial farming fish species in China, is often infected by a variety of viruses. As the first line of defence against microbial pathogens, the innate immune system plays a crucial role in teleost fish, which are lower vertebrates. Interferon (IFN) regulatory factor 5 (IRF5) is a key molecule in antiviral immunity that regulating the expression of IFN and other pro-inflammatory cytokines. It is necessary to gain more insight into the common carp IFN system and the function of fish IRF5 in the antiviral and antibacterial response., Results: In the present study, we characterized the cDNA and genomic sequence of the IRF5 gene in common carp, and analysed tissue distribution and expression profile of this gene in response to polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharides (LPS) treatment. The common carp IRF5 (ccIRF5) gene is 5790 bp in length and is composed of 9 exons and 8 introns. The open reading frame (ORF) of ccIRF5 is 1554 bp, and encodes 517 amino acid protein. The putative ccIRF5 protein shares identity (65.4-90.0 %) with other fish IRF5s and contains a DNA binding domain (DBD), a middle region (MR), an IRF-associated domain (IAD), a virus activated domain (VAD) and two nuclear localization signals (NLSs) similar to those found in vertebrate IRF5. Phylogenetic analysis clustered ccIRF5 into the IRF5 subfamily with other vertebrate IRF5 and IRF6 genes. Real-time PCR analysis revealed that ccIRF5 mRNA was expressed in all examined tissues of healthy carps, with high levels observed in the gills and the brain. After poly I:C challenge, expression levels of ccIRF5, tumour-necrosis factor α (ccTNFα) and two IFN stimulated genes [ISGs (ccISG5 and ccPKR)] were up-regulated in seven immune-related tissues (liver, spleen, head kidney, foregut, hindgut, skin and gills). Furthermore, all four genes were up-regulated in vitro upon poly I:C and LPS challenges., Conclusions: Our findings suggest that IRF5 might play an important role in regulating the antiviral and antibacterial response in fish. These results could provide a clue for preventing common carp infection by pathogenic microorganisms present in the aquatic environment.

β-defensins are a group of cysteine-rich cationic antimicrobial peptides that play antibacterial and antiviral roles in immune systems of vertebrates. Here, we report the cloning and identification of a β-defensin 3 cDNA sequence from the common carp (Cyprinus carpio L.). Sequence alignment and phylogenetic analysis indicated that this β-defensin 3 belonged to the BD-2 group of fish. Real-time PCR showed that the β-defensin 3 mRNA was expressed in all the tissues of normal common carp that we examined and was highly expressed in the spleen and gills. When challenged with Vibrio anguillarum, the expression level of common carp β-defensin 3 mRNA was quickly upregulated in various tissues. Our results indicate that the β-defensin 3 showed markedly high constitutive expression in the gills, and significantly upregulated expression in the hindgut of the common carp after infection, suggesting it plays an important role in the innate and mucosal immunity of common carp.