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Krüppel-like factor 2a (KLF2A), a transcription factor of the krüppel-like family, is involved in regulating the immune molecules and is associated with viral infection. However, the function of KLF2A during viral infections in fish remains unclear. In this study, grass carp (Ctenopharyngodon idellus) was used to predict the target genes regulated by KLF2A. The results showed that the candidate target genes included four members of the serpin gene family (serpinb1l2 , serpinc1 , serpinh1a , and serpinh1b). Dual-luciferase experiments showed that klf2a positively regulates serpinc1 expression. Dose-dependent klf2a overexpression in C. idellus kidney (CIK) cells significantly upregulated the expression of serpinc1. Overexpressing klf2a or serpinc1 in CIK cells activated interferon responses and suppressed grass carp reovirus (GCRV) replication. Klf2a and serpinc1 co-expression inhibited GCRV replication. These results show that klf2a upregulates serpinc1 mRNA expression, promotes type 1 interferon responses, and suppresses GCRV infection. This study provides insights into the regulatory role and biological functions of KLF2A in host-virus interactions in fish. • Grass carp KLF2A targets the activation of serpinc1. • KLF2A and serpinc1 are involved in regulating virus-induced interferon responses. • KLF2A upregulates serpinc1 , and suppresses GCRV replication. [ABSTRACT FROM AUTHOR]

Peroxiredoxins (Prxs) are a family of antioxidant enzymes crucial for shielding cells against oxidative damage from reactive oxygen species (ROS). In this study, we cloned and analyzed two grass carp peroxiredoxin genes, CiPrx5 and CiPrx6. These genes exhibited ubiquitous expression across all sampled tissues, with their expression levels significantly modulated upon exposure to grass carp reovirus (GCRV). CiPrx5 was localized in the mitochondria, while CiPrx6 was uniformly distributed in the whole cells. Transfection or transformation of CiPrx5 and CiPrx6 into fish cells or E. coli significantly enhanced host resistance to H 2 O 2 and heavy metals, leading to increased cell viability and reduced cell apoptosis rates. Furthermore, purified recombinant CiPrx5 and CiPrx6 proteins effectively protected DNA against oxidative damage. Notably, overexpression of both peroxiredoxins in fish cells effectively inhibited GCRV replication, reduced intracellular ROS levels induced by GCRV infection and H 2 O 2 treatment, and induced autophagy. Significantly, these functions of CiPrx5 and CiPrx6 in GCRV replication and ROS mitigation were abolished upon treatment with an autophagy inhibitor. In summation, our findings suggest that grass carp Prx5 and Prx6 promote autophagy to inhibit GCRV replication, decrease intracellular ROS, and provide protection against oxidative stress. • CiPrx5 and CiPrx6 enhance host resistance to H 2 O 2 and heavy metals. • Recombinant CiPrx5 and CiPrx6 proteins protect DNA against oxidative damage. • CiPrx5 and CiPrx6 inhibit GCRV replication, reduce ROS, and induce autophagy. • CiPrx5 and CiPrx6 mediate autophagy to inhibit GCRV replication and ROS. [ABSTRACT FROM AUTHOR]

In mammal, CYP1A has attracted special attention due to its important roles in the oxidative metabolism. In fish, the researches on CYP1A are more focus on its roles in pollution in water environments, but the immune function is unclear. In the study, CiCYP1A gene was cloned from grass carp (Ctenopharyngodon idella). Tissue distribution exhibited an overwhelmingly high basal expression levels in the liver. After GCRV infection, CiCYP1A showed a potent response, indicating CiCYP1A was involved in GCRV-induced immunity. Subcellular localisation showed CiCYP1A was distributed in the cytoplasm. Besides, dual-luciferase activity assays revealed CYP1A was relevant for IFN-I signaling pathway modulation, furthermore, overexpressed CYP1A potently suppressed the mRNA expression of IRF3 and IFN-I but not IRF7. The results provide new sights into exploring immune function of CiCYP1A in teleosts. • The grass carp (Ctenopharyngodon idella) CYP1A was identified and analysed. • Tissue distribution of CiCYP1A and the expression pattern in response to GCRV infection were analysed. • CiCYP1A suppressed the promoter activity of IFN-I signaling pathway genes. • CiCYP1A suppressed mRNA expression levels of IFN-I. [ABSTRACT FROM AUTHOR]

Galectins, as an evolutionary conserved group of lectin superfamily, has the functions of pathogen recognition, anti-bacteria and anti-virus. In this study, a 405 bp cDNA sequence of galectin 1-like 2 (CiGal1-L2) was obtained from grass carp (Ctenopharyngodon idella), which encoded 134 amino acids with a predicted molecular mass of 15.143 kDa and an isoelectric point of 5.33. The sugar binding motifs (H–N-R, V–N and W--E-R) were detected in carbohydrate-binding domain (CRD). The amino acid sequence similarity showed that CiGal1-L2 was 40.30–42.54% and 66.42–81.20% similarity to mammalian and fish counterparts, respectively. The phylogenetic tree showed that CiGal1-L2 was clustered with fish galectin-1s and closely related to Cyprinus carpio. Real-time quantitative PCR (RT-qPCR) analysis revealed that CiGal1-L2 was widely expressed in all tested tissues. In addition, the expression of CiGal1-L2 was differentially up-regulated challenged with grass carp reovirus (GCRV), lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C). The fluorescence of CiGal1-L2-GFP was distributed in the cytoplasm and nucleus of HEK 293T cells and showed a trend of nuclear translocation after LPS and poly I:C treatment. Finally, the recombinant CiGal1-L2 (rCiGal1-L2) protein showed strong binding ability to LPS. In conclusion, the results provided further insight into the immune roles of galectin-1 in teleost. • CiGal1-L2 was widely expressed in all tested tissues. • CiGal1-L2 transcripts were induced by various stimulations. • The fluorescence of CiGal1-L2-GFP showed a trend of nuclear translocation after LPS and poly I:C treatment. • The recombinant CiGal1-L2 protein showed strong binding ability to LPS. [ABSTRACT FROM AUTHOR]

Autophagy is an essential and conserved process that plays an important role in physiological homeostasis, adaptive response to stress and the immune response. Autophagy-related proteins (ATGs) are key components of the autophagic machinery. In the study, grass carp (Ctenopharyngodon idella) autophagy-related gene 5 (ATG5) and 12 (ATG12) were identified. In the gill and intestine, ATG5 and ATG12 were highly expressed, but after grass carp reovirus (GCRV) infection, they were decreased significantly. In Ctenopharyngodon idella kidney (CIK) cells, the sharp variation of ATG5 and ATG12 expression was observed after poly(I:C) infection. Subcellular localisation showed that ATG5 and ATG12 were evenly distributed in the cytoplasm and nucleus. However, the interaction between ATG5 and ATG12 was only found in cytoplasm in both 293T cells and CIK cells. In addition, the overexpression of ATG5 or ATG12 in 293T cells showed enhanced autophagy, and autophagic process was facilitated when ATG5 and ATG12 were simultaneously overexpressed. Dual-luciferase activity assay indicated that both ATG5 and ATG12 remarkably suppressed the promoter activity of IRF3 , IRF7 , and IFN-I. Further, ATG5 and ATG12 conjugate showed far stronger inhibitory affection on the expression of IFN-I than either ATG5 or ATG12 in response to poly(I:C) or GCRV infection. Taken together, the results demonstrate that grass carp ATG5 and ATG12 play an important role in innate immunity and autophagy. • The grass carp ATG5 and ATG12 were identified and analysed. • ATG5 interacted with ATG12 in CIK and 293T cells. • ATG5 and ATG12 proteins promoted autophagy. • ATG5 and ATG12 repressed the promoter activity of IRF3 , IRF7 and IFN-I. • ATG5 and ATG12 conjugate suppressed the expression of IFN-I signaling genes. [ABSTRACT FROM AUTHOR]

Peroxiredoxin (Prx), also named thioredoxin peroxidase (TPx), is a selenium independent antioxidant enzyme that can protect organisms from oxidative damage caused by reactive oxygen species (ROS) and is important for immune responses. In this study, the molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiPrx2 was 1163 bp containing 5ʹ-untranslated region (UTR) of 52 bp, a 3′-UTR of 517 bp with the putative polyadenylation consensus signal (AATAAA), an open reading frame (ORF) of 594 bp encoding polypeptides of 197 amino acids with a predicted molecular mass of 21.84 kDa and theoretical isoelectric point of 5.93. The analysis results of multiple sequence alignment and phylogenetic tree confirmed that CiPrx2 belong to the typical 2-Cys Prx subfamily. The CiPrx2 mRNA was ubiquitously expressed in all tested tissues. The temporal expression of CiPrx2 were differentially induced infected with grass carp reovirus (GCRV), polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) in liver and spleen. Subcellular localization of CiPrx2-GFP fusion proteins were only distributed in the cytoplasm. The purified recombinant CiPrx2 possessed an apparent antioxidant activity and could protect DNA against oxidative damage. Finally, CiPrx2 proteins could obviously inhibit H 2 O 2 and heavy metal toxicity. However, further researches are needed to better understand the regulation of CiPrx2 under oxidative stresses. • The molecular cloning and characterization of a Prx2 homologue (CiPrx2) were described from grass carp. • The CiPrx2 mRNA was ubiquitously expressed in the various tissues. • CiPrx2 was likely to be involved in the immune response against viral and bacterial infection. • Subcellular localization of CiPrx2-GFP fusion protein were only distributed in the cytoplasm. • CiPrx2 proteins could obviously inhibit H 2 O 2 and heavy metal toxicity and protect DNA against oxidative damage. [ABSTRACT FROM AUTHOR]

Peroxiredoxin (Prx) family are known as an important antioxidant enzyme as the first line of defense against oxidative damage, and also involved in immune responses following viral and bacterial infection. Here, a full-length Prx1 cDNA sequence (CiPrx1) was cloned from grass carp (Ctenopharyngodon idella), which was 1029 bp, including a 5′-terminal untranslated region (UTR) of 121 bp, a 3′-UTR of 272 bp, an open reading frame of 600 bp encoding 199 amino acids with molecular weight of 22.21 kDa and isoelectric point of 6.30. CiPrx1 shares 80.8–99% protein sequence similarity with Prx1 of other fishes. The conserved peroxidase catalytic center "FYPLDFTFVCPTEI" and "GEVCPA" were observed in the sequence of CiPrx1; this indicated that it was a member of 2-Cys Prx. Subcellular localization of CiPrx1 was only strongly distributed in the cytoplasm. Quantitative real-time PCR (RT-qPCR) assays revealed that CiPrx1 mRNA was ubiquitously detected in all tested tissues, and the expression was comparatively high in liver, gill and spleen. Further, the expression of CiPrx1 can be induced by grass carp reovirus (GCRV), lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (Poly I:C) infection in the different tissues. Moreover, the recombinant CiPrx1 (rCiPrx1) protein was found a potential antioxidant enzyme, that could inhibit DNA damage from oxidants. Altogether, our results imply that CiPrx1 is associated with defending against virus and bacteria pathogens and oxidants in grass carp. • Peroxiredoxin 1 from grass carp was cloned and characterized. • CiPrx1 mRNAs was ubiquitously detected in all tested tissues. • The expression of CiPrx1 can be induced by GCRV, LPS and Poly I:C infection in the different tissues. • Subcellular localization of Prx1-pEGFP was only strongly distributed in the cytoplasm. • The antioxidant activity of the recombinant rCiPrx1 was assessed by mixed-function oxidase assay. [ABSTRACT FROM AUTHOR]

Scavenger receptor class B type 1 (SRB1) is a transmembrane protein belonging to the scavenger receptors (SRs) family and it plays an important role in viral entry. Not much is known on SRB1 in teleost fish. Grass carp reovirus (GCRV) cause huge economic losses in grass carp industry. In this study, rare minnow (Gobiocypris rarus) was used as a model fish to investigate the mechanism of GCRV infection, which is sensitive to GCRV. The structure of SRB1 gene in G. rarus (GrSRB1) was cloned and elucidated. GrSRB1 is composed of 13 exons and 12 introns, and its full-length cDNA is 2296 bp in length, with 1521 bp open reading frame (ORF) that encodes a 506 amino acid protein. The Gr SRB1 protein is predicted to contain a typical CD36 domain and two transmembrane regions. In G. rarus , GrSRB1 is expressed strongly in the liver (L), intestines (I), brain (B) and muscle (M), while it is expressed poorly in the heart (H), middle kidney (MK), head kidney (HK) and gills (G). After infection with GCRV, GrSRB1 expression was up-regulated in main immune tissues during the early infection period. Moreover, co-immunoprecipitation assays revealed that Gr SRB1 could interact with the outer capsid protein of GCRV (VP5 and VP7). These results suggest that Gr SRB1 could be a receptor for GCRV. We have managed to characterize the GrSRB1 gene and provide evidence for its potential functions for GCRV entry into host cells. • SRB1 gene in Gobiocypris rarus (GrSRB1) was cloned and elucidated. • GrSRB1 expression was up-regulated in main immune tissues after GCRV challenge. • Gr SRB1 could interacted with the outer capsid protein of GCRV (VP5 and VP7). • Gr SRB1 might play important role in GCRV entry into host cells. [ABSTRACT FROM AUTHOR]

Abstract Tripartite motif (TRIM) proteins are key components of the innate immune system, functioning as antiviral restriction factors or modulating signaling cascades that lead to proinflammatory cytokine induction. In the present study, the TRIM family gene TRIM23 from grass carp (Ctenopharyngodon idella) was cloned and characterised. TRIM23 was moderately expressed in the examined tissues, and the significantly altered expression was observed after grass carp reovirus (GCRV) and poly(I:C) infection. Dual-luciferase activity assay showed that TRIM23, especially its C-terminal domain ARF, depressed the promoter activity of IRF3 and IRF7. The subcellular localisation showed that TRIM23 protein was located in the cytoplasm and could be recruited by both TRAF6 and MyD88. Furthermore, TRIM23 was confirmed to interact with either TRAF6 or MyD88 by the bimolecular fluorescence complementation (BiFC) system in CIK cells. Additionally, autophagy was enhanced by over-expressed TRIM23 in 293T cells. Taken together, our results demonstrate that TRIM23 gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the TRIM23 in teleosts. Highlights • The TRIM23 gene in grass carp (Ctenopharyngodon idella) was identified and analysed. • Expression and transcriptional regulation of TRIM23 gene were investigated. • TRIM23 involved in the regulation of the promoter activity of IRF3 and IRF7. • The TRIM23 protein could colocalise and interact with TRAF6 and MyD88 in CIK cells. • The TRIM23 protein could induce autophagy. [ABSTRACT FROM AUTHOR]

Abstract Haemorrhagic disease caused by grass carp reovirus (GCRV) can result in large-scale death of young grass carp, leading to irreparable economic losses that seriously affect large-scale breeding. Protein kinase C (PKC, also known as PRKC) represents a family of serine/threonine protein kinases that includes multiple isozymes in many species. Among these, PKC-θ (PKC theta, also written as PRKCQ) is a novel isoform, mainly expressed in T cells, that is known to be involved in immune system function in mammals. To date, no research on immunological functions of fish Pkc-θ has been reported. To address this issue, we cloned the grass carp pkc-θ gene. Phylogenetic and syntenic analysis showed that this gene is the most evolutionarily conserved relative to zebrafish. Real-time quantitative PCR (RT-qPCR) indicated that pkc-θ was expressed at high levels in the gills and spleen of healthy grass carp. Infection with GCRV down regulated pkc-θ expression in the gills and spleen. Gene products that function upstream and downstream of pkc-θ were up regulated in the gill, but were down-regulated in the spleen. These results suggest that direct or indirect targeting of pkc-θ by GCRV may help the virus evade host immune defences in the spleen. Phorbol ester (PMA) treatment of Jurkat T cells induced translocation of grass carp Pkc-θ from the cytoplasm to the plasma membrane. This response to PMA suggests evolutionary conservation of an immune response function in fish Pkc-θ, as well as conservation of its sequence and structural domains. This study expanded our knowledge of the fish PKC gene family, and explored the role of pkc-θ in function of the grass carp immune system, providing new insights which may facilitate further studies of its biological functions. Highlights • Grass carp Pkc-θ was structurally conserved compared with other species. • Grass carp pkc-θ was enriched in the gill and spleen. • Grass carp pkc-θ and its related genes underwent similarily significant changes in gill and spleen after GCRV infection. • Phorbol ester (PMA) made grass carp Pkc-θ translocated from- cytoplasm to the membrane in Jurkat T cells. [ABSTRACT FROM AUTHOR]

Abstract Ubiquitination is a post-translational modification of proteins that is widely present in eukaryotic cells. There is increasing evidence that ubiquitinated proteins play crucial roles in the immune response process. In mammals, RING-between-RING (RBR) proteins play a key role in regulating immune signaling as the important E3 ubiquitin ligases during ubiquitination. However, the function of RBR in fish is still unclear. In the present study, six RBR genes (RNF19A , RNF19B , RNF1 44AA, RNF1 44AB, RNF144B and RNF217) of grass carp (Ctenopharyngodon idellus) were cloned and characterized. Similar to mammals, all six members of RBR family contained RING, in-between-ring (IBR) and transmembrane (TM) domains. These genes were constitutively expressed in all studied tissues, but the relative expression level differed. Following grass carp reovirus(GCRV) infection, the expression of six RBR genes in liver, gill, spleen and intestine significantly altered. Additionally, their expression in Ctenopharyngodon idellus kidney (CIK) cells was significantly increased after GCRV infection. And deficiency of RNF144B in CIK with small interference RNA (siRNA) up-regulated polyinosinic:polycytidylic acid poly(I:C))-induced inflammatory cytokines production, including IFN-I , TNF-α , IL-6 , and transcription factor IRF3 , which demonstrated that RNF144B was a negative regulator of inflammatory cytokines. Our results suggested that the RBR might play a vital role in regulating immune signaling and laid the foundation for the further mechanism research of RBR in fishes. Highlights • Six RING-between-RING (RBR) genes from Ctenopharyngodon idellus were cloned for the first time. • RNF144AA , RNF144AB , and RNF144B responded substantially after GCRV challenge. • Poly (I: C)-induced proinflammatory cytokines were up-regulated by RNF144B silencing. • The expression of RNF144B in liver, especially in intestine was intensely up-regulated at 1 day post–infection. [ABSTRACT FROM AUTHOR]

Abstract Grass carp is an important fish species in Chinese aquaculture, and can be afflicted by a hemorrhagic disease caused by the grass carp reovirus (GCRV). Interestingly, the affects of GCRV infection of grass carp are age-restricted, meaning that one-year-old grass carp can be infected and can suffer hemorrhagic disease, but three-year-old carp are not so afflicted. In this study, we investigated the mechanism responsible for this age-restricted pathology. We evaluated the relative copy number of GCRV RNA, the expression levels of proteins in blood, and changes in DNA methylation in carp from the two age groups after infection with GCRV. After GCRV infection, the relative copy number of GCRV RNA in three-year-old grass carp was significantly lower than in one-year-old carp. The differences in circulating protein levels mainly occurred in concentrated in complement and coagulation proteins, and the expression levels of these proteins were significantly higher in three-year-old grass carp than in one-year-old carp. Moreover, the expression levels of DNA methylation-related genes in the liver and spleen of one-year-old grass carp were significantly higher than those of three-year-old carp. These results suggested that as age of grass carp increases, faster and more efficient response of the immune system after viral infection, especially the complement system, and differences in DNA methylation may be important factors that affect the age restriction observed in GCRV infection. Our study provides new insights into the mechanisms underlying age restriction of GCRV infection. Highlights • The affects of GCRV infection of grass carp are age-restricted. • The relative copy number of GCRV in three-year-old grass carp was significantly lower than in one-year-old carp. • The expression level of proteins involved in complement and blood coagulation were higher in three-year-old carp. • More efficient and faster response of the immune system may contribute to the age restriction of GCRV infection. [ABSTRACT FROM AUTHOR]

Abstract T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins from the High Mobility Group (HMG) box family act as the main downstream effectors of the Wnt signaling pathway. HMGB proteins play multifaceted roles in the immune system of mammals. To clarify the immunological characteristics of LEF/TCF genes in grass carp (Ctenopharyngodon idella) , five LEF/TCF genes (TCF7, LEF1, TCF7L1A, TCF7L1B, and TCF7L2) were identified and characterized. All five LEF/TCF proteins contained two characteristic domains: a HMG-BOX domain and a CTNNB1_binding region. Phylogenetic tree analysis revealed that the LEF/TCF proteins were represented different lineages. These results of subcellular localization showed that four of the LEF/TCF genes were localized exclusively within the nucleus, while TCF7L2 was localized in the cytoplasm and nucleus. The mRNA expression profiles of these LEF/TCF family genes differed across different tissues. The mRNA expression levels of TCF7, TCF7L1A, and TCF7L2 changed significantly in liver after grass carp reovirus (GCRV) challenge; TCF7 and TCF7L1A responded early while TCF7L2 responded late. This suggests that these genes may participate in GCRV-related immune responses. Moreover, TCF7 promoted Bcl6 transcription in response to the GCRV challenge. These findings further our understanding of the function of LEF/TCF genes in teleosts. Highlights • All LEF/TCF family genes in C. idella were cloned and analyzed. • Four LEF/TCF proteins localized in the nucleus while the TCF7L2 localized in the cytoplasm and nucleus. • The TCF7, TCF7L1A and TCF7L2 of grass carp participate in the GCRV-related immune responses. • TCF7 promoted Bcl6 transcription to respond the GCRV challenge. [ABSTRACT FROM AUTHOR]

Abstract Grass carp, an economically important aquaculture fish, is very sensitive to Grass Carp Reovirus (GCRV). Haemorrhagic disease caused by GCRV infection can cause large-scale death of first-year grass carp, thereby severely restricting the intensive culture. Serpins (serine protease inhibitors) belong to the protease inhibitor gene family and are involved in numerous physiological and pathological processes, particularly coagulation and anticoagulation. Reports on grass carp serpins are scarce. Thus, we cloned six grass carp serpin genes (serpinb1 , serpinc1 , serpind1 , serpinf1 , serpinf2b and serping1) in this study. Molecular evolution showed that serpins between grass carp and zebrafish or carp are the closest relatives. SERPIN domains in these 6 serpins and reactive centre loop (RCL) along with their cleavage sites of 5 serpins (serpinb1 , serpinc1 , serpind1 , serpinf2b and serping1) were predicted. Real-time quantitative PCR (RT-qPCR) showed that these serpins displayed tissue significance. Among them, serpinc1 , serpind1 , s erpinf2b and serping1 had the highest expression levels in the liver. After GCRV infection, RT-qPCR showed that the liver-enriched serpins were significantly changed. Key procoagulant factor genes (kng-1 , f2 , f3a , f3b and f7) and anticoagulant genes (tpa , plg , thbd , proc and pros) also showed significant changes on the mRNA level. Comprehensive comparative analysis showed that the up-regulated expression of key clotting factor genes was more prominent than that of main anti-coagulation factor genes. Thus, the function of coagulation may be more dominant in grass carp during the GCRV infection, which may cause overproduction of thrombi. The serpins were involved in GCRV infection and liver-enriched serpins participate in the interaction between coagulation and anticoagulation. This study provided new insights into further research on the biological functions of grass carp serpins and clarifying the molecular mechanism of GCRV affecting the homeostasis of grass carp blood environment. Highlights • Six serpin genes in grass carp (Ctenopharyngodon idella) were cloned, and all the serpin proteins contained an SERPIN domain. • Liver-enriched serpins (serpinc1 , serpind1 , serpinf2b and serping1) may participate in the interaction between coagulation and anticoagulation. • The function of coagulation was more dominant in grass carp during the GCRV infection, which may cause overproduction of thrombi. • Serpins may affect the homeostasis of blood environment in grass carp during GCRV infection. [ABSTRACT FROM AUTHOR]

Abstract Interleukin-1 receptor-associated kinase (IRAK) family members play important roles in myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor (TLR) signaling, the crucial innate immune pathway in vertebrates. In the present study, the IRAK family gene IRAK-M (also called IRAK3) from grass carp (Ctenopharyngodon idella) was cloned and characterised. IRAK-M was mainly enriched in the spleen, and the significantly altered expression was observed after grass carp reovirus (GCRV) infection. Subcellular localisation showed that IRAK-M protein distributed uniformly in the entire cell and co-localised with MyD88 in the cytoplasm of transfected cells. Additionally, the interaction between IRAK-M and MyD88 was confirmed by bimolecular fluorescence complementation (BiFC) system. Moreover, deficient of IRAK-M in C. idella kidney cell line (CIK) with small interference RNA (siRNA) upregulated polyinosinic:polycytidylic acid (poly(I:C))-induced inflammatory cytokines production, including interleukin 8 (IL-8), IL-6 , and tumour necrosis factor α (TNF-α), which reveals that IRAK-M functions as a negative regulator of inflammatory cytokines. Taken together, our results demonstrate that IRAK-M gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the IRAK-M in teleosts. Highlights • The IRAK-M gene from Ctenopharyngodon idella was identified and analysed. • Expression and transcriptional regulation of IRAK-M gene were investigated. • Poly(I:C)-induced proinflammatory cytokines are upregulated by IRAK-M silencing. • The IRAK-M protein could colocalise and interact with MyD88 in CIK cells. [ABSTRACT FROM AUTHOR]

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. In the present study, the NLR family gene NLRX1 from grass carp ( Ctenopharyngodon idella ) was cloned and characterised. NLRX1 was widely expressed in all tissues examined, albeit at varying levels. After exposure to the grass carp reovirus (GCRV), NLRX1 mRNA expression levels were altered in immune organs, and dramatically altered in liver. Subcellular localisation indicated that NLRX1 protein co-localised with the mitochondria in the transfected cells. Additionally, the bimolecular fluorescence complementation (BiFC) system was introduced to detect the interaction between tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) and NLRX1. Moreover, deficient of NLRX1 in CIK cells with small interference RNA (siRNA) promoted polyinosinic:polycytidylic acid (poly (I:C))-induced IFN -related genes production, including IRF3 , IRF7 , and IFN-I , which reveals that NLRX1 is a negative regulator of IFN. Taken together, our results demonstrate that NLRX1 gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the NLRX1 in teleosts. [ABSTRACT FROM AUTHOR]

The mammalian sterile 20-like (MST) family, which belongs to the serine/threonine protein kinase superfamily, has five members that can be found in mammals: STK3 (also called MST2), STK4 (MST1), STK24 (MST3), STK25 (YSK1 or SOK1), and STK26 (MST4). The MST kinases have key roles in apoptosis, immune regulation, inflammatory responses, cancer, and cell proliferation in mammals, whereas the roles and transcriptional regulatory mechanism of these kinases in teleost fish are still unclear. In this study, four STK genes ( CiSTK3 , CiSTK24 , CiSTK25 , and CiSTK26 ) were cloned and analyzed in grass carp ( Ctenopharyngodon idella ). All four STK genes were broadly expressed in the examined tissues, while their relative expression levels differed. In addition, after exposure to the grass carp reovirus, mRNA expression levels of the four STK genes were altered to different levels in the immune organs, and the levels were dramatically altered in the blood. Subcellular localization indicated that all four STK proteins were localized in the cytoplasm of transfected cells. Moreover, bimolecular fluorescence complementation analysis revealed that mouse protein-25 could interact with CiSTK3, CiSTK24, CiSTK25, and CiSTK26 independently in grass carp. Thus, our findings provide new insights for understanding the functions of the MST family in teleosts. [ABSTRACT FROM AUTHOR]

Grass carp ( Ctenopharyngodon idellus ) is an economical aquaculture species in China, and the Grass Carp Reovirus (GCRV) that causes hemorrhagic disease seriously affects the grass carp cultivation industry. Substantial evidence indicates that there is an association between the membrane-associated RING-CH family of E3 ligase (MARCH) family and immune defense in mammals, while functional studies on non-mammalian MARCH proteins are limited. In order to know the characteristics of the MARCH genes in C. idellus , eight MARCH genes ( MARCH1, 2, 5, 6, 7, 8, 9 and 11 ) were cloned and the open reading frames (ORF) were identified in grass carp. All MARCH proteins in grass carp contained an RING-CH domain, which is characteristic of the MARCH protein. The phylogenetic analysis revealed that different MARCH proteins gathered into their separate clusters. All eight members of the MARCH gene family were detected in all tissues sampled, but the relative expression level differed. In addition, the mRNA expression of all the MARCHs was regulated at different levels in the immune organs after a GCRV challenge, and they responded robustly in both the intestine and liver. The mRNA expression of MARCH8, MHC II, TfR, IL1RAP, EGR1, and DUSP1 in the intestine after GCRV infection was analyzed, and the results showed that MARCH8 could negatively regulate TfR, IL1RAP, EGR1, and DUSP1, which signaled via the MAPK or NF-κB-activation pathways that play vital roles in immunity. Our findings identified a novel gene family in C. idellus and provided novel evidence that MARCH genes are inducible and involved in the immune response. Moreover, MARCH8 might function to negatively regulate immune receptors in C. idellus . Therefore, the MARCH might play a vital role in regulating the immune response of C. idellus . [ABSTRACT FROM AUTHOR]