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Viral seasonality in the aquaculture industry is an important scientific issue for decades. While the molecular mechanisms underpinning the temperature-dependent pathogenesis of aquatic viral diseases remain largely unknown. Here we report that temperature-dependent activation of IL6-STAT3 signaling was exploited by grass carp reovirus (GCRV) to promote viral entry via increasing the expression of heat shock protein 90 (HSP90). Deploying GCRV infection as a model system, we discovered that GCRV induces the IL6-STAT3-HSP90 signaling activation to achieve temperature-dependent viral entry. Further biochemical and microscopic analyses revealed that the major capsid protein VP7 of GCRV interacted with HSP90 and relevant membrane-associated proteins to boost viral entry. Accordingly, exogenous expression of either IL6, HSP90, or VP7 in cells increased GCRV entry in a dose-dependent manner. Interestingly, other viruses (e.g., koi herpesvirus, Rhabdovirus carpio, Chinese giant salamander iridovirus) infecting ectothermic vertebrates have evolved a similar mechanism to promote their infection. This work delineates a molecular mechanism by which an aquatic viral pathogen exploits the host temperature-related immune response to promote its entry and replication, instructing us on new ways to develop targeted preventives and therapeutics for aquaculture viral diseases. Author summary: Viral seasonality in the aquaculture industry has been an important scientific issue for decades, which is causing billions of economic losses every year worldwide. However, there is a lack of understanding of how temperature influences the pathogenesis of aquatic viruses infecting fish, shellfish, and other ectotherms. Here, deploying aquatic grass carp reovirus (GCRV) as a model system, we demonstrate for the first time that the temperature-dependent IL6-STAT3-HSP90 signaling axis is exploited by GCRV to promote viral entry. We found that HSP90 interacted with viral structural proteins and membrane-associated proteins to enable viral entry. Furthermore, our study indicates other aquatic viruses might evolve a similar mechanism to promote their infection. By elucidating the molecular mechanism of temperature-dependent aquatic viral pathogenesis, our work may help to develop targeted prevention and control strategies against aquatic viral diseases. [ABSTRACT FROM AUTHOR]

Interaction between the chemokine receptor XCR1 and its ligand is closely related to the immune function in animals; however, there are only a few reports on role of XCR1 in the immune system of fish. We aimed to analyze the expression of XCR1 in various organs or tissues of grass carp before and after Grass Carp Reovirus (GCRV) infection to better understand the function of XCR1 in resistance to GCRV infection. We cloned and sequenced the cDNA of grass carp XCR1 and analyzed the molecular structure of XCR1 based its amino acid sequence. Further, we analyzed the relative expression levels of XCR1 in different organs or tissues of male parent grass carp with GCRV resistance (P1) and their first-generation offspring (F1) before and after GCRV infection. Our results show that the total length of cDNA of the grass carp XCR1 gene is 1659 bp and encodes 365 amino acids. XCR1 contains seven conserved transmembrane helical domains. The homologous tertiary structure of XCR1 is similar to its homologs in other species. After artificial GCRV infection, there were significant differences in the expression of the grass carp XCR1 gene in different tissues, at different time points, and between P1 and F1 fish. These results will contribute to our understanding of the role of XCR1 in fish immune responses and contribute to the development of GCRV-resistant grass carp. [ABSTRACT FROM AUTHOR]

Our aim was to verify whether genes in the coagulation complement and cytokine interaction pathways are crucial to the resistance to grass carp hemorrhagic disease in grass carps (Ctenopharyngodon idella). To evaluate this, we acquired male grass carps with grass carp reovirus (GCRV) resistance, F1 generation, ordinary male fish, control F1 progenies (hybridized ordinary fish), and barbel chub (Squaliobarbus curriculus) as a positive control. We compared the expression variation of 13 immune factors in different tissues using real-time fluorescent quantitative PCR. Our results showed that the expression of the transforming growth factor-β3, nerve growth factor receptor, coagulation factor D, chemokine receptor XCR1, profibrinolysin, γ-globulin of complement component 1, complement component 2, and complement component 5 in the different tissues of the barbel chub were significantly higher than those in the grass carps populations under normal conditions. Except for a limited number of genes, their expression exhibited significant differences. There was no significant difference in these genes among grass carp populations with different GCRV resistance. These results indicate that the high gene expression in the coagulation complement and cytokine interaction pathways may be the main reason for the strong resistance of barbel chub to GCRV. However, high gene expression was not a factor affecting GCRV resistance between grass carp populations. [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the effects of diet 7.5 g/kg α-ketoglutarate (AKG) on the growth performance, antioxidant defense system, digestive enzymes, and immune response of grass carp (Ctenopharyngodon idellus). A total of 400 grass carp with an average body weight 10.81 ± 0.68 g was randomly allocated into 2 groups with 4 replicates of 50 fish respectively. The experiment was conducted in net cages (1.5 m × 1.5 m × 1.5 m) suspended in an indoor cement pool. Fish were fed a basic diet containing either 0 (control) or 7.5 g/kg AKG (supplemented diet). The experiment lasted for 8 weeks (56 days). Results indicated that compared with the control group, the final weight (FW), weight gain rate (WGR), specific growth rate (SGR), and protein efficiency ratio (PER) in the AKG group were increased significantly (P < 0.05). However, the feed conversion ratio (FCR) was decreased significantly (P < 0.05). The 7.5 g/kg AKG supplementation significantly increased the activities of glutamine synthetase (GS), glutathione peroxidase (GSH-Px), catalase (CAT), total superoxide dismutase (T-SOD), and hexokinase (HK), as well as the concentrations of glutathione (GSH), total antioxidant capacity (T-AOC), and complement 3 (C3) in blood (P < 0.05), while significantly decreased the concentrations of malondialdehyde (MDA) and hemoglobin (Hb) (P < 0.05). The GS activity and GSH concentration in hepatopancreas were increased significantly (P < 0.05), whereas the glycogen concentration in hepatopancreas, and the glycogen concentration and GS activity in the muscle were significantly decreased (P < 0.05). In addition, 7.5 g/kg AKG supplementation significantly increased the concentration of GSH and the activities of amylase, protease, and lipase in fore-gut, alkaline phosphates (ALP) in the mid-gut, and Na-ATP and Ca-ATP in the gill (P < 0.05), as well as γ-glutamyl transpeptidase (γ-GT) both in fore-gut and mid-gut (P < 0.05), whereas the activity of acid phosphatase (ACP) in the mid-gut was decreased significantly (P < 0.05). In conclusion, diet 7.5 g/kg AKG supplementation in grass carp may improve the growth performance and immune response and play crucial roles in regulating the activities of GS, antioxidant defense system, and digestive enzymes. [ABSTRACT FROM AUTHOR]

Current intensive fish farming usually causes high environmental ammonia (HEA) in ponds that is toxic to fish. α‐Ketoglutarate (α‐KG) can be rapidly transaminated to glutamic acid and further aminated to glutamine. Therefore, we hypothesized that dietary α‐KG supplementation would alleviate HEA toxicity to fish. To test the hypothesis, 270 healthy grass carp (Ctenopharyngodon idella) juveniles were randomly assigned to control, HEA (18.37 mg/L ammonia) and HEA + α‐KG (0.75% of α‐KG) groups. Ammonia and free amino acid content in plasma and brain, liver glutamic pyruvic transaminase and glutamic oxaloacetic transaminase activity, and urea and glycogen content were measured on the first, seventh and 42nd days. Our results showed short‐term HEA exposure (1 day) led to a significant ammonia accumulation in the brain and plasma and significantly decreased glutamic and aspartic acid content in the brain and increased glutamine content in the brain and plasma. The long‐term HEA exposure (42 days) caused significant reductions in glycine and arginine content in the brain tissue. In most cases, dietary α‐KG supplementation alleviated the fluctuations in FAA content in the brain and plasma. Our results suggested dietary α‐KG alleviated HEA toxicity to grass carp. [ABSTRACT FROM AUTHOR]

Abstract The grass carp reovirus (GCRV) has been shown to cause lethal infections in the grass carp Ctenopharyngodon idella (C. idella). In order to investigate the immune response to GCRV infection, the full-length cDNA sequences of coagulation factor VIII (CiFVIII) and plasminogen (CiPLG) from C. idella were cloned and their involvement in the immune response was studied. The immunity factor levels in C. idella with different GCRV resistances were also analyzed. The full-length 2478 bp cDNA of CiFVIII contained an open reading frame of 1965 bp and encoded a putative polypeptide of 654 amino acid residues. The full-length 2907 bp cDNA of CiPLG contained an open reading frame of 2133 bp and encoded a putative polypeptide of 710 amino acid residues. CiFVIII was closely clustered with that of Clupea harengus. CiPLG was first clustered with those of Cyprinus carpio and Danio rerio. CiFVIII transcripts were most abundant in the liver and least in the skin. The highest expression level of CiPLG was observed in liver and the lowest in muscle. Expression levels of CiFVIII in gill, head kidney and spleen, and expression levels of CiPLG in gill, intestine and liver all reached the maximum at 72 h post GCRV infection. In spleen, expression levels of CiFVIII and CiPLG were significantly positively correlated. The activities of T-AOC, LSZ and IgM in R ♂ were significantly higher than those in O ♂. Likewise, T-AOC and LSZ activities in F1 were significantly higher than f1 individuals (P < 0.01). These results indicated that CiFVIII and CiPLG may play important roles in the immune response to GCRV infection. In addition, antioxidant ability and serum immune factor activity may confer a different viral resistance to C. idella. Highlights • The cDNAs of FVIII and PLG of Ctenopharyngodon idella were cloned. • Highest expression of CiFVIII and CiPLG were both observed in liver. • Transcription of both CiFVIII and CiPLG were up-regulated after GCRV infection. • Expression levels of CiFVIII and CiPLG after infection were positively correlated. • GCRV-resistance fish owns stronger immune activity than ordinary fish. [ABSTRACT FROM AUTHOR]

Complement factor D (Df) is a serine protease well known for activating the alternative pathway (AP) in mammals by promoting the cleavage of complement component 3 (C3), thus becoming involved in innate defense. In teleost fish, however, the functional mechanisms of Df in the AP and against pathogen infection are far from clear. In the present study, we cloned and characterized the Df gene, CiDf, from grass carp (Ctenopharyngodon idella) and analyzed its function in promoting C3 cleavage and expression changes after grass carp reovirus (GCRV) infection. The open reading frame of CiDf was found to be 753 bp, encoding 250 amino acids with a molecular mass of 27.06 kDa. CiDf harbors a conserved Tryp_SPc domain, with three conserved residues representing the catalytic triad and three conserved binding sites in the substrate specificity pocket. Pairwise alignment showed that CiDf shares the highest identity (96%) and similarity (98%) with Df from Anabarilius grahami. Phylogenetic analysis indicated that CiDf and other fish Dfs formed a distinct evolutionary branch. Similar to most Dfs from other vertebrates, the CiDf gene structure is characterized by four introns and five exons. The incubation of recombinant CiDf protein with grass carp serum significantly increased the C3b content, demonstrating the conserved function of CiDf in the AP in promoting C3 cleavage, similar to Dfs in mammals. CiDf mRNA expression was widely detected in various tissues and levels were relatively higher in the liver, spleen, and intestine of grass carp. During GCRV infection over a 168-hour period, a high level of CiDf mRNA expression in the liver, spleen, and intestine was maintained at 144 and 168 h, suggesting AP activity at the late stage of GCRV infection. Collectively, the above results reveal the conserved structure and function of CiDf and its distinct expression patterns after GCRV infection, which provide a key basis for studying the roles of Df and AP during GCRV infection in the grass carp C. idella. [ABSTRACT FROM AUTHOR]

Disease resistance varies among fish species. The genetic factors contributing to disease resistance are largely unknown. Here, by comparative analysis on the disease resistance trait of grass carp (Ctenopharyngodon idellus, Ci) and barbel chub (Squaliobarbus curriculus, Sc), we report that the genes encoding SIDT2, MDA5 and IFN are associated with the resistance to viral infection. We identified the amino acid difference of SIDT2s, the existing or lacking of dsRNA binding loci in the carboxy-terminal domain (CTD) of MDA5s, and the promoter difference of ifn 2s between the two fish species. Both CiSIDT2 and ScSIDT2 negatively regulate the MDA5-IRF3/7 mediated type I IFN reaction, and ScSIDT2 exhibited a stronger ability than CiSIDT2 to induce Ciirf 7, Ciifn 1, Ciifn 2 and Cimx expressions, and to resist GCRV infection. ScMDA5 showed a greater ability than CiMDA5 in the induction of IRF7-mediated type I interferon and anti-GCRV function. CiMDA5 CTD inhibited Ciifn 1 while enhanced Ciifn 2 exrpessions, and ScMDA5 CTD exerts opposite effects on Ciifn 1 and Ciifn 2 expressions. ScMDA5 CTD possesses the ability to bind with GCRV nucleic acid fragments, while CiMDA5 lacking binding with virus genome. The promoter activities of Ciifn 2 is significantly higher than that of Scifn 2 owning to their differences in promoter sequences and binding proteins. These data revealed the mechanism of SIDT2-MDA5-IFN genetic architecture contributing to the difference in interspecies resistance and opening the possibility of molecular breeding for fish resistance. • Fish SIDT2/MDA5/IFN axis existed genetic structure difference. • ScSIDT2 exhibited a stronger ability than CiSIDT2 to resist GCRV infection. • ScMDA5 showed a greater ability than CiMDA5 in anti-GCRV function. • ScMDA5 CTD bind with GCRV genome while CiMDA5 not. • SIDT2-MDA5-IFN genetic architecture contributes to interspecies resistance. [ABSTRACT FROM AUTHOR]

IL-1β is an important proinflammatory cytokine with multifaceted modulatory roles in immune responses. In fish, recombinant IL-1β has been employed in the control of bacterial diseases, while the antiviral mechanisms of IL-1β remain largely unknown, and the efficacy of recombinant IL-1β as an immunomodulator to prevent viral diseases is still not determined. This study evaluated the immunomodulatory effects of recombinant grass carp IL-1β against grass carp reovirus (GCRV) in vitro and in vivo. Firstly, the mature form (Ser111-Lys270) of grass carp IL-1β was identified, and its recombinant protein (designated as rgcIL-1β) was prepared through prokaryotic expression. Then, an in vitro evaluation model for rgcIL-1β activity was established in the CIK cells, with the appropriate concentration (600 ng/mL) and effect time (1 h). In vitro , rgcIL-1β could not only induce the production of proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α but also a series of antiviral factors including IFN-1, IFN-2, IFN-γ, and ISG15. Mechanistically, transcriptome analysis and western blotting confirmed that rgcIL-1β activated multiple transcriptional factors, including NF-κB, IRF1, IRF3, and IRF8, and the signal pathways associated with inflammatory cytokines and antiviral factors expression. Expectedly, rgcIL-1β treatment significantly inhibited GCRV replication in vitro. In vivo administration of rgcIL-1β via intraperitoneal pre-injection significantly aroused an antiviral response to restrict GCRV replication and intense tissue inflammation in grass carp, demonstrating the immunomodulatory effects of rgcIL-1β. More importantly, rgcIL-1β administrated with 10 ng/g and 1 ng/g could improve the survival rate of grass carp during GCRV infection. This study represents the first time to comprehensively reveal the immunomodulatory and antiviral mechanisms of IL-1β in fish and may also pave the way for further developing recombinant IL-1β as an immunotherapy for the prevention and control of fish viral diseases. [ABSTRACT FROM AUTHOR]

Abstract The barbel chub (Squaliobarbus curriculus) is a kind of small size commercial fish species that is widely spread in Asia and has shown significant resistance to disease. In this study, the full-length cDNA sequences of Toll-like receptor (TLR) 7 and 8 from S. curriculus , designated as ScTLR7 and ScTLR8 , were cloned, and their differences in the structure and the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation were investigated. The full-length 3715 base pair (bp) cDNA of ScTLR7 contained a complete open reading frame of 3162 bp and encoded a putative polypeptide of 1053 amino acid residues. The full-length 4624 base pair (bp) cDNA of ScTLR8 contained a complete open reading frame of 3072 bp and encoded a putative polypeptide of 1023 amino acid residues. ScTLR7 and ScTLR8 consisted of N-terminal signal peptide, leucine-rich repeats (LRRs), and Toll/IL-1 Receptors domain. LRR motifs of ScTLR7 and ScTLR8 bend into horseshoe-like solenoid structure, while the number of LRRs between the two genes is different. Phylogenetic analysis showed that both the ScTLR7 and ScTLR8 were closely clustered with Ctenopharyngodon idellus and Megalobrama amblycephala. Quantitative real-time polymerase chain reaction analysis showed that the expression levels of ScTLR7 in S. curriculus were most abundant in the brain followed by the spleen and heart, and the lowest in the intestine. The highest expression level of ScTLR8 was observed in spleen and the lowest in the liver. After LPS stimulation, the relative expression levels of both ScTLR7 and ScTLR8 exhibited an overall trend of up-regulation. The expression levels of type I-IFN showed an overall trend of down-regulation at time points of 12, 24, 72 and 168 h compared to that of 6 h after LPS stimulation. Compared to 6 h post GCRV infection, the transcription level of ScTLR7 was up-regulated from 12 to 168 h, and transcription levels of ScTLR8 , MyD88 , and type I-IFN were firstly up-regulated from 12 to 72 h, and then down-regulated from 72 to 168 h. Correlation analysis showed that expression level of ScTLR7 in the spleen was significantly positively correlated with that of MyD88 (Pearson correlation coefficient: 0.909, P : 0.033), and a significantly positive correlation was also observed between expression levels of MyD88 and type I IFN (Pearson correlation coefficient: 0.962, P : 0.009), after GCRV stimulation. These results indicate that ScTLR7 and ScTLR8 may play important roles in the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation and trigger different downstream immune signal pathways. Highlights • The TLR7 and 8 of Squaliobarbus curriculus was cloned and characterized. • ScTLR7 and 8 transcriptions were up-regulated after GCRV or LPS stimulation. • ScTLR7 and MyD88 expressions were significantly positively correlated. [ABSTRACT FROM AUTHOR]