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Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of double-stranded RNA (dsRNA) that activates anti-infective innate immunity. The underlying mechanisms are identified as targeting pattern recognition receptors and Th1-inducing. However, whether poly I:C manipulates metabolism to implement this anti-infective function is unknown. Here, GC-MS based metabolomics was used to characterize metabolic profiles induced by different doses of poly I:C. Analysis on the dose-dependent metabolomes shows that elevation of the TCA cycle and malate with the increasing dose of ploy I:C forms the most characteristic feature of the poly I:C stimulation. Exogenous malate activates the TCA cycle and elevates survival of zebrafish infected with Vibrio alginolyticus , which is related to the elevated expression of il-1b, il-6, il-8, tnf-a , and c3b. These results reveal a previously unknown regulation of poly I:C that boosts the TCA cycle to enhance innate immunity against bacterial infection. • Metabolic profiles induced by different doses of poly I:C. was characterized. • Elevated TCA cycle and malate forms the most characteristic feature. • Malate boosts TCA cycle and elevates zebrafish survival post bacterial infection. • This is related to the elevated expression of il-1b, il-6, il-8, tnf-a , and c3b. • These reveal a metabolic modulation of poly I:C against bacterial infection. [ABSTRACT FROM AUTHOR]

Control of bacterial infection resides in the core of human health and sustainable animal breeding. Vaccines as an economic and efficient immunoprophylaxis have been widely accepted, but mechanisms for vaccines do not fully understand. Information regarding to metabolome in response to vaccines is not available. Here we explore the metabolic features by using GC/MS based metabolic profile and trace metabolic mechanisms in zebrafish ( Dario rerio ) in response to live Edwardsiella tarda vaccine. Pathway enrichment analysis shows that live vaccine activates biosynthesis of unsaturated fatty acids and the TCA cycle and reduces aminoacyl-tRNA biosynthesis, suggesting a metabolic characteristic feature in response to the live vaccine. We further demonstrate that hydroxyl radical is limited during stimulation. Finally, we reveal oleate induces effective protection against E. tarda infection. These results have implications for immunity study that metabolic regulation contributes to immune protection. Our findings enable us to propose novel therapeutic strategies on metabolism against bacterial infections. [ABSTRACT FROM AUTHOR]

Abstract: The JAK/STAT signal transduction pathway plays a critical role in host defence against viral and bacterial infections. In the present study, we report cDNA cloning and characterization of the JAK family (mJAK1-3 and mTYK2) and STAT family members (mSTAT1, mSTAT3-6) from the mandarin fish Siniperca chuatsi. To our knowledge, JAK2, TYK2 and STAT6 genes were cloned from fish for the first time. The mJAK family proteins consist of 1112–1177 residues with a FERM domain, an SH2 domain, a pseudokinase domain, and a tyrosine kinase domain. The mSTAT family members contain 716–786 residues with similar architecture, including an N-terminal domain, a coiled coil domain, a DNA binding domain, a linker domain, an SH2 domain, and a transcription activation domain. Multiple sequence alignments of mJAKs/mSTATs and phylogenetic analysis showed that mJAK1 was closed to mTYK2, and mJAK2 was closed to mJAK3. Quantitative real-time PCR results revealed that mJAK/mSTAT family members were expressed in most tissues examined except muscle. In mandarin fish fry cells, the expressions of IRF-1, Mx, SOCS1 and SOCS3 genes were significantly induced by poly(I:C) stimulation, indicating that the mJAK/mSTAT signal pathway is activated by poly(I:C). Furthermore, expressions of all four mJAKs and four mSTATs were all up-regulated after poly(I:C) stimulation, but expression of mSTAT5 was inhibited by poly(I:C). These results suggest that mandarin fish has the JAK/STAT signal transduction pathways similar to those in mammals, and these signalling pathways may play an important role in regulation of antiviral responses in fish. [Copyright &y& Elsevier]

Mechanisms by which vaccines enhance immunity to combat bacterial pathogens are not fully understood. Recently, we have found that live Edwardsiella tarda vaccine enhances ability against the bacterial challenge by metabolic modulation in zebrafish. Here we first explored the metabolic modulation promoted by inactivated E. tarda to eliminate the pathogen. Inactivated E. tarda vaccine modulated a similar metabolome to combat with the pathogen in zebrafish as live E. tarda vaccine did. Specifically, both vaccines promoted biosynthesis of unsaturated fatty acids and the TCA cycle. However, due to relatively higher activated TCA cycle in inactivated vaccine than live vaccine, live vaccine promoted higher abundance of palmitate than inactivated vaccine. Consistently, the protection against E. tarda challenge was palmitate dose-dependent. Live vaccine activated higher expression of IL-1β, IL-8,Cox-2 genes and lower expression of IL-15, IL-21 genes than inactivated vaccine, which is similar to the results stimulated by high and low doses of palmitate, respectively. These findings indicate live and inactivated E. tarda vaccines stimulate differential abundances of palmitate that contribute to differential innate immunities against bacterial infection. Thus, metabolic environment contributes to immune response. Image 1 • Metabolic feature is revealled in zebrafish immunized with dead E. tarda vaccine. • Similar metabolic profile but low palmitate is detected compared with live vaccine. • Palmitate induces immune effective protection in a dose-dependent manner. • High and low palmitate induce altered innate immunity as live and dead vaccines do. • Metabolic environment contributes to immune response. [ABSTRACT FROM AUTHOR]

Abstract: The NF-κB/IκBα pathway plays an important role in the regulation of immune and inflammatory responses. IκBα is an inhibitory molecule that sequesters transcription activator NF-κB dimer in the cytoplasm of unstimulated cells. Here, we isolated the full-length cDNAs of the mandarin fish (Siniperca chuatsi) alpha inhibitor of NF-κB (ScIκBα) and p65 NF-κB (Scp65). Multiple sequence alignments showed that the amino acid sequences of both ScIκBα and Scp65 contain conserved domains similar to those of mammalian counterparts. Protein pull-down and coimmunoprecipitation assays showed that ScIκBα directly bound with Scp65. Real-time quantitative PCR analysis showed that ScIκBα mRNA was constitutive in all mandarin fish tissues detected. After challenge with infectious spleen and kidney necrosis virus (ISKNV), the mRNA level of ScIκBα was decreased nearly 6 fold in the spleen. This result suggests that the NF-κB/IκBα pathway in mandarin fish may play a role in the immune response against ISKNV. [Copyright &y& Elsevier]

Tiger frog virus (TFV) belongs to the genus Ranavirus (family Iridoviridae) and causes significant harm in cultured frogs, resulting in substantial losses in ecological and economic field in Southern China. Attachment is the first step in viral life cycle, which is dependent on the interactions of virions with extracellular matrix (ECM) components. Studying this process will help in understanding virus infection and controlling viral diseases. In this study, the roles of primary ECM components in TFV attachment were investigated. The results on the kinetics of virus attachment showed TFV successful attachment to the cell surface as a relatively rapid process after TFV was used to inoculate cells for 10 min at 4 °C. Western blot and quantitative PCR analyses results showed that soluble fibronectin, collagen IV, laminin, or hyaluronic acid treatment with TFV caused no significant effect on virus attachment. Soluble heparin, heparan sulfate and chondroitin sulfate A/B could inhibit TFV attachment in a dose-dependent manner. Enzymic digestion by cell surface heparin/heparan sulfate using heparinase I, II, and III could significantly prevent TFV attachment, suggesting that heparan sulfate plays an important role in TFV attachment. Furthermore, the binding assays of heparin–agarose beads and virion showed that TFV virions specifically bound with heparin in a dose-dependent manner. Given that heparin is a structural analogue of heparan sulfate, the above results suggest that heparan sulfate might serve as an attachment factor of TFV infection. Our work would be beneficial to understand the mechanisms of TFV attachment and the interactions of TFV with cellular receptor(s). • Kinetics of virus attachment showed that TFV attachment is a relatively rapid process. • Extracellular matrix glycosaminoglycans play an important role in TFV attachment. • Heparan sulfate may be an attachment factor for TFV infection. [ABSTRACT FROM AUTHOR]

The mandarin fish Siniperca chuatsi is a cultured freshwater fish species that is popular in China because of its high market value. With the development of high-density cultural mode in mandarin fish, viral diseases such as Infectious spleen and kidney necrosis virus (ISKNV) are becoming increasingly serious. Stimulator of interferon genes (STING) is a central component in the innate immune response to cytosolic DNA and RNA derived from different pathogens. However, the roles of STING in innate immune response of mandarin fish remain unknown. In the present study, S. chuatsi STING (sc STING)-mediated host immune response against ISKNV infection was investigated. Sc STING transcription level increased remarkably in response to ISKNV infection, LPS, PMA, or poly (I:C) stimulation in mandarin fish fry (MFF-1) cells. Immunofluorescence results showed that sc STING localized majorly in the endoplasmic reticulum. sc STING overexpression remarkably increased the expression levels of scIFN-h , scMx , scISG15 , scPKR , scViperin , scIL-1β , scIL-18 , and scTNF-α genes. IFN-β-luciferase report assay results showed that the relative expressions of luciferin were remarkably increased in MFF-1 cells. Site mutation of serine (S) on C-terminus of sc STING showed that both S388 and S396 were important for mediated signaling. Furthermore, sc STING overexpression inhibited ISKNV infection, and knockdown of sc STING promoted ISKNV infection, indicating that sc STING could suppress ISKNV infection in MFF-1 cells. These observations suggested that the sc STING played an important role in innate immune against ISKNV infection. Our work would help elucidate the roles of teleost fish STING in innate immunity. • sc STING can evoke interferon and inflammatory immune responses. • The Ser388 and Ser396 sites are crucial for sc STING-mediated signaling. • sc STING mediates host antiviral immune response against ISKNV infection. [ABSTRACT FROM AUTHOR]

Mandarin fish (Siniperca chuatsi) is a significant cultured species with high added value in China. With the expansion of farming, diseases of mandarin fish such as Infectious spleen and kidney necrosis virus (ISKNV) diseases are becoming more and more serious. Human endogenous retrovirus subfamily H long terminal repeat associating protein 2 (HHLA2) is a type 1 transmembrane molecule with three extracellular Ig domains (IgV-IgC-IgV) and plays important roles in the T cell proliferation and tumorigenesis. The HHLA2-homologues have not been found in virus. In this study, a viral HHLA2 protein encoded by ISKNV ORF069L was identified and the virulence of the deleted ORF069L reconstruction ISKNV strain (Δ ORF069L) was investigated. ISKNV ORF069L gene was predicted to encode a 222-amino acids peptide. The bioinformation analysis revealed that ISKNV ORF069L contained an Ig HHLA2 domain and was homologous to vertebrate B7-CD28 family proteins. The recombinant virus strain of Δ ORF069L was constructed by homologous recombination technology. The virus titer and growth curves between ISKNV wild type (WT) and Δ ORF069L on cellular level showed no significant differences indicating that the ORF069L did not influence the ISKNV replication. The expression levels of immune-related genes (Mx1, IL-1β, IL-8, TNF-a and IgM) were increased in fish infected with Δ ORF069L , compared to those in fish infected with ISKNV WT. Furthermore, the lethality caused by Δ ORF069L declined by 40% compared with ISKNV WT, indicating that ORF069L was a virulence gene of ISKNV. Most importantly, the protection rate was nearly 100% for fish immunized with Δ ORF069L strain. Those results suggested that Δ ORF069L could be developed as a potential attenuated vaccine against ISKNV. Our work will be beneficial to promote the development of gene deletion attenuated vaccines for ISKNV disease. • A viral HHLA2 protein encoded by ISKNV ORF069L was identified. • A recombinant virus of ISKNV ORF069L deletion (Δ ORF069L) was constructed. • ISKNV ORF069L was an important virulence gene. • The protection rate was nearly 100% for fish immunized with Δ ORF069L strain. • Δ ORF069L could be developed as an attenuated vaccine against ISKNV. [ABSTRACT FROM AUTHOR]

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (sc YB-1) and its roles in cold stress and virus infection were investigated. The sc YB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the sc YB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of sc YB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a , scHsc70b , and scp53. Furthermore, the role of sc YB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of sc YB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of sc YB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α , and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with sc YB-1 compared with those in cells overexpressed with control plasmid. These results indicate that sc YB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed sc YB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of sc YB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that sc YB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that sc YB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity. • S. chuatsi YB-1 (sc YB-1) is involved in the cold stress response in mandarin fish. • sc YB-1 can regulate the NF-κB signaling pathway in MFF-1 cells. • sc YB-1 suppresses ISKNV infection but enhances SCRV infection. [ABSTRACT FROM AUTHOR]

Ranaviruses belong to the family Iridoviridae , and have become a serious threat to both farmed and natural populations of fish and amphibians. Previous reports showed that ranaviruses could encode viral Bcl-2 family-like proteins (vBcl-2), which play a critical role in the regulation of cell apoptosis. However, the mechanism of ranaviruses vBcl-2 interactions with host protein in mediating apoptosis remains unknown. Tiger frog virus (TFV) belonging to the genus Ranavirus has been isolated from infected tadpoles of Rana tigrina rugulosa , and it causes a high mortality rate among tiger frog tadpoles cultured in southern China. This study elucidated the molecular mechanism underlying the interaction of TFV ORF104R with the VDAC2 protein to regulate cell apoptosis. TFV ORF104R is highly similar to other ranaviruses vBcl-2 and host Mcl-1 proteins, indicating that TFV ORF104R is a postulate vBcl-2 protein. Transcription and protein expression levels showed that TFV orf104r was a late viral gene. Western blot results suggested that TFV ORF104R was a viral structural protein. Subcellular localization analysis indicated that TFV ORF104R was predominantly colocalized with the mitochondria. Overexpressed TFV ORF104R could suppress the release of cytochrome C and the activities of caspase-9 and caspase-3. These results indicated that TFV ORF104R might play an important role in anti-apoptosis. Furthermore, the interaction between TFV ORF104R and VDAC2 was detected by co-immunoprecipitation in vitro. The above observations suggest that the molecular mechanism of TFV-regulated anti-apoptosis is through the interaction of TFV ORF104R with the VDAC2 protein. Our study provided a mechanistic basis for the ranaviruses vBcl-2-mediated inhibition of apoptosis and improved the understanding on how TFV subverts host defense mechanisms in vivo. • TFV ORF104R was a late viral protein as viral structural protein of virion particles. • TFV ORF104R interacted with cellular VDAC2 protein. • TFV ORF104R inhibited cell apoptosis though intrinsic pathway. [ABSTRACT FROM AUTHOR]

Mandarin fish (Siniperca chuatsi) is a popular cultured freshwater fish species due to its high market value in China. With increasing density of breeding, mandarin fish is often cultured under low environmental oxygen concentrations (hypoxia). In this study, the relative expression levels of hypoxia response element (HRE)-luciferase reporter and the HIF signaling pathway downstream genes (scldha , scvegf , and scglut-1) were significantly increased by hypoxic stress, thereby indicating that mandarin fish has an HIF signaling pathway. The mandarin fish HIF-1α (sc HIF-1α) was also characterized. Multiple sequence alignments showed that sc HIF-1α presented similar architectures to other known vertebrates. Subcellular localization analysis showed that sc HIF-1α was mainly located in the nucleus of the mandarin fish fry-1 (MFF-1) cells. The role of sc HIF-1α in the regulation of the HIF signaling pathway was confirmed. Overexpression of sc HIF-1α could induce the HIF signaling pathway, whereas knockdown of sc HIF-1α inhibited the activity of the HIF-1 signaling pathway. Tissue distribution analysis showed that schif-1α was significantly highly expressed in the blood, heart, and liver, which indicated that the main function of sc HIF-1α was closely related to the circulatory system. Furthermore, sc HIF-1α expression was significantly induced by poly I:C, poly dG:dC or PMA, thereby indicating that sc HIF-1α was involved in the immune response. HIF-1α plays an important role in pathogen infections in mammals, but its role in fish is rarely investigated. Overexpression of sc HIF-1α could inhibit MRV and SCRV infections, whereas knockdown of sc HIF-1α could promote such infections. Those results suggested that sc HIF-1α played an important role in fish virus infection. Our study will help understand the hypoxia associated with the outbreaks of aquatic viral disease. • The sc HIF signaling pathway exists in MFF-1 cells. • The expression of sc HIF-1α was up-regulated in response to the immune challenge. • sc HIF-1α could inhibit MRV and SCRV infections. [ABSTRACT FROM AUTHOR]