Defensins are a group of small cationic and cysteine-rich peptides that are important components of the innate immune system. However, studies on defensins in teleosts are very limited, particularly studies on defensin functions through in vivo assays. In this study, we cloned and identified one β-defensin (TroBD) the golden pompano, Trachinotus ovatus, and analyzed the functions of TroBD in both in vivo and in vitro assays. TroBD is composed of 63 amino acids and shares high sequence identities (27.27-98.41%) with known β-defensins of other teleosts. The protein has a signature motif of six conserved cysteine residues within the mature peptide. The expression of TroBD was most abundant in the head kidney and spleen and was significantly upregulated following infection by Vibrio harveyi and viral nervous necrosis virus (VNNV). Purified recombinant TroBD (rTroBD) inhibited the growth of V. harveyi, and its antimicrobial activity was influenced by salt concentration. TroBD was found to have a chemotactic effect on macrophages in vitro. The results of an in vivo study demonstrated that TroBD overexpression/knockdown in T. ovatus significantly reduced/increased bacterial colonization or viral copy numbers in tissues. Taken together, these results indicate that TroBD plays a significant role in both antibacterial and antiviral immunity and provide new avenues for protection against pathogen infection in the aquaculture industry.

The double-stranded RNA-activated protein kinase (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, PKR inhibits or promotes viral replication. In the present study, PKR homologues of orange-spotted grouper (Epinephelus coioides) (EcPKR) were cloned and the involvement of EcPKR during Red-spotted grouper nervous necrosis virus (RGNNV) infection was investigated. EcPKR encodes a 621-amino acid polypeptide that is closely related to the equivalent protein in Larimichthys crocea. EcPKR encoded two dsRNA binding domains and a Serine/Threonine protein kinase domain. Quantitative real-time PCR (qRT-PCR) analysis indicated that EcPKR was present in all examined tissues, with higher expression in spleen, intestine and gill. When stimulated with poly(I:C), the expression of EcPKR in the grouper spleen was increased, with highest expression 12 h post stimulation. EcPKR concentration was significantly increased in RGNNV-infected cells, with highest expression at 36 h post stimulation. EcPKR is mainly present in the cytoplasm. Overexpression of EcPKR in grouper spleen (GS) cells inhibits the transcription of the RGNNV genes. Furthermore, our results show that EcPKR overexpression significantly enhances the immune response of interferon and the activation of interferon-beta (IFN-β), interferon stimulated response element (ISRE) and nuclear factor-kappa B (NF-κB). Taken together, these results are important for better understanding of the function of PKR in fish and reveal its involvement in host response to immune challenges in RGNNV.

Cystatin B is a cysteine protease inhibitor that plays a crucial role in immune response. Nevertheless, the molecular mechanism of fish Cystatin B in virus replication remains obscure. In this study, we identified and characterized Cystatin B (Ec-CysB) in the orange-spotted grouper (Epinephelus coioides). The Ec-CysB encoded a 100-amino acid protein with the conserved QXVXG motif, PC motif and cysteine protease inhibitory motif, which shared high identities with reported Cystatin B. The abundant transcriptional level of Ec-CysB was found in gill, intestine and head kidney. And the Ec-CysB expression was significantly up-regulated in spleen after infection with Singapore grouper iridovirus (SGIV) in vitro. Subcellular localization analysis revealed that Ec-CysB was distributed mainly in the cytoplasm and nucleus. Further studies showed that overexpression of Ec-CysB in vitro significantly increased SGIV replication and virus-induced cell apoptosis, but replication of SGIV was inhibited by knockdown or mutant of Ec-CysB. Moreover, overexpression of Ec-CysB significantly inhibited the interferon (IFN), interferon-stimulated response element (ISRE) promoter activities, and enhanced apoptosis-related transcription factors p53 promoter activities. Collectively, our results suggest that Ec-CysB affect viral replication and virus-induced cell apoptosis, which will help us to explore its potential functions during SGIV infection.

In this study, the transcriptional response of grouper to Singapore grouper iridovirus (SGIV) stimulation was characterized using RNA sequencing. Transcriptome sequencing of three test groups in the grouper was performed using the Illumina MiSeq platform. The three test groups were a control group, which was injected with PBS buffer; a high-susceptible (HS) group, which died shortly after the SGIV injection; and a high-resistance (HR) group, which survived the SGIV injection. In total, 38,253 unigenes were generated. When the HS group was compared with the control group, 885 unigenes were upregulated and 487 unigenes were downregulated. When the HR and control groups were compared, 1114 unigenes were upregulated and 420 were downregulated, and when the HR and HS groups were compared, 1010 unigenes were upregulated and 375 were downregulated. In the KEGG analysis, two immune-related pathways, the p53 and peroxisome proliferator-activated receptor pathways, were detected with highly significant enrichment. In addition, 7465 microsatellites and 22,1569 candidate single nucleotide polymorphisms were identified from our transcriptome data. The results suggested several pathways that are associated with traits of disease susceptibility or disease resistance, and provided extensive information about novel gene sequences, gene expression profiles, and genetic markers. This may contribute to vaccine research and a breeding program against SGIV infection in grouper.

The Lymphocyte antigen-6 (Ly-6) superfamily has been considered to play an important role in the innate immunity of mammals. The functions of Ly-6 proteins are diverse since their low sequence homology. Currently, the function of Ly-6D, a member of Ly-6 family proteins, is completely unknown in teleost. In the present study, we identified and characterized a Ly-6D homologue (named PoLy-6D) from the teleost fish Paralichthys olivaceus and examined its immune function. PoLy-6D possesses a hydrophobic signal peptide, a LU domain including a conserved "LXCXXC" motif in N-terminus and a "CCXXXXCN" motif in C-terminus. Under normal physiological condition, PoLy-6D expression distributes in all the examined tissues, the highest three tissues are successively spleen, head kidney, and blood. When infected by extracellular and intracellular bacterial pathogens and viral pathogen, PoLy-6D expression was induced and the patterns vary with different types of microbial pathogens infection and different immune tissues. In vitro experiment showed recombinant PoLy-6D (rPoLy-6D) inhibited the lysis of rabbit red blood cells by serum and selectively improved bacterial survival in serum. After serum were treated by antibody of rPoLy-6D, bacteriostatic effect of serum was obviously enhanced. These results indicate the importance of PoLy-6D as a complement regulator. rPoLy-6D possessed the binding activity to multiple bacteria but did not exhibit antimicrobial activities. The interaction between rPoLy-6D and bacteria suggests that PoLy-6D is involved in host clearance of pathogens probably by serving as a receptor for pathogens. Overexpression of PoLy-6D in vivo promoted the host defense against invading E. piscicida. These findings add new insights into the regulation mechanism of the complement system in teleost and emphasize the importance of Ly-6D products for the control of pathogen infection.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with a unique structure involved in immune regulation and inflammation. In the present study, we identified a MIF from Trachinotus ovatus (golden pompano) and analyzed its function. TroMIF shares high homology (58.26%-94.78%) with the other known MIF sequences of vertebrates. TroMIF is most closely related to large yellow croaker (Larimichthys crocea). The expression of TroMIF was most abundant in the liver and head kidney, and was significantly up-regulated after Edwardsiella tarda infection. The subcellular localization of TroMIF was mostly distributed in the cytoplasm. In vitro results revealed that the recombinant protein rTroMIF could inhibit the migration of head kidney lymphocytes (HKLs) and macrophages (HKMs) and enhance the phagocytic activity of HKMs. As a pro-inflammatory cytokine, rTroMIF could increase the expression levels of some pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), IL-6, IL-8, and interferon-gamma (IFN-γ) and decrease the expression of IL-10. The rTroMIF was proved to have enzymatic redox activity in vitro. Furthermore, overexpression of TroMIF in the head kidney cell line of golden pompano could significantly enhance its ability to resist E. tarda infection from 1 h to 4 h. The knockdown of TroMIF expression induced a significant increase in the number of bacteria after E. tarda infection at 1, 2, and 4 hpi. Our results suggest that TroMIF is an essential effector of the innate immune system and plays a pivotal role in antibacterial immunity.

Cystatin C is an endogenous inhibitor of cysteine proteases and widely exist in organisms. Several studies in mammals have showed that Cystatin C plays critical role in the immune defense against microorganisms. It is also well known that some fish Cystatin C have important immune regulation functions in inflammatory responses. However, the function of fish Cystatin C in virus infection as well as its underlying molecular mechanisms remain to be elucidated. In the present study, a Cystatin C gene termed Ec-CysC was identified from orange-spotted grouper, Epinephelus coioides. The full-length of Ec-CysC cDNA was 817 bp with a 387 bp open reading frame (ORF) that encoded a 129-amino acid (aa) protein, including 18-aa signal peptide and 111-aa mature polypeptide. The deduced amino acid of Ec-CysC shared three conserved domains containing Glycine at the N-terminus region, QVVAG motif in the middle and PW motif near the C-terminus region. Transcription analysis of the Ec-CysC gene showed its expression in all twelve examined tissues including liver, spleen, kidney, brain, intestine, heart, skin, muscle, fin, stomach, gill and head kidney. Its expression following stimulation with Singapore grouper iridovirus (SGIV) was further tested in spleen, the relative expression of Ec-CysC was significantly up-regulated at 12 h post-infection. The subcellular localization experiment revealed that Ec-CysC was mainly distributed in the cytoplasm in Grouper Spleen (GS) cells. In vitro, Overexpression of Ec-CysC in GS cells significantly reduced the expression of viral genes, namely, ORF162, ORF049 and ORF072. Meanwhile, we found that overexpression of Ec-CysC resulted in upward trend of expression of inflammatory cytokines TNF-a, IL-1β and IL8 during SGIV infection. Further, SGIV-inducible apoptosis and Caspase-3 activity were also weakened by overexpression Ec-CysC in fathead minnow (FHM) cells. These results indicated that Ec-CysC might have a deeper involvement in fish immune defense, and played important roles in inflammation and apoptosis induced by SGIV.

Chemokines comprise a group of small molecular weight (6-14 kDa) cytokines; chemokine receptors are a superfamily of seven transmembrane domain G-coupled receptors. Both chemokines and their receptors have important roles in immune surveillance, inflammation, and development. Recently, 9 CXC chemokine ligands (CXCLs) and 8 CXC chemokine receptors (CXCRs) were identified and cloned from orange-spotted grouper (Epinephelus coioides) and annotated by phylogenetic and syntenic analyses. We detected mRNA transcripts for CXCLs and CXCRs in healthy tissues of E. coioides. Our data show that CXCL genes are highly expressed in the spleen, kidney and liver and that CXCR genes are ubiquitously expressed, rather than being expressed only in immune organs. Analysis of gene expression after Singapore grouper iridovirus infection indicated that CXCL and CXCR genes are regulated in a gene-specific manner. CXCL8 and CXCL12a were significantly upregulated in the spleen, kidney and liver of resistant fish, indicating potential roles in immunity against the pathogen. Additionally, CXCR4a was upregulated in all three organs in resistant fish, suggesting that CXCL8 or CXCL12a may participate in the immune response via interaction with CXCR4a. In addition, the new orange-spotted grouper receptor CXCR1b was found to be upregulated in the spleen and kidney of resistant fish, indicating that this receptor plays an important role in immune responses to viral infection. These results are valuable for comparative immunological studies and provide insight into the roles of these genes in viral infection.

Interferon regulatory factor (IRF) 7 plays a crucial role in modulating cellular responses to viral infection and cytokines, including interferons (IFNs). In the present study, a novel IRF7 gene (designated as EcIRF7) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length EcIRF7 cDNA is composed of 2089 bp and encodes a polypeptide of 433 amino acids with 81% identity to IRF7 of Siniperca chuatsi, and the genomic DNA of EcIRF7 consists of 9 exons and 8 introns, with a length of approximately 5629 bp. EcIRF7 contains three conserved domains including a DNA-binding domain (DBD), an IRF associated domain (IAD) and a serine-rich domain, all of which are highly conserved across species. Recombinant EcIRF7 was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-EcIRF7 serum preparation. Realtime quantitative PCR (RT-qPCR) analysis revealed a broad expression of EcIRF7, with a relative strong expression in spleen, kidney, skin and intestine. The expression of EcIRF7 was differentially up-regulated after stimulation with Vibrio vulnificus, Staphylococcus aureus and Singapore grouper iridovirus (SGIV). EcIRF7 showed similar intracellular localization pattern to those of mammalian and chicken, and translocated into nucleus after SGIV infection. Further more, EcIRF7 was proved to be capable of activating zebrafish type I IFN promoter and inhibiting the replication of SGIV in grouper spleen (GS) cells. These results suggest that EcIRF7 is potentially involved in grouper immune responses to invasion of viral and bacterial pathogens.

C-Jun N-terminal kinases (JNKs), a subgroup of serine-threonine protein kinases that activated by phosphorylation, are involve in physiological and pathophysiological processes. JNK3 is one of JNK proteins involved in JNK3 signaling transduction. In the present study, two JNK3 isoforms, Ec-JNK3 X1 and Ec-JNK3 X2, were cloned from orange-spotted grouper, Epinephelus coioides. Both Ec-JNK3 X1 and Ec-JNK3 X2 were mainly expressed in liver, gill, skin, brain and muscle of juvenile grouper. The relative expression of Ec-JNK3 X2 mRNA was much higher in muscle and gill than that of Ec-JNK3 X1. Isoform-specific immune response to challenges was revealed by the expression profiles in vivo. Immunofluorescence staining indicated that JNK3 was localized in the cytoplasm of grouper spleen (GS) cells and shown immune response to SGIV infection in vitro. Over-expressing Ec-JNK3 X1 and/or Ec-JNK3 X2 inhibited the SGIV infection and replication and the SGIV-induced apoptosis. To achieve the antiviral and anti-apoptosis activities, JNK3 promoted the activation of genes ISRE and type I IFN in the antiviral IFN signaling pathway, and inhibited the activation of transcription factors NF-κB and p53 relating to apoptosis, respectively. Ec-JNK3 X2 showed stronger activities in antivirus and anti-apoptosis than that of Ec-JNK3 X1. Our results not only define the characterization of JNK3 but also reveal new immune functions and the molecular mechanisms of JNK3 on iridoviruses infection and the virus-induced apoptosis.

Lysozyme acts as an innate immunity molecule against pathogen infection. In this study, a new G-type lysozyme gene with a typical G-type lysozyme domain (designated as Ec-lysG) was cloned and characterized from the orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysG cDNA contains 1419 bp and encodes a 256-residue protein containing a 25-residue signal peptide at the N-terminus. BLAST analysis reveals Ec-lysG shares 64% identity with Siniperca chuatsi, but 63% to another reported G-type lysozyme from orange-spotted grouper (OSG-lysG). The genomic DNA of Ec-lysG contains four exons and three introns, with a total length of 2062 bp. An amino acid sequence alignment showed that Ec-lysG shares the fundamental structural features of G-type lysozyme, including the catalytic residues, substrate binding sites, and soluble lytic transglycosylase domain. Quantitative PCR showed that Ec-lysG transcript is most abundant in the head kidney, and less abundant in the heart. The expression of Ec-lysG was differentially upregulated in the head kidney after stimulation with lipopolysaccharide, Vibrio alginolyticus, and Singapore grouper iridovirus (SGIV). A subcellular localization analysis showed that Ec-lysG is distributed predominantly in the cytoplasm. Recombinant Ec-lysG (rEc-lysG) has optimal activity at pH 7.5 and 35°C. rEc-lysG showed lytic activities against Gram-positive bacterium Streptococcus iniae, Staphylococcus aureus, and Micrococcus lysodeikticus, and the Gram-negative bacterium V. alginolyticus. Scanning electron microscopy (SEM) showed that rEc-lysG acts on M. lysodeikticus cell walls. The overexpression of Ec-lysG in grouper cells did not significantly delay the occurrence of the cytopathic effect (CPE) induced by SGIV, and did not inhibit viral gene transcription. In conclusion, Ec-lysG might be a potent antibacterial protein, with a role in innate immunity.

Vibrio harveyi is regarded as serious pathogen for marine fishes. However, host defense mechanisms involved in V. harveyi infection remain incompletely defined. The transcription factor IFN regulatory factor 7 (IRF7) is largely associated with host defense against viral infections, and the role of IRF7 during V. harveyi infection in fish has not been well illuminated previously. In this study, IRF7 from golden pompano (Trachinotus ovatus) was characterized (TroIRF7). The TroIRF7 gene is 1323 bp, which encodes 440 amino acid residues. Multiple amino acid alignments of TroIRF7 shows 30.37%-80.18% identity with other fish IRF7s, including Epinephelus coioides (80.18%), Larimichthys crocea (79.72%), Collichthys lucidus (79.26%), Miichthys miiuy (79.26%), Channa argus (78.77%), Cynoglossus semilaevis (72.67%), and Gadus morhua (65.23%). Like other IRF7s, TroIRF7 also contains 3 conserved domains: an N-terminal DNA-binding domain (DBD), an IRF association domain (IAD), and a C-terminal serine-rich domain (SRD). In the DBD, 4-5 conserved tryptophans were observed, which is a characteristic unique to all fish IRF7 members. TroIRF7 was constitutively expressed, with high levels in gill, head kidney, spleen, skin, and intestine. V. harveyi infection-induced TroIRF7 transcripts significantly up-regulation and translocation to the nucleus. TroIRF7 overexpression promote the fish to inhibit the replication of V. harveyi. And TroIRF7 knockdown led to decreased bacterial clearance in fish tissue. Furthermore, over-expression of TroIRF7 resulted in an increased production of interferon a3 and IFN signaling molecule in the spleen, suggesting that V. harveyi activates the IRF7- IFN pathway. These results suggest that TroIRF7 is an important component of immune responses against V. harveyi infection.

microRNAs (miRNAs) are an evolutionarily conserved class of non-coding RNA molecules that participate in various biological processes. Employment of high-throughput screening strategies greatly prompts the investigation and profiling of miRNAs in diverse species. In recent years, grouper (Epinephelus spp.) aquaculture was severely affected by iridoviral diseases. However, knowledge regarding the host immune responses to viral infection, especially the miRNA-mediated immune regulatory roles, is rather limited. In this study, by employing Solexa deep sequencing approach, we identified 116 grouper miRNAs from grouper spleen-derived cells (GS). As expected, these miRNAs shared high sequence similarity with miRNAs identified in zebrafish (Danio rerio), pufferfish (Fugu rubripes), and other higher vertebrates. In the process of Singapore grouper iridovirus (SGIV) infection, 45 and 43 miRNAs with altered expression (>1.5-fold) were identified by miRNA microarray assays in grouper spleen tissues and GS cells, respectively. Furthermore, target prediction revealed 189 putative targets of these grouper miRNAs.

Cathepsin B (CTSB) is one of the typical representatives of cysteine protease family. It has the activity of both exopeptidase and endopeptidase. It plays an important role in antigen presentation, degradation, apoptosis, inflammatory response and physiological process of many diseases. In this study, CTSB of Trachinotus ovatus (TroCTSB) was cloned, and its structure and function were analyzed. The results showed that the coding region of TroCTSB was 993 bp, encoding 330 amino acid residues. The homology analysis showed that the amino acid sequence of TroCTSB was similar to that in other teleosts and mammals (68.69%-88.48%). Under normal physiological conditions, TroCTSB was widely distributed in various tissues with the highest expression level in stomach, followed by liver, and the lowest expression level in blood. The optimal pH and temperature of purified recombinant protein rTroCTSB were 5.5 and 40 °C, respectively. The toxicity test of metal ions showed that Fe2+, Cu2+, Ca2+ and Zn2+ could all inhibit the activity of TroCTSB, with Zn2+ ranking the first. In addition, after Edwardsiella tarda infection, the expression of TroCTSB was significantly up-regulated in liver, spleen and head kidney. The overexpression of TroCTSB significantly inhibited the infection of E. tarda in golden pompano tissues, and the knockdown of TroCTSB remarkably promoted the reproduction of E. tarda in golden pompano tissues in vivo. This study suggests that TroCTSB was involved in the antibacterial immune response of T. ovatus, and provided a reference for further research in elucidating the resistance mechanism of TroCTSB.

Cystatin A (CyA), an inhibitor of cysteine protease, was widely studied in immune defense and cancer therapy. However, the function of CyA and its potential molecular mechanism during virus infection in fish remain unknown. In our study, we cloned the open reading frame (ORF) of CyA homology from orange-spotted grouper (Ec-CyA) consisting of 303 nucleotides and encoding a 101-amino acid protein. Ec-CyA included two conserved sequences containing one N-terminal glycine fragment and one QXVXG sequence (48aa-52aa) without the signal peptide. Tissue distribution analysis showed that Ec-CyA was highly expressed in spleen and head kidney. Moreover, further analysis indicated that the expression of Ec-CyA increased during SGIV simulation in grouper spleen (GS) cells. Subcellular localization assay demonstrated that Ec-CyA was mainly distributed in cytoplasm in GS cells. Overexpressed Ec-CyA promoted the mRNA level of viral genes MCP, VP19 and LITAF. Meanwhile, SGIV-induced apoptosis in fat head minnow (FHM) cells was facilitated, as well as the activation of caspase-3/7, caspase-9. In addition, Ec-CyA overexpression down-regulated the expression of interferon (IFN) related molecules including ISG15, IFN, IRF3, MAVS, MyD88, TRAF6 and up-regulated proinflammatory factors such as IL-1β, IL-8 and TNF-α. At the same time, Ec-CyA-overexpressing inhibited the activity of IFN and ISRE promoter, but induced NF-κB promoter activity by luciferase reporter gene assay. In summary, our findings suggested that Ec-CyA was involved in innate immune response and played a key role in DNA virus infection.

Mammalian studies have shown that the nuclear transcription factor Y (NFYC) regulates the expression of major histocompatibility complex (MHC) by binding to CCAAT-box on promoters. However, few studies have focused on the regulatory mechanisms of NFYC in MHC pathway in fish. To explore the transcriptional regulatory mechanism of MHCIa in fish, we characterized NFYC and MHCIa of red-spotted grouper (Epinephelus akaara) (named EaNFYC and EaMHCIa, respectively). The EaNFYC genome sequence is 13,796 bp and contains 1,065 bp open reading frame. It is composed of ten exons and nine introns and encode a 354 amino acid sequence. The putative EaNFYC protein sequence shared 67.2-99.4% identity to vertebrate NFYC and possesses a typically conserved domain (histone- or haem-associated protein 5 domain (HAP5)) at the N-terminus. Transcripts of both EaNFYC and EaMHCIa were ubiquitously expressed in all detect tissues, and higher mRNA levels were detected in immune-relevant tissues (middle-kidney). EaNFYC expression increased after treatment with polyinosinic: polycytidylic acid, lipopolysaccharide, nervous necrosis virus, zymosan A, and Singapore grouper iridovirus. Analysis of subcellular localization indicated that EaNFYC was localized at the cell nucleus only. Furthermore, overexpression of EaNFYC significantly stimulated the expression of EaMHCIa, interferon signalling molecules and inflammatory cytokine. The region -878 bp to +82 bp of EaMHCIa promoter was identified to be the core promoter which EaNFYC take effect on. Additionally, point mutations and electrophoretic mobility shift assays verified that NFYC activate MHCIa expression by binding at the M1 and M2 binding sites that do not contain CCAAT-box. These results contribute to elucidating the function of fish NFYC on MHC transcriptional mechanisms, and provide the first evidence of positive regulation of MHCIa expression by NFYC in fish.