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Transcription factor ATF1 is a member of the ATF/CREB family of the CREB subfamily and is involved in physiological processes such as tumorigenesis, organ development, reproduction, cell survival, and apoptosis in mammals. However, studies on ATF1 in fish have been relatively poorly reported, especially on its role in antiviral immunity in fish. In this study, ATF1 from orange-spotted grouper (named EcATF1) were cloned and characterized. Molecular characterization analysis showed that EcATF1 encodes a 307-amino-acid protein, containing PKID and bZIP_CREB1 domains. Homology analysis showed that had the highest homology with E. lanceolatus(88.93%). Tissue expression pattern showed that EcATF1 was extensively distributed in twelve selected tissues, with higher expression in the skin, gill, liver and spleen. Subcellular localization analysis showed that EcATF1 was distributed in the nucleus of GS cells. EcATF1 overexpression inhibits Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by a diminished degree of CPE induced by SGIV and RGNNV and a reduction in the level of viral gene transcription and viral capsid protein expression. Furthermore, EcATF1 overexpression upregulated interferon pathway-related genes and proinflammatory factors, and increased the promoter activities of IFN, IFN stimulated response element (ISRE), and nuclear factor κB(NFκB). Meanwhile, EcATF1 overexpression positive regulate the MHC-I signaling pathway, and upregulated the promoter activity of MHC-I. Collectively, these data demonstrate that EcATF1 plays an important role during the host antiviral immune response. This study provides insights into the function of ATF1 in the immune system of lower vertebrates.

Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor that plays a crucial role in the immune response against microbial infections. To clarify the roles of fish MARCO in Singapore grouper iridovirus (SGIV) infection, we identified and characterized Ec-MARCO in the orange-spotted grouper (Epinephelus coioides). The Ec-MARCO encoded a 370-amino acid protein with transmembrane region, coiled coil region and SR domain, which shared high identities with reported MARCO. The abundant transcriptional level of Ec-MARCO was found in spleen, head kidney and blood. And the Ec-MARCO expression was significantly up-regulated in grouper spleen (GS) cells after infection with SGIV in vitro. Subcellular localization analysis revealed that Ec-MARCO was mainly distributed in the cytoplasm and on the cell membrane. Ec-MARCO knockdown in vitro significantly inhibited SGIV infection in GS cells, as evidenced by reduced decreased SGIV major capsid protein (MCP) transcription and MCP protein expression. Further studies showed that Ec-MARCO knockdown positively regulated proinflammatory cytokines and interferon-stimulated genes, and enhanced IFN and ISRE promoter activities. However, overexpression of Ec-MARCO did not affect SGIV entry into host cells. In summary, our results suggested that Ec-MARCO affected SGIV infection by regulating antiviral innate immune response.

Cystatin F (CyF), an inhibitor of cysteine protease, was widely studied in immune defense and cancer therapy. However, the function of CyF and its latent molecular mechanism during virus infection in fish remain vacant. In our research, we cloned the open reading frame (ORF) of CyF homology from orange-spotted grouper (Ec-CyF) consisting of 342 nucleotides and encoding a 114-amino acid protein. Ec-CyF included two cystatins family sequences containing one KXVXG sequence without the signal peptide, and a hairpin ring containing proline and tryptophan (PW). Tissue distribution analysis indicated that Ec-CyF was highly expressed in spleen and head kidney. Besides, further analysis showed that the expression of Ec-CyF increased during SGIV infection in grouper spleen (GS) cells. Subcellular localization assay demonstrated that Ec-CyF was mainly distributed in cytoplasm in GS cells. Overexpressed Ec-CyF demoted the mRNA level of viral genes MCP, VP19 and LITAF. Meanwhile, SGIV-induced apoptosis in fat head minnow (FHM) cells was impeded, as well as the restraint of caspase 3/7 and caspase 8. In addition, Ec-CyF overexpression up-regulated the expression of IFN related molecules including ISG15, IFN, IFP35, IRF3, IRF7, MYD88 and down-regulated proinflammatory factors such as IL-1β, IL-8 and TNF-α. At the same time, Ec-CyF-overexpressing increased the activity of IFN3 and ISRE promoter, but impeded NF-κB promoter activity by luciferase reporter gene assay. In summary, our findings suggested that Ec-CyF was involved in innate immunity response and played a key role in DNA virus infection.

(1) Background: Lysosomal aspartic protease Cathepsin D (CD) is a key regulator and signaling molecule in various biological processes including activation and degradation of intracellular proteins, the antigen process and programmed cell death. However, the function of fish CD in virus infection remains largely unknown. (2) Methods: The functions of the CD gene response to SGIV infection was determined with light microscopy, reverse transcription quantitative PCR, Western blot and flow cytometry. (3) Results: In this study, Ec-Cathepsin D (Ec-CD) was cloned and identified from the orange-spotted grouper

Peroxisome proliferative–activated receptor α (PPARα) belongs to the superfamily of nuclear receptors (NR). Studies have demonstrated that PPARα functions in energy metabolism, hepatic function, immune response, cell cycle, and apoptosis. In teleost fish, few studies have investigated the role of PPARα in the immune response. In this study, the grouper PPARα gene (EcPPARα) was investigated for its role in viral infection. The open reading frame of EcPPARα encoded a protein of 469 amino acids and contained an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD). Phylogenetic analysis revealed that EcPPARα was most closely related to homologous genes in Sander lucioperca and Perca flavescens. Upon challenge with SGIV (Singapore grouper iridovirus) and RGNNV (Red-spotted grouper nervous necrosis virus), EcPPARα expression levels were significantly upregulated in different tissues. Subcellular localization analysis showed that the EcPPARα protein localized throughout the cytoplasm and nucleus with diffuse intracellular expression patterns, which is consistent with the localization pattern of mammalian PPARs. Based on morphological observation of cytopathic effect (CPEs), viral gene expression mRNAs, and virus titer assays, the results presented here showed that an overexpression of EcPPARα promoted SGIV production in grouper spleen cells. Overexpression of EcPPARα significantly inhibited the expression of several cytokines, including interferon-related genes (IFN-γ, ISG15, MXI, MXII, MAVS and MDA5), inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and Toll like receptor adaptors (TRAF6 and MyD88). Luciferase activity of IFN-α, IFN-γ, ISRE and NF-κB promoters was also significantly decreased in EcPPARα overexpression cells. Due to these detected interferon-related genes and inflammatory cytokines play important antiviral effect against SGIV in grouper, we speculated that the promotion effect of EcPPARα on SGIV replication may be caused by down-regulation of interferon and inflammatory response. In addition, through apoptotic body observation, capspase-3 activity detection, and flow cytometry analysis, it was found that overexpression of EcPPARα promoted SGIV-induced apoptosis in fathead minnow (FHM) cells. These data may increase an understanding of the role of PPARα in fish antiviral immune responses and apoptosis.

The transcription factor nuclear factor kappa B (NF-κB) is a critical regulator of immune and inflammatory responses with crucial roles in various pathophysiologic conditions involving cell survival and death. Recent studies in mammals showed that NF-κB was also involved in peroxisome proliferator-activated receptors (PPARs)-mediated immune responses However, the mechanism by which NF-κB regulates PPARδ in teleosts remains unclear. In the present study, we analyzed the potential role of NF-κB/p65 (Ecp65) in the immune response stimulated by various pathogens in the grouper Epinephelus coioides. Ecp65 expression was significantly induced soon after infection with lipopolysaccharide, nervous necrosis virus, poly(I:C), and zymosan A. We also analyzed the promoter to determine the regulatory effect of Ecp65 on PPARδ expression, using progressive EcPPARδ promoter deletion mutations. Among the five truncated mutants, the luciferase reporter activity of the PPARδ-5 promoter region was highest in response to Ecp65, indicating that the core p65-binding region was located in the PPARδ-5 promoter region (+122 bp to +383 bp). Mutation analyses indicated that the luciferase reporter activity of the EcPPARδ promoter was dramatically decreased by mutation of the M3 (+305 bp to +324 bp) and M4 (+346 bp to +365 bp) binding sites, respectively. We further confirmed that Ecp65 bound to the M3 and M4 binding sites in the 5'-untranslated region of EcPPARδ by electrophoretic mobility shift assay. Finally, overexpression of Ecp65 in vitro notably promoted the transcription of EcPPARδ, interferon-related genes, and several inflammatory cytokines. This study demonstrated that Ecp65 plays an important role in modulating the innate immune responses in groupers. These results also further our understanding of the mechanisms involved in the transcriptional regulation of PPARs by p65 in bony fish.

The Kruppel-like factor 6 (KLF6) is a member of Kruppel-like factor family, which belong to the Zinc finger family of transcription factors that mediates various cellular processes, such as proliferation, differentiation, development, and programmed cell death. Peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily and they regulate numerous genes through ligand-dependent transcriptional activation and repression. In this study, we focus on the role of KLF6 gene in virus infection and the regulation of KLF6 on PPAR-δ in orange-spotted grouper (Epinephelus coioides). The ORF sequence of EcKLF6 was 846 bp, encoding a polypeptide of 282 amino acids with three conserved Zinc finger (type Cys2-His2) domain in the C-terminal region. Basing on the detection of the mRNA levels of viral genes, western blotting of MCP protein, and morphological CPEs, we found that the overexpression of EcKLF6 suppressed the replication of Singapore grouper iridovirus (SGIV), exerting its antiviral activity against fish virus. Moreover, promoter analysis was performed to investigate whether EcKLF6 was a regulator of EcPPAR-δ. The luciferase reporter assay and real time PCR results indicated a negative regulatory role of EcKLF6 on EcPPAR-δ transcription in grouper. Further experimental analysis shows that the potential EcKLF6 binding sites may locate in the EcPPAR-δ-4-M3 (+133 to +154) and EcPPAR-δ-4-M4 (+354 to +368) region of the EcPPAR-δ promoter. Electrophoretic mobile shift assays (EMSAs) verified that EcKLF6 interacted with the binding site of the EcPPAR-δ-4-M4 promoter region. In addition, we also found that KLF6 promotes inflammatory responses in GS cells. Considering that KLF6 and PPAR-δ play opposite roles in regulating inflammatory responses, we speculated the promoting effect of KLF6 on inflammatory response may be related to its negative regulation on EcPPAR-δ. In conclusion, the present study provides the first evidence of the negative regulation of EcPPAR-δ transcription by EcKLF6 and contributes to a better understanding of the transcriptional mechanisms of EcKLF6 in fish.

Lipid metabolism plays an important role in viral infections, and it can directly or indirectly affect various stages of viral infection in cells. As an important component of lipid metabolism, high-density lipoprotein (HDL) plays crucial roles in inflammation, immunity, and viral infections. Scavenger receptor B type 1 (SR-B1), a receptor of HDL, cannot be ignored in the regulation of lipid metabolism. Here, we investigate, for the first time, the role of Epinephelus coioides SR-B1 (Ec-SR-B1) in red-spotted grouper nervous necrosis virus (RGNNV) infection. Our results indicate that Ec-SR-B1 could promote RGNNV infection. We also demonstrate that Ec-SR-B1 could facilitate viral entry and interact with capsid protein (CP) of RGNNV. As the natural ligand of SR-B1, HDL significantly increased RGNNV entry in a dose-dependent manner. However, we observed no effect of HDL on Ec-SR-B1 expression. The results of the micro-scale thermophoresis assay did not reveal an association between HDL and CP, suggesting that RGNNV does not enter target cells by using HDL as a ligand to bind to its receptor. In addition, block lipid transport-1, a compound that inhibits HDL-mediated cholesterol transfer, reduced the HDL-induced enhancement of RGNNV infection, indicating a role for lipid transfer in facilitating RGNNV entry. Furthermore, HDL inhibited the expression of pro-inflammatory factors and antiviral genes in a dose-dependent manner. These findings suggest that the HDL-induced enhancement of RGNNV entry involves the complex interplay between Ec-SR-B1, HDL, and RGNNV, as well as the regulation of innate antiviral responses by HDL. In summary, we highlight the crucial role of HDL in RGNNV entry, identify a possible molecular connection between RGNNV and lipoprotein metabolism, and indicate the role of Ec-SR-B1 in RGNNV infection.

Beclin-1 is an essential autophagic regulator that plays diverse roles in physiology and disease. However, reports about the function of fish Beclin-1 during pathogen infection are still very limited. In this study, a Beclin-1 homolog (EcBeclin-1) from orange-spotted grouper (Epinephelus coioides) was identified and its roles in viral infection were investigated. EcBeclin-1 encoded 447amino acids protein with a BH3 domain, a CCD domain and an ECD domain, which shared high identities (97%–82%) with reported Beclin-1 proteins from mammal to fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBeclin-1 was predominantly expressed in brain and muscle of healthy grouper. Using fluorescence microscopy, we found that EcBeclin-1 was co-localized with endoplasmic reticulum (ER) in grouper spleen cells (EAGS). After red-spotted grouper nervous necrosis virus (RGNNV) infection in vitro, EcBeclin-1 transcript was significantly up-regulated, implying that EcBeclin-1 might be involved in viral infection. Furthermore, the in vitro studies of EcBeclin-1 overexpression promoted RGNNV induced autophagy, as well as the expression of coat protein (CP) and RNA-dependent RNA polymerase (RdRp). The overexpression of EcBeclin-1 suppressed the expressions of interferon pathway-related factors, inflammatory-related factors and activities of NF-κB and ISRE. Additionally, EcBeclin-1 could interact with EcBcl-xL in vitro. These data suggest that EcBeclin-1 affect viral replication through modulating IFN and inflammatory responses, as well as virus-induced cell death, which will help us to further explore the immune response of fish during viral infection.

Peroxisome proliferator-activated receptor δ (PPAR-δ), also called PPAR-β or PPAR-β/δ, is a member of the peroxisome proliferator-activated receptor (PPAR) family, which belongs to the nuclear steroid receptor superfamily. Activated PPARs participate in the regulation of lipid and glucose metabolism and also affect cellular proliferation, differentiation, and apoptosis, and the immune responses. To investigate the roles of PPAR-δ in Singapore grouper iridovirus (SGIV) infection, we cloned and characterized the gene encoding a PPAR-δ homologue from the orange-spotted grouper, Epinephelus coioides (EcPPAR-δ). EcPPAR-δ encodes a 514-amino-acid polypeptide, with 95.29% and 74.76% homologue to the Seriola dumerili and human proteins, respectively. EcPPAR-δ contains a typical DNA-binding domain and a ligand-binding domain. Its expression was induced by SGIV infection in vitro. A subcellular localization analysis showed that EcPPAR-δ localizes throughout the cytoplasm and nucleus, with a diffuse intracellular expression pattern. SGIV replication was reduced by EcPPAR-δ overexpression, which was evident in the reduced severity of the cytopathic effect, reduced viral gene transcription, and the reduced expression of the viral capsid protein. The replication of SGIV increased with the knockdown of EcPPAR-δ. The overexpression and silencing of EcPPAR-δ in grouper spleen cells showed that EcPPAR-δ plays a positive role in the regulation of the interferon signaling pathway, but has an anti-inflammatory effect on the inflammatory response. The anti-inflammatory effect of EcPPAR-δ may be related to its function in maintaining cell homeostasis. Because the interferon signaling pathway plays an important role in antiviral immune responses, we speculate that the activation of the interferon signaling pathway by EcPPAR-δ overexpression underlies its inhibitory effect on SGIV replication. Together, our data greatly extend our understanding of the roles of the EcPPAR-δ family members in the pathogenesis of fish viruses.

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.

Krϋppel-like factor 9 (KLF9) is a member of the SP/KL family, which are transcription factors implicated in several biological processes, including cell proliferation, differentiation, development and apoptosis. Studies have focused on the function of KLF9 in mammalian disease and the immune system, such as its regulatory role in the growth of tumors and its impact on interferon-related genes and inflammatory cytokines. In fish, little is known about the role of KLF9, especially its regulatory function in the innate antiviral immune response. In this study, we characterized the grouper KLF9 gene (EcKLF9) and investigated its role in viral infection. Amino acid alignment analysis showed that EcKLF9 was approximately 228 amino acids long and contained a typical three-tandem Krϋppel-like zinc fingers. Phylogenetic tree analysis revealed that EcKLF9 clustered with three fish species: Amphiprion ocellaris, Acanthochromis pollyacanthus and Stegastes partitus. Comparison analyses showed that the three Kruppel-like zinc finger domains of KLF9 were highly conserved in different fish species. Tissue expression analysis showed that EcKLF9 was constitutively expressed in all 12 tissues tested, in the healthy grouper, the highest expression being detected in the gonads. The relative expression levels of EcKLF9 in the head kidney, spleen and brain was significantly increased during red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infections. Using fluorescence microscopy, EcKLF9 was primarily localized to the nucleus and cytoplasm. The in vitro ectopic expression of EcKLF9 significantly increased the severity of vacuoles induced by RGNNV and the cytopathic effect progression evoked by SGIV infection. Real-time PCR results showed that the transcription levels of viral genes, such as the Singapore grouper iridovirus infection genes, MCP (major capsid protein), LITAF (lipopolysaccharide induced TNF-α factor), VP19 (envelop protein) ICP-18 (infected cell protein-18) and the red-spotted grouper nervous necrosis virus genes, CP (coat protein), RdRp (RNA-dependent RNA polymerase), were all significantly increased in EcKLF9 overexpressing cells, when compared to control cells. Furthermore, western blotting analyses showed that protein levels of the RGNNV gene, CP and the SGIV gene, MCP were also increased in EcKLF9 overexpressing cells, suggesting EcKLF9 may promote viral activity against iridovirus and nodavirus, in vitro. Moreover, the overexpression of EcKLF9 significantly inhibited the expression of several interferon related cytokines and several inflammatory cytokines. Accordingly, we speculate that EcKLF9 may exert stimulatory effects on RGNNV and SGIV replication, through the negative regulation of host immune and inflammation responses.

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.

Palmitic acid is the most common saturated fatty acid in animals, plants, and microorganisms. Studies highlighted that palmitic acid plays a significant role in diverse cellular processes and viral infections. Accumulation of palmitic acid was observed in fish cells (grouper spleen, GS) infected with Singapore grouper iridovirus (SGIV). The fluctuated content levels after viral infection suggested that palmitic acid was functional in virus-cell interactions. In order to investigate the roles of palmitic acid in SGIV infection, the effects of palmitic acid on SGIV induced cytopathic effect, expression levels of viral genes, viral proteins, as well as virus production were evaluated. The infection and replication of SGIV were increased after exogenous addition of palmitic acid but suppressed after knockdown of fatty acid synthase (FASN), of which the primary function was to catalyze palmitate synthesis. Besides, the promotion of virus replication was associated with the down-regulating of interferon-related molecules, and the reduction of IFN1 and ISRE promotor activities by palmitic acid. We also discovered that palmitic acid restricted TBK1, but not MDA5-induced interferon immune responses. On the other hand, palmitic acid decreased autophagy flux in GS cells via suppressing autophagic degradation, and subsequently enhanced viral replication. Together, our findings indicate that palmitic acid is not only a negative regulator of TBK1-IRF3/7 pathway, but also a suppressor of autophagic flux. Finally, palmitic acid promotes the replication of SGIV in fish cells.

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.

BNIP3 is a kind of BH3-only protein that induces both cell death and autophagy. Here, a BNIP3 gene (EcBNIP3) was identified from orange spotted grouper, Epinephelus coioides. EcBNIP3 possessed 236 amino acids residues, contained a conservative BNIP3 domain and a transmembrane region. Besides, EcBNIP3 expressed at a relative high level in heart and spleen. EcBNIP3 transcript was up-regulated after SGIV infection in vitro. Subcellular localization analysis revealed that EcBNIP3 was predominantly localized in the cytoplasm and co-localized with mitochondria. In addition, overexpression EcBNIP3 accelerated SGIV infection induced cell death but inhibited viral genes transcription. Taken together, these results provided new evidence that fish BNIP3 might involved in response to virus infection.

Autophagy is an important biological activity that maintains homeostasis in eukaryotic cells. However, little is known about the functions of fish autophagy-related genes (Atgs). In this study, we cloned and characterized Atg5, a key gene in the autophagy gene superfamily, from orange-spotted grouper (Epinephelus coioides) (EcAtg5). EcAtg5 encoded a 275-amino acid protein that shared 94 and 81% identity to seabass (Lates calcarifer) and humans (Homo sapiens), respectively. The transcription level of EcAtg5 was significantly increased in cells infected with red-spotted grouper nervous necrosis virus (RGNNV). In cells infected with Singapore grouper iridovirus (SGIV), EcAtg5 expression declined during the early stage of infection and increased in the late stage. Fluorescence microscopy revealed that EcAtg5 mainly localized with a dot-like pattern in the cytoplasm of grouper cells. Overexpression of EcAtg5 significantly increased the replication of RGNNV and SGIV at different levels of detection, as indicated by increased severity of the cytopathic effect, transcription levels of viral genes, and levels of viral proteins. Knockdown of EcAtg5 decreased the replication of RGNNV and SGIV. Further studies showed that overexpression EcAtg5 activated autophagy, decreased expression levels of interferon related cytokines or effectors and pro-inflammatory factors, and inhibited the activation of nuclear factor κB, IFN-sensitive response element, and IFNs. In addition, ectopic expression of EcAtg5 affected cell cycle progression by hindering the G1/S transition. Taken together, our results demonstrated that fish Atg5 exerted a crucial role in virus replication by promoting autophagy, down-regulating antiviral IFN responses, and affecting the cell cycle.

Nervous necrosis virus (NNV), is one of the most fatal viruses in marine fish aquaculture, and is capable of infecting over 50 different fish species. Trachinotus ovatus NNV (GTONNV) was isolated from diseased golden pompano. This T. ovatus strain was isolated from Guangxi, China. Single-stranded DNA (ssDNA) aptamers with high specificity for GTONNV-infected T. ovatus cerebellum cells (TOCC) were produced by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The characterization of these aptamers was performed using flow cytometry and laser scanning confocal microscopy. The selected aptamers showed significant specificity for GTONNV-infected cells. Based on MFOLD prediction, aptamers formed distinct stem-loop structures that could form the basis for the aptamers' specific binding to their cellular targets. Protease treatment results revealed that the target molecules for aptamers TNA1, TNA4 and TNA19 within GTONNV-infected cells may be membrane proteins that were trypsin-sensitive. Specific endocytosis of aptamer TNA1, TNA4 and TNA19 into GTONNV-infected cells was also shown. The selected aptamers demonstrated antiviral effects against GTONNV both in vitro and in vivo. This is the first time that aptamers targeting GTONNV-infected T. ovatus cells have been selected and characterized. These aptamers hold promise as rapid diagnostic reagents or targeted therapeutic drugs against GTONNV.

Golden pompano Trachinotus ovatus, a popularly cultured and commercially important marine fish worldwide, has been recognized as a promising candidate for mariculture. However, outbreaks of infectious bacterial or viral diseases and environmental deterioration have led to great economic losses in T. ovatus aquaculture recently. In our research, we established a new mid-kidney cell line, designated as TOK, from golden pompano, T. ovatus. The optimized growth temperature and working concentration of fetal bovine serum (FBS) were 28°C and 10-20%, respectively. Foreign genes could express well in TOK cells. The modal number of TOK cells was 54. The TOK cells were susceptive to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV), and the virus could propagate in cells. Propagation was verified by qRT-PCR, and virions were observed under electron microscopy. Cytotoxicity analysis revealed that TOK cells were sensitive to different concentrations of extracellular products (ECPs) from Vibrio alginolyticus and V. anguillarum. Moreover, heavy metals (Cd, Cu, and Hg) also showed dose-dependent cytotoxicity to the TOK cell line. We established a mid-kidney cell line from T. ovatus which could be applied to cytotoxicity assays of heavy metals. The newly established TOK cell line possesses great application potential in genetic manipulation, virus-host interaction studies, and toxicity assays of bacterial extracellular products and heavy metals.

Singapore grouper iridovirus (SGIV) is a devastating aquaculture virus responsible for heavy economic losses to grouper, Epinephelus sp. aquaculture. The aim of this study was to develop a rapid and sensitive detection method for SGIV infections in infected groupers.We previously generated DNA aptamers against SGIV-infected cells. In this study, we established and characterized a novel aptamer (Q3)-based enzyme-linked apta-sorbent assay (ELASA) for the detection of SGIV infection in Epinephelus coioides. The Q3-based ELASA could detect SGIV infection rapidly in vitro and in vivo, with high specificity and stability. Q3-based ELASA specifically recognized SGIV-infected cells, but not other-virus-infected cells or uninfected cells. Q3-based ELASA detected SGIV infection in a dose-dependent manner at Q3 concentrations as low as 125 nmol l(-1) . The results in relation to SGIV-infected cells (5 × 10(4) ), incubation time (1 min) and incubation temperature (37°C) demonstrated that Q3-based ELASA could detect SGIV infection quickly and stably, superior to antibody-based enzyme-linked immunosorbent assay. Q3-based ELASA could detect the presence of SGIV infection in kidney, liver and spleen samples in vivo, at dilutions of 1/50, 1/100 and 1/50 respectively. The complete detection process took 1-2 h.Q3-based ELASA could be a useful tool for diagnosing SGIV infection.This is the first developed aptamer-based ELASA for detecting SGIV infection, and is widely applicable in grouper aquaculture industry in light of its rapidity, and high specificity and stability.

Apoptosis plays vital roles in many physiological process and immune response. BOK is one of the central regulators in apoptosis. In this study, a new BOK homolog (Ec-BOK) was cloned and characterized from Orange-spotted grouper, Epinephelus coioides. Ec-BOK encoded a 210 amino acid peptides which shared 97% identity to Stegastes partitus BOK protein, contained four BH domains and one transmembrane region. Ec-BOK widely expressed in all analyzed tissues with the higher expressions in kidney and spleen. Its expression level was up-regulated after SGIV infection in vitro. Further analysis revealed that overexpression of Ec-BOK inhibited viral genes transcriptions and virus replication in fish cell. Our findings suggested that Ec-BOK might play a role in the immune response against virus.

Nervous necrosis virus (NNV) infected larvae and juveniles of more than 50 fish species, resulting in mortality rates of greater than 95%. However, there is no efficient method to control NNV infections. Aptamers generated by selective evolution of ligands by exponential enrichment (SELEX) are short, single-stranded nucleic acid oligomers. They display a high degree of affinity and specificity for many targets, such as viruses and viral proteins. In this study, three novel DNA aptamers (A5, A10, and B11) that specifically target the coat protein (CP) of grouper nervous necrosis virus (GNNV) were selected using SELEX. Secondary structures and minimum free energy (ΔG) predictions indicated that these aptamers could form stable, secondary stem-loop structures. Electrophoretic mobility shift assays, enzyme-linked immunosorbent assays, Kd measurements, the co-localization of tetramethylrhodamine (TAMRA) labeled-aptamers with the CP and flow cytometry analysis revealed that these aptamers could specifically bind the CP with high (nanomolar) affinities. In addition, competition analysis suggested the aptamers shared some common CP binding sites with the anti-CP antibody. Moreover, all three aptamers did not show any cytotoxic effects in vitro or in vivo, and anti-viral analysis indicated the selected aptamers could inhibit NNV infection in vitro and in vivo. Compared with controls, mortality of GNNV-infected fish decreased by 40% and 80% after 10 days infection, when the GNNV was pre-incubated with the 1000 nM A10 and B11, respectively. TAMRA-labeled aptamers could bind to NNV virions and directly enter NNV-infected cells, suggesting they could be used as tracers to study the mechanism of viral infection, as well as for targeted therapy. This is the first time that aptamers targeting a viral protein of marine fish have been generated and characterized. These aptamers hold promise as diagnostic, therapeutic, and targeted drug delivery agents for controlling NNV infections.

Tripartite motif-containing 32 (TRIM32) has been demonstrated to pay vital roles in cancer, genetic disorders and antiviral immunity. However, the molecular functions of fish TRIM32 still remained largely unknown. Here, a novel TRIM32 gene from orange spotted grouper (EcTRIM32) was cloned and characterized. EcTRIM32 encoded a 685-aa protein which showed 93%, and 60% identity to large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Amino acid alignment showed that EcTRIM32 contained a conserved RING-finger domain, a BBOX domain and NHL domain. In healthy grouper, the transcript of EcTRIM32 was predominantly detected in brain, liver, intestine, spleen and skin. After injection with Singapore grouper iridovirus (SGIV) and polyI:C, the relative expression of EcTRIM32 in grouper spleen was differently regulated, suggested that EcTRIM32 was involved in antiviral immune response. In transfected grouper spleen (GS) cells, EcTRIM32 displayed bright fluorescence aggregates or spots in the cytoplasm. Notably, the deletion RING domain altered its precise localization and distributed throughout the cytoplasm in GS cells. In EcTRIM32 overexpressing cells, the replication of SGIV or red-spotted grouper nervous necrosis virus (RGNNV) was significantly inhibited compared to the vector control cells. Moreover, the overexpression of EcTRIM32 positively regulated the interferon immune response, evidenced by the significant increase of the expression level of interferon related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon-stimulated gene 15 (ISG15), interferon-induced 35-kDa protein (IFP35), MXI, TIR-domain-containing adaptor-inducing interferon-β (TRIF) and melanoma differentiation-associated protein 5 (MDA5). Further studies showed that overexpression of EcTRIM32 significantly enhanced the MDA5-mediated interferon immune response, but decreased stimulator of interferon genes (STING)-mediated interferon immune response. Meanwhile, the expression levels of pro-inflammation cytokines, including TNFα, IL-6 and IL-8 were up-regulated by the ectopic expression of EcTRIM32. We speculated that the regulation of IRF7, and pro-inflammation cytokines by EcTRIM32 overexpression might contribute critical roles in SGIV infection. In addition, the deletion of RING domain not only significantly weakened the antiviral roles of EcTRIM32, but also obviously affected the regulatory effects of EcTRIM32 on interferon immune and inflammation response. Together, our results firstly demonstrated that fish TRIM32 acted as an antiviral factor against both DNA and RNA virus infection.

Bcl-2 family proteins play essential roles in modulating immune response and controlling cells' fate. Bcl-xL is one of anti-apoptotic protein in this family. In this study, a new Bcl-xL homolog (EcBcl-xL) was identified and characterized from orange-spotted grouper, Epinephelus coioides. EcBcl-xL encoded a 221 amino acid peptides that shared 86% identity to Larimichthys crocea Bcl-xL protein, contained four conserved BH domains and one transmembrane region. The predicted three-dimensional structure of EcBcl-xL was similar with Homo sapiens Bcl-xL. EcBcl-xL widely expressed in all tested tissues with highest expression in head kidney. Its expression level was significantly up-regulated after SGIV infection in vivo. Furthermore, overexpression of EcBcl-xL could inhibit SGIV-induced nonapoptotic cell death and suppressed viral genes transcriptions in GS cells. Our findings suggested that EcBcl-xL might play a role during virus infection through modulating SGIV-induced nonapoptotic cell death.

The lysosomal cysteine protease cathepsin C plays a pivotal role in regulation of inflammatory and immune responses. However, the function of fish cathepsin C in virus replication remains largely unknown. In this study, cathepsin C gene (Ec-CC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CC cDNA was composed of 2077 bp. It contained an open reading frame (ORF) of 1374 bp and encoded a 458-amino acid protein which shared 89% identity to cathepsin C from bicolor damselfish (Stegastes partitus). Amino acid alignment analysis showed that Ec-CC contained an N-terminal signal peptide, the propeptide region and the mature peptide. RT-PCR analysis showed that Ec-CC transcript was expressed in all the examined tissues which abundant in spleen and head kidney. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the relative expression of EC-CC was significantly increased at 24 h post-infection. Subcellular localization analysis revealed that Ec-CC was distributed mainly in the cytoplasm. Further studies showed that overexpression of Ec-CC in vitro significantly delayed the cytopathic effect (CPE) progression evoked by SGIV and inhibited the viral genes transcription. Moreover, overexpression of Ec-CC significantly increased the expression of proinflammatory cytokines during SGIV infection. Taken together, our results demonstrated that Ec-CC might play a functional role in SGIV infection by regulating the inflammation response.

Bax inhibitor-1 (BI-1) is a conserved anti-apoptotic protein that suppresses endoplasmic reticulum (ER) stress-induced cell death. However, the function of fish BI-1 is not quite clear. In the present study, a bi-1 homolog (Ecbi-1) from orange spotted grouper, Epinephelus coioides was identified and its roles in viral infection were investigated. EcBI-1 encoded 237 amino acids protein, contained six transmembrane regions and a conservative C-terminus motif. Ecbi-1 predominantly expressed in kidney and spleen of healthy grouper. After SGIV stimulation, Ecbi-1 transcript was significantly increased in vitro. Subcellular localization analysis revealed that EcBI-1 was localized throughout the cytoplasm and co-localized with ER. Furthermore, overexpression of EcBI-1 suppressed SGIV infection induced cell death, caspase-3 activity and viral genes transcription. And C-terminus motif was critical for regulation roles of EcBI-1 during SGIV infection. In addition, EcBI-1 could interact with EcBNIP3 in vitro. Together, our data firstly demonstrated that fish BI-1 play important roles in response to viral infection.

Singapore grouper iridovirus (SGIV) is a major viral pathogen of grouper aquaculture, and has caused heavy economic losses in China and South-east Asia. In this study, we generated four ssDNA aptamers against SGIV-infected grouper spleen (GS) cells using SELEX (systematic evolution of ligands by exponential enrichment) technology. Four aptamers exhibited high affinity to SGIV-infected GS cells, in particular the Q2 aptamer. Q2 had a binding affinity of 12.09 nM, the highest of the four aptamers. These aptamers also recognized SGIV-infected tissues with high levels of specificity. Protease treatment and flow cytometry analysis of SGIV-infected cells revealed that the target molecules of the Q3, Q4 and Q5 aptamers were trypsin-sensitive proteins, whilst the target molecules of Q2 might be membrane lipids or surface proteins that were not trypsin-sensitive. The generated aptamers appeared to inhibit SGIV infection in vitro. Aptamer Q2 conferred the highest levels of protection against SGIV and was able to inhibit SGIV infection in a dose-dependent manner. In addition, Q2 was efficiently internalized by SGIV-infected GS cells and localized at the viral assembly sites. Our results demonstrated that the four novel aptamers we generated were specific for SGIV-infected cells and could potentially be applied as rapid molecular diagnostic test reagents or therapeutic drugs targeting SGIV.

Apolipoproteins, synthesized mainly in liver and intestine and bounded to lipids, play important roles in lipid transport and uptake through the circulation system. In this study, an apolipoprotein A-I gene homologue was cloned from orange-spotted grouper Epinephelus coioides (designed as Ec-ApoA-I) by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-ApoA-I was comprised of 1278 bp with a 792 bp open reading frame (ORF) that encodes a putative protein of 264 amino acids. Quantitative real-time PCR (qPCR) analysis revealed that Ec-ApoA-I was abundant in liver and intestine, and the expression in liver was significantly (P

Interferon regulatory factor 3 (IRF3) is an important transcription factor which regulates the expression of interferon (IFN) and IFN-stimulated genes (ISGs) following virus recognition. In this study, a novel IRF3 gene was cloned from grouper Epinephelus coioides (EcIRF3) and its effects against Singapore grouper iridovirus (SGIV) and red spotted grouper nervous necrosis virus (RGNNV) was investigated. The full-length of EcIRF3 cDNA was composed of 2513 bp and encoded a polypeptide of 458 amino acids which shared 82% identity with European seabass (Dicentrarchus labrax). EcIRF3 contained three conserved domains including a DNA-binding domain (DBD), an IRF associated domain (IAD) and a serine-rich domain. Expression profile analysis revealed that EcIRF3 was abundant in head kidney, kidney, spleen and gill. Upon different stimuli in vitro, the transcript of EcIRF3 was significantly up-regulated after RGNNV infection or treatment with polyinosin-polycytidylic acid (poly I:C). During SGIV infection, the increase of the EcIRF3 transcription was only detected at the late stage, suggesting that EcIRF3 was differently regulated by different stimuli. Immune fluorescence assay indicated that the fluorescence signal of EcIRF3 was increased significantly after infection with RGNNV or treatment with poly I:C, but moderately at the late stage of SGIV infection. Reporter gene assay showed that EcIRF3 activated zebrafish type I IFN and type III IFN promoter in vitro. The viral gene transcription and virus production of RGNNV were significantly decreased in EcIRF3 overexpressing cells. However, the ectopic expression of EcIRF3 did not affect the gene transcription and virus production of SGIV. Moreover, the mRNA expression levels of type I IFN and IFN-inducible genes (MxI, ISG15 and ISG56) were increased in RGNNV infected EcIRF3 overexpressing cells compared to empty vector transfected cells. Together, our results demonstrated that IFN immune response mediated by grouper IRF3 was exerted crucial roles for fish RNA virus, but not for DNA virus replication.

As the major viral pathogen of grouper aquaculture, Singapore grouper iridovirus (SGIV) has caused great economic losses in China and Southeast Asia. In the previous study, we have generated highly specific ssDNA aptamers against SGIV-infected grouper spleen cells (GS) by Systematic Evolution of Ligands by Exponential Enrichment technology (SELEX), in which Q2 had the highest binding affinity of 16.43 nM. In this study, we would try to identify the specific sequences in the aptamer Q2 that exhibited the high binding affinity to SGIV-infected cells by truncating the original Q2 into some different specific segments. We first evaluated the specificity and binding affinity of these truncated aptamers to SGIV-infected cells by flow cytometry, fluorescent imaging of cells and aptamer-based enzyme-linked apta-sorbent assay (ELASA). We then performed cytotoxicity analysis, assessment of the inhibitory effects upon SGIV infection and the celluar internalization kinetics of each truncated aptamer. Compared to the initial Q2, one of the truncated aptamer Q2-C5 showed a 3-fold increase in the binding affinity for SGIV-infected cells, and held more effective inhibitory effects, higher internalization kinetics and stability. Hence, the aptamer’s truncated methods could be applied in the research of identifying aptamer’s key sequences. The shorter, structure optimizing aptamer showed more excellent performance over the originally selected aptamer, which could potentially be applied in developing commercial detection probes for the early and rapid diagnosis of SGIV infection, and highly specific therapeutic drugs against SGIV infection.

MicroRNA-146a (miR-146a) has been demonstrated to function as a negative regulator of cellular immune responses against pathogens in mammals, however, little information focused on its functions in lower vertebrates. In this study, we investigated the regulatory roles of orange spotted grouper, Epinephelus coioides miR-146a during red spotted grouper nervous necrosis virus (RGNNV) infection. During RGNNV infection in grouper spleen (GS) cells, the endogenous expression level of miR-146a and tumor necrosis factor receptor-associated factor 6 (TRAF6) significantly increased along with the infection time. Overexpression of miR-146a significantly facilitated viral infection, evidenced by the increased transcription of viral CP and RdRp genes, while miR-146a knockdown by specific inhibitors decreased RGNNV replication. Using pMIR-REPORT Luciferase system, we found that the 3' untranslated region (UTR) of grouper TRAF6 could be specifically targeted by miR-146a. Further studies showed that its downstream target gene pro-inflammatory cytokines, including TNF-α, IL-8 and IL-1β, were all significantly decreased in miR-146a mimic transfected cells, but increased in miR-146a inhibitors transfected cells during RGNNV infection. Thus, our results suggested and verified that holding the level of miR-146a exerted crucial roles in RGNNV infection through TRAF6-mediated inflammatory response.

Redspotted grouper nervous necrosis virus (RGNNV) is one of the most devastating pathogens in the aquaculture of the grouper, Epinephlus sp., worldwide. The early and rapid diagnosis of RGNNV is important for the prevention of RGNNV infection. In this study, an aptamer (A10)-based sandwich enzyme-linked apta-sorbent assay (ELASA) was developed for RGNNV diagnosis. This sandwich ELASA showed high specificity for the RGNNV coat protein (CP) and virions in virus-infected cells and tissues. At the optimized working concentration of 200 nM of aptamer, the ELASA could detect RGNNV in the lysates of as few as 4 × 103 RGNNV-infected GB cells. Incubation for 10 min was sufficient to produce accurate results. The sandwich ELASA was most stable at incubation temperatures of 4-25°C, but could still distinguish RGNNV-infected samples from the controls at 37°C. It could detect RGNNV infection in brain lysates diluted 1/10, with results consistent with those of reverse transcription PCR, although with 10% less sensitivity. The main equipment required includes dissection tools, a water bath, Pierce™ Streptavidin Coated Plates and a microplate reader. The sandwich ELASA has great potential utility for the rapid and sensitive diagnosis of RGNNV in its early stages by fish farmers.

The Singapore grouper iridovirus (SGIV), a member of the genus Ranavirus, is a major viral pathogen that has caused heavy economic losses to the grouper aquaculture industry in China and Southeast Asia. No efficient method of controlling SGIV outbreaks is currently available. Systematic evolution of ligands by exponential enrichment (SELEX) is now widely used for the in vitro selection of artificial ssDNA or RNA ligands, known as aptamers, which bind to targets through their stable three-dimensional structures. In our current study, we generated ssDNA aptamers against the SGIV, and evaluated their ability to block SGIV infection in cultured fish cells and cultured fish in vivo. The anti-SGIV DNA aptamers, LMB-761, LMB-764, LMB-748, LMB-439, LMB-755, and LMB-767, were selected from a pool of oligonucleotides randomly generated using a SELEX iterative method. The analysis of the secondary structure of the aptamers revealed that they all formed similar stem-loop structures. Electrophoretic mobility shift assays showed that the aptamers bound SGIV specifically, as evidenced by a lack cross-reactivity with the soft shell turtle iridovirus. The aptamers produced no cytotoxic effects in cultured grouper spleen cells (GS). Assessment of cytopathic effects (CPE) and viral titer assays showed that LMB-761, LMB-764, LMB-748, LMB-755, and LMB-767 significantly inhibited SGIV infection in GS cells. The in vivo experiments showed that LMB-761 and LMB-764 reduced SGIV-related mortality, and no negative effects were observed in orange-spotted grouper, Epinephelus coioides, indicating that these DNA aptamers may be suitable antiviral candidates for controlling SGIV infections in fish reared in marine aquaculture facilities.

Grouper Epinephelus spp. is one of the most important mariculture fish species in China and South-East Asian countries. The emerging viral diseases, evoked by iridovirus which belongs to genus Megalocytivirus and Ranavirus, have been well characterized in recent years. To date, few data on lymphocystis disease in grouper which caused by lymphocystis disease virus (LCDV) were described. Here, a novel LCDV isolate was identified and characterized. Based on the sequence of LCDV major capsid protein (MCP) and DNA polymerase gene, we found that the causative agents from different species of diseased groupers were the same one and herein were uniformly defined as grouper LCDV (GLCDV). Furthermore, H&E staining revealed that the nodules on the skin were composed of giant cells that contained inclusion bodies in the cytoplasm. Numerous virus particles with >210 nm in diameter and with hexagonal profiles were observed in the cytoplasm. In addition, phylogenetic analysis based on four iridovirus core genes, MCP, DNA polymerase, myristoylated membrane protein (MMP) and ribonucleotide reductase (RNR), consistently showed that GLCDV was mostly related to LCDV-C, followed by LCDV-1. Taken together, our data firstly provided the molecular evidence that GLCDV was a novel emerging iridovirus pathogen in grouper culture.

Vaccination is one of the best methods against viral diseases. In this study, experimental inactivated Singapore grouper iridovirus (SGIV) vaccines were prepared, and immunogenicity and protection against virus infection of the vaccines were investigated in orange-spotted grouper, Epinephelus coioides. Two kinds of vaccines, including,beta-propiolactone (BPL) inactivated virus at 4 degrees C for 12 h and formalin inactivated virus at 4 degrees C for 12 d, was highly protective against the challenge at 30-day post-vaccination and produced relative percent of survival rates of 91.7% and 100%, respectively. These effective vaccinations induced potent innate immune responses mediated by pro-inflammatory cytokines and type! interferon (IFN)-stimulated genes (ISGs). It is noteworthy that ISGs, such as Mx and ISG15, were up-regulated only in the effective vaccine groups, which suggested that type 1 IFN system may be the functional basis of early anti-viral immunity. Moreover, effective vaccination also significantly up-regulated of the expression of MHC class I gene and produced substantial amount of specific serum antibody at 4 weeks post-vaccination. Taken together, our results clearly demonstrated that effective vaccination in grouper induced an early, nonspecific antiviral immunity, and later, a specific immune response involving both humoral and cell-mediated immunity. (C) 2012 Elsevier Ltd. All rights reserved.

It has been demonstrated that tumour necrosis factor receptor (TNFR) homologues encoded by viruses are usually involved in virus immune evasion by regulating the host immune response or mediating apoptotic cell death. Here, a novel TNFR-like protein encoded by Singapore grouper iridovirus (SGIV VP51) was cloned and characterized. Amino acid analysis showed that VP51 contained three cysteine-rich domains (CRDs) and a transmembrane domain at its C terminus. The expression of VP51 in vitro enhanced cell proliferation, and affected cell cycle progression via altering the G1/S transition. Furthermore, VP51 overexpression improved cell viability during SGIV infection via inhibiting virus-induced apoptosis, evidenced by the reduction of apoptotic bodies and the decrease of caspase-3 activation. In addition, overexpression of VP51 increased viral titre and the expression of viral structural protein gene MCP and cell proliferation promoting gene ICP-18. In contrast, the expression of the viral apoptosis inducing gene, LITAF, was significantly decreased. Although all three CRDs were essential for the action of VP51, CRD2 and CRD3 exerted more crucial roles on virus-induced apoptosis, viral gene transcription and virus production, while CRD1 was more crucial for cell proliferation. Together, SGIV TNFR-like products not only affected cell cycle progression and enhanced cell growth by increasing the expression of the virus encoded cell proliferation gene, but also inhibited virus-induced apoptotic cell death by decreasing the expression of the viral apoptosis inducing gene. Our results provided new insights into understanding the underlying mechanism by which iridovirus regulated the apoptotic pathway to complete its life cycle.

As a member of animal lectin family, galectin has the functions of pathogen recognition, anti-bacteria and anti-virus. In the present study, a galectin-1 homolog (EcGel-1) from grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcGel-1 is 504 bp, including a 408 bp open reading frame (ORF) which encodes 135 amino acids with a molecular mass of 15.19 kDa. Quantitative real-time PCR analysis indicated that EcGel-1 was constitutively expressed in all analyzed tissues of healthy grouper. The expression of EcGel-1 in the spleen of grouper was differentially up-regulated challenged with Singapore grouper iridovirus (SGIV), poly (I:C), and LPS. EcGel-1 was abundantly distributed in the cytoplasm in GS cells. Recombinant EcGel-1(rEcGel-1) protein can make chicken erythrocyte aggregation, and combine with gram negative bacteria and gram positive bacteria in the presence of 2-Mercaptoethanol (β-ME). Taken together, the results showed that EcGel-1 may be an important molecule involved in pathogen recognition and pathogen elimination in the innate immunity of grouper.

Toll-interacting protein (Tollip) is one of the important regulatory proteins of Toll-like receptor (TLR) signaling pathways. In previous studies, a Tollip sequence of grouper (Epinephelus coioides) was identified and the signal transduction functions of Tollip were studied. However, the response of Tollip to virus infection has not been characterized from grouper. In the present paper, the Tollip homolog (EtTollip) from grouper (Epinephelus tauvina) was cloned and its immune response to Singapore grouper iridovirus (SGIV) was investigated. EtTollip shares significant similarities to other mammalian Tollips, which contain a centrally localized protein kinase C conserved region 2 (C2) domain and a C-terminal CUE domain. After challenging with SGIV, the expression levels of EtTollip were altered in the spleen and head kidney of grouper. EtTollip mainly aggregated in the cytoplasm in a condensed state and was also distributed on the membranes of GS cells. EtTollip significantly inhibited the activities of NF-κB and IFN-β luciferase reporter when transfected into grouper spleen (GS) cells. SGIV can increase the activities of NF-κB and IFN-β luciferase reporter, especially to IFN-β. When transfected EtTollip with EcMyd88, the activity of NF-κB was increased, while transfected EtTollip with EcIRF3, the activity of IFN-β was significantly increased. Over-expressed EtTollip inhibited the transcription of SGIV genes significantly in GS cells, and silencing of EtTollip with siRNA led to increase of SGIV genes loads. Taken together, the results provide new insights in to the importance of Tollip as evolutionarily conserved molecule for grouper innate immunity against virus infection.

Stimulator of interferon genes (STING, also known as MITA, ERIS, MPYS or TMEM173) has been identified as a central component in the innate immune response to cytosolic DNA and RNA derived from different pathogens. However, the detailed role of STING during fish iridovirus infection still remained largely unknown. Here, the STING homolog from grouper Epinephelus coioides (EcSTING) was cloned and its effects on IFN response and antiviral activity were investigated. The full-length EcSTING cDNA was composed of 1590 bp and encoded a polypeptide of 409 amino acids with 80% identity to STING homolog from large yellow croaker. Amino acid alignment analysis indicated that EcSTING contained 4 predicated transmembrane motifs (TMs) in the N terminal, and a C-terminal domain (CTD) which consisted of a dimerization domain (DD), c-di-GMP-binding domain (CBD) and a C-terminal tail (CTT). Expression profile analysis revealed that EcSTING was abundant in gill, spleen, brain, skin, and liver. Upon different stimuli in vivo, the EcSTING transcript was dramatically up-regulated after challenging with Singapore grouper iridovirus (SGIV), lipopolysaccharide (LPS) and polyinosin-polycytidylic acid (poly I:C). Reporter gene assay showed that EcSTING activated ISRE, zebrafish type I IFN and type III IFN promoter in vitro. Mutant analysis showed that IFN promoter activity was mostly mediated by the phosphorylation sites at serine residue S379 and S387. Moreover, EcSTING induced type I and III IFN promoter activity could be impaired by overexpression of EcIRF3-DN or EcIRF7-DN, suggesting that EcSTING mediated IFN response in IRF3/IRF7 dependent manner. In addition, the cytopathic effect (CPE) progression of SGIV infection and viral protein synthesis was significantly inhibited by overexpression of EcSTING, and the inhibitory effect was abolished in serine residue S379 and S387 mutant transfected cells. Together, our results demonstrated that EcSTING might be an important regulator of grouper innate immune response against iridovirus infection.

TTRAP (TRAF and TNF receptor-associated protein) is latest identified cytosolic protein that serves as a negative regulator for TNF signaling pathway. In this study, a member of TNF superfamily, TTRAP gene (designed as EcTTRAP) was cloned from grouper, Epinephelus coioides. There was an Exo_endo_phos type domain in EcTTRAP, and it was well conserved when compared with other TTRAPs, especially the endonuclease activity related motifs. EcTTRAP exhibited prominent endonuclease activity against the genome DNA from Escherichia coli, Vibrio vulnificus and E. coli JM109. Intracellular localization revealed that EcTTRAP expression distributed in both cytoplasm and nucleus. Real-time PCR analysis indicates that EcTTRAP is expressed in all selective grouper tissues, with the higher expression level in muscle, skin and gills. EcTTRAP was identified as a remarkably (P < 0.01) up-regulated protein responding to Singapore grouper iridovirus (SGIV) infection. Overexpression of EcTTRAP inhibited NF-κB activation, meanwhile the C terminal portion of the protein was found to be responsive domain for the inhibition. Stable transfection of FHM cells with EcTTRAP inhibited apoptosis induced by SGIV. Overexpression of EcTTRAP in grouper spleen (GS) cells inhibited the replication of SGIV. The present results provided new evidences for the potential roles of such molecule in E. coioides, and further confirmed the existence of TTRAP modulated TNF signaling pathway in grouper.

Singapore grouper iridovirus (SGIV) encodes a number of microRNAs (miRNAs) during infection. Among these, SGIV-miR-13 has robust expression at early stage after SGIV inoculation, raising a huge possibility that it participates in the viral infection. In the present study, we found that SGIV-miR-13 overexpression led to a significant reduction in viral load in cultured fish cells with SGIV infection, as demonstrated by less level of viral transcripts, viral-induced cytopathic effect (CPE) and assembled viral particles. In silico analysis showed that SGIV-miR-13 maps antisense to the coding region of SGIV major capsid protein (SGIV-MCP), suggesting it to be a potential target of SGIV-miR-13. Coincidently, SGIV-miR-13 showed an inverted expression profile with SGIV-MCP during SGIV infection, and luciferase reporter assay further demonstrated SGIV-MCP as the direct target of SGIV-miR-13. Functionally, overexpression of SGIV-miR-13 inhibited, whereas knockdown of SGIV-miR-13 restored the expression of SGIV-MCP during viral infection, resulting in altered viral progeny emergences. In conclusion, our data suggest that SGIV-miR-13 functions in a negative regulatory mechanism to restrict early viral replication, and thus prevents excessive cellular antiviral responses during SGIV infection. The detailed investigation of SGIV encoded miRNAs may provide new insights into the mechanism of iridovirus pathogenesis.

The nuclear phosphoprotein c-Jun is a member of the AP1 family of transcription activating complex, can be induced by various extracellular stimuli such as virus infection. In this study, the c-Jun gene (Ec-c-Jun) was cloned from orange-spotted grouper, Epinephelus coioides. The full-length Ec-c-Jun cDNA is composed of 2046 bp and encodes a polypeptide of 328 amino acids with 81% identity of zebrafish. Amino acid alignment analysis indicated that Ec-c-Jun contained three conserved domains including a transactivation domain (TAD), a DNA-binding domain (DBD) and leucine zipper domain (LZD). RT-PCR results showed that Ec-c-Jun transcript was most abundant in spleen, kidney, heart and gill. The expression of Ec-c-Jun was up-regulated after challenged with Singapore grouper iridovirus (SGIV). To investigate the roles of Ec-c-Jun during SGIV infection, we constructed its dominant-negative mutant (DN-Ec-c-Jun) by deleting the major TAD that lacks amino acids 3-122. Fluorescence microscopy observation revealed that Ec-c-Jun and DN-Ec-c-Jun were expressed predominantly in the nucleus in transfected cells. Interestingly, the green fluorescence of Ec-c-Jun was congregated and co-localized with virus assembly sites at the late stage of SGIV infection. However, in DN-Ec-c-Jun transfected cells, no virus assembly sites were observed, and the distribution of fluorescence remained unchanged. Moreover, overexpression of DN-Ec-c-Jun in vitro delayed the occurrence of CPE induced by SGIV infection and inhibited the virus gene transcription. In addition, ectopic expression of DN-Ec-c-Jun was able to inhibit SGIV induced c-Jun/AP1 promoter activity in GS cells. Thus, we proposed that c-Jun transcription factor was essential for SGIV replication in vitro. Our results will contribute to understanding the crucial roles of JNK signaling pathway in fish virus infection.

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor which plays crucial roles in immune regulation, inflammation, cell proliferation, transformation, and other physiological processes of the organism. In this study, a novel STAT3 gene from orange spotted grouper (Ec-STAT3) was cloned and characterized. Bioinformatic analysis revealed that full-length of Ec-STAT3 was 3105-bp long and contained a 280-bp 5′UTR, a 470-bp 3′UTR, and a 2355-bp open reading frame (ORF) that encoded a 784-amino acid peptide. The deduced protein of Ec-STAT3 showed 98% identity to that of turbot (Scophthalmus maximus). Amino acid alignment showed that Ec-STAT3 contained four conserved domains, including a protein interaction domain, a coiled coil domain, a DNA binding domain, and an SH2 domain. Quantitative real-time PCR analysis showed that the highest expression level was detected in the liver, followed by skin and spleen. After injection with Singapore grouper iridovirus (SGIV), the transcript of Ec-STAT3 in spleen was increased significantly. To further explore the function of Ec-STAT3, we investigated the roles of Ec-STAT3 in SGIV infection in vitro. Immune fluorescence analysis indicated that SGIV infection altered the distribution of phosphorylated Ec-STAT3 in nucleus, and a small part of phosphorylated Ec-STAT3 was associated with virus assembly sites, suggesting that Ec-STAT3 might be important for SGIV infection. Using STAT3 specific inhibitor, S3I-201, we found that inhibition of Ec-STAT3 activation decreased the SGIV replication significantly. Moreover, inhibition of Ec-STAT3 activation obviously altered SGIV infection induced cell cycle arrest and the expression of pro-survival genes, including Bcl-2, Bcl-xL and Bax inhibitor. Together, our results firstly demonstrated the critical roles of fish STAT3 in DNA virus replication and virus induced paraptosis, but also provided new insights into the mechanism of iridovirus pathogenesis.

The lysosomal cysteine protease cathepsin B of papain family is a key regulator and signaling molecule that involves in various biological processes, such as the regulation of apoptosis and activation of virus. In the present study, cathepsin B gene (Ec-CB) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CB cDNA was composed of 1918 bp and encoded a polypeptide of 330 amino acids with higher identities to cathepsin B of teleosts and mammalians. Ec-CB possessed typical cathepsin B structural features including an N-terminal signal peptide, the propeptide region and the cysteine protease domain which were conserved in other cathepsin B sequences. Phylogenetic analysis revealed that Ec-CB was most closely related to Lutjanus argentimaculatus. RT-PCR analysis showed that Ec-CB transcript was expressed in all the examined tissues which abundant in spleen, kidney and gill. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the mRNA expression of cathepsin B in E. coioides was up-regulated at 24 h post-infection. Subcellular localization analysis revealed that Ec-CB was distributed predominantly in the cytoplasm. When the fish cells (GS or FHM) were treated with the cathepsin B specific inhibitor CA-074Me, the occurrence of CPE induced by SGIV was delayed, and the viral gene transcription was significantly inhibited. Additionally, SGIV-induced typical apoptosis was also inhibited by CA-074Me in FHM cells. Taken together, our results demonstrated that the Ec-CB might play a functional role in SGIV infection.

Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. During the virus-host co-evolution, host Rab7 was also exploited by virus to complete their life cycle. To date, however, the roles of fish Rab7 in virus infection remained largely unknown. Here, we cloned and characterized a Rab7 gene from grouper, Epinephelus coioides (Ec-Rab7). The full-length Ec-Rab7 cDNA was composed of 1182 bp and encoded a polypeptide of 207 amino acids which shared 99% identity with that from Anoplopoma fimbria or Oreochromis niloticus. Ec-Rab7 contained five conserved domains of Rab GTPase family including GTP-binding or GTPase regions as well as an effector site. RT-PCR analysis revealed that Ec-Rab7 ubiquitously expressed in all detected tissues and its transcript in spleen was up-regulated after challenge with Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-Rab7 was distributed in the cytoplasm as spots and mostly colocalized with lysosomes. Notably, the ectopic expressed Ec-Rab7 partly aggregated into the viral factories in cells infected by SGIV. Furthermore, overexpression of Ec-Rab7 accelerated the occurrence of cytopathic effect (CPE) induced by SGIV infection and promoted viral gene transcription. In addition, far western blotting assay revealed that Ec-Rab7 might interact with viral proteins, including SGIV VP69 and VP101. Taken together, our data suggested that Ec-Rab7 might be potentially involved in SGIV replication. (C) 2013 Elsevier Ltd. All rights reserved.

p38 mitogen-activated protein kinases (MAPKs) are broadly expressed signaling molecules that involves in the regulation of cellular responsible for various extracellular stimuli. In this study, three p38 MAPK genes (Ec-p38a, p38b and p38β) were cloned from grouper, Epinephelus coioides and their characteristics were investigated in vitro. Although Ec-p38a, p38b and p38β showed high homologies to other fish p38a MPAK, p38b MAPK and p38β MAPK, respectively, they all contained the conserved structures of Thr-Gly-Tyr (TGY) motif and substrate binding site Ala-Thr-Arg-Trp (ATRW). Phylogenetic analysis indicated that Ec-p38a, p38b and p38β are more closely related to those from fish than mammals. The tissue distribution patterns of Ec-p38a, p38b and p38β were different, and Ec-p38β was up-regulated most obviously in head kidney after Singapore grouper iridovirus (SGIV) infection. Overexpression of Ec-p38β in FHM cells delayed the occurrence of CPE induced by SGIV infection. Further analysis indicated that overexpression of Ec-p38β inhibited viral gene transcription and protein synthesis, as well as SGIV induced typical apoptosis in fish cells. Taken together, our data indicated that Ec-p38β played a crucial role in regulating apoptosis and virus replication during iridovirus infection.