In Vitro Antiviral Activity of Eugenol on Singapore Grouper Iridovirus.

The high mortality rate of Singapore grouper iridovirus (SGIV) posing a serious threat to the grouper aquaculture industry and causing significant economic losses. Therefore, finding effective drugs against SGIV is of great significance. Eugenol (C10H12O2) is a phenolic aromatic compound, has been widely studied for its anti-inflammatory, antioxidant and antiviral capacity. In this study, we explored the effect of eugenol on SGIV infection and its possible mechanisms using grouper spleen cells (GS) as an in vitro model. We found that treatment of GS cells with 100 µM eugenol for 4 h exhibited the optimal inhibitory effect on SGIV. Eugenol was able to reduce the expression level of inflammatory factors by inhibiting the activation of MAPK pathway and also inhibited the activity of NF-κB and AP-1 promoter. On the other hand, eugenol attenuated cellular oxidative stress by reducing intracellular ROS and promoted the expression of interferon-related genes. Therefore, we conclude that eugenol inhibits SGIV infection by enhancing cellular immunity through its anti-inflammatory and antioxidant functions.

Development and immune evaluation of LAMP1 chimeric DNA vaccine against Singapore grouper iridovirus in orange-spotted grouper, Epinephelus coioides.

Grouper is one of the most important and valuable mariculture fish in China, with a high economic value. As the production of grouper has increased, massive outbreaks of epidemic diseases have limited the development of the industry. Singapore grouper iridovirus (SGIV) is one of the most serious infectious viral pathogens and has caused huge economic losses to grouper farming worldwide due to its rapid spread and high lethality. To find new strategies for the effective prevention and control of SGIV, we constructed two chimeric DNA vaccines using Lysosome-associated membrane protein 1 (LAMP1) fused with major capsid proteins (MCP) against SGIV. In addition, we evaluated the immune protective effects of vaccines including pcDNA3.1-3HA, pcDNA3.1-MCP, pcDNA3.1-LAMP1, chimeric DNA vaccine pcDNA3.1-MLAMP and pcDNA3.1-LAMCP by intramuscular injection. Our results showed that compared with groups injected with PBS, pcDNA3.1-3HA, pcDNA3.1-LAMP1 or pcDNA3.1-MCP, the antibody titer significantly increased in the chimeric vaccine groups. Moreover, the mRNA levels of immune-related factors in groupers, including IRF3, MHC-I, TNF-α, and CD8, showed the same trend. However, MHC-II and CD4 were significantly increased only in the chimeric vaccine groups. After 28 days of vaccination, groupers were challenged with SGIV, and mortality was documented for each group within 14 days. The data showed that two chimeric DNA vaccines provided 87 % and 91 % immune protection for groupers which were significantly higher than the 52 % protection rate of pcDNA3.1-MCP group, indicating that both forms of LAMP1 chimeric vaccines possessed higher immune protection against SGIV, providing the theoretical foundation for the creation of novel DNA vaccines for fish.

Berberine inhibits SGIV replication by suppressing inflammatory response and oxidative stress.

Singapore grouper iridovirus (SGIV) is one of the major infectious diseases responsible for high mortality and huge economic losses in the grouper aquaculture industry. Berberine (BBR), a naturally occurring plant alkaloid, is a phytochemical having a variety of biological properties, such as antiviral, antioxidant, and anti-inflammatory effects. In this work, we used an in vitro model based on Western blot, ROS fluorescence probe, and real-time quantitative PCR (qRT-PCR) to examine the antiviral qualities of BBR against SGIV. The outcomes demonstrated that varying BBR concentrations could significantly inhibit the replication of SGIV. In addition, BBR greatly inhibited the production of genes associated with pro-inflammatory cytokines in SGIV-infected or SGIV-uninfected GS cells based on qRT-PCR data. Subsequent investigations demonstrated that BBR suppressed the expression of the promoter activity of NF-κB and NF-κB-p65 protein. Additionally, BBR reduced the phosphorylation of ERK 1/2, JNK, and p38. Furthermore, BBR also inhibits SGIV-induced ROS production by upregulating the expression of antioxidant-related genes. In conclusion, BBR is a viable therapy option for SGIV infection due to its antiviral properties.

Common carp (Cyprinus carpio) CD81 promoting CyHV-3 virus replication via regulating autophagy and RLRs-interferon signaling pathway.

Cyprinid herpesvirus type 3 (CyHV-3), also called Koi herpesvirus (KHV), which leads to mass cyprinid mortality and enormous economic losses. To establish an infection, CyHV-3 needs to counteract host antiviral responses. CD81 belongs to the evolutionary conserved tetraspanin family of proteins. Several studies have shown that different members of the tetraspanin superfamily modulates different virus infectious processes. Here we aimed at analysing the role of CD81 in CyHV-3 infection. In this study, we cloned and characterized the CD81 of Common Carp, the open reading frame of CcCD81 gene was 702 bp, which encoded 234 amino acids with four transmembrane domains (TM1 to TM4), a small extracellular loop (SEL), and a large extracellular loop (LEL). Tissue distribution analysis showed that CcCD81 was widely expressed in all the tested tissues with the highest expression in head kidney, followed by a high expression in brain. Subsequently, expression levels of CcCD81 were significantly increased in CCB cells within the first 3h after infection, meanwhile, the expression of viral gene VP136 was reduced after CcCD81 knockdown in CCB cells post CyHV-3 infection. Furthermore, CcCD81 knockdown can significantly reduce the autophagy process and increase the promoter activity of ISRE and IFN-1 in the CCB cells after viral infection, as well as other genes involved in the IFN signaling pathway, including RIG-1、MDA5、MAVS、TBK1 and IRF3. Taking the data together, we revealed that CcCD81 mediates autophagy and blocks RIG-1-mediated antiviral signaling and negatively regulates the promoter activity of type I interferon (IFN) promoting virus replication. These results reveal a new link between autophagy and four-transmembrane-domain protein superfamily and contribute to elucidate the mechanism of CyHV-3 infection.

Functional analysis of the Cystatin F gene response to SGIV infection in orange-spotted grouper, Epinephelus coioides.

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-1ß, 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.

Characterization of scavenger receptor MARCO in orange-spotted grouper, Epinephelus coioides.

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.

Modulatory effects of curcumin on Singapore grouper iridovirus infection-associated apoptosis and autophagy in vitro.

Singapore grouper iridovirus (SGIV) with high pathogenicity can cause great economic losses to aquaculture industry. Thus, it is of urgency to find effective antiviral strategies to combat SGIV. Curcumin has been demonstrated effective antiviral activity on SGIV infection. However, the molecular mechanism behind this action needs to be further explanations. In view of the fact that apoptosis (type I programmed cell death) and autophagy (type II programmed cell death) were key regulators during SGIV infection, we aimed to investigate the relevance between antiviral activity of curcumin and SGIV-associated programmed and clarify the role of potential signaling pathways. Our results showed that curcumin suppressed SGIV-induced apoptosis. At the same time, the activities of caspase-3/8/9 and activating protein-1 (AP-1), P53, nuclear factor-κB (NF-ΚB) promoters were inhibited. Besides, the activation of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen activate protein kinase (p38 MAPK) signal pathways were suppressed in curcumin-treated cells. On the other hand, curcumin down-regulated protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway to promote autophagy representing by increased LC3 II and Beclin1 expression. Curcumin also hindered the transition of cells from G1 to S phase, as well as down-regulating the expression of CyclinD1. Our findings revealed the resistance curcumin induced to the effects of DNA virus on cell apoptosis and autophagy and the insights gained from this study may be of assistance to understand the molecular mechanism of curcumin against DNA virus infection.

The transcription factor RFX5 positively regulates expression of MHCIa in the red-spotted grouper (Epinephelus akaara).

Regulatory factor X 5 (RFX 5) is a member of the RFX family, and it forms the transcription factor complex RFX with RFXANK/B and RFXAP. The RFX complex can activate MHC expression by binding to the MHC promoter. However, the regulate mechanism of RFX in fish species is not been fully elucidated. In this study, we investigated the transcriptional regulation of Epinephelus akaara RFX5 (EaRFX5) on EaMHCI, and its effect on immune pathways. The genomic sequence of EaRFX5 was 35,774 bp and consisted of ten exons and nine introns. The length of EaRFX5 ORF sequence is 2,160 bp, encoding 719 amino acids. By qRT-PCR, EaRFX5 was detected constitutively expressed in twelve selected tissues, showing a wide range of expression. EaRFX5 expression parttern in response to poly (I:C), LPS, Zymosan A, SGIV, and NNV challenges showed that EaRFX5 plays a differentiated immunomodulatory role in response to various stimuli in different tissues, and EaRFX5 was most significantly upregulated in the kidney after challenge with SGIV. Subcellular localization assays showed that EaRFX5 is a typical nuclear protein. Based on the in vitro overexpression experiments, EaRFX5 appeared to promote the expression of EaMHCIa gene, interferon signalling pathway and inflammatory cytokine. Luciferase reporter assay showed that the -267 bp to +82 bp region of EaMHCIa promoter was the core region where EaRFX5 modulated. Additionally, point mutations and electrophoretic mobility shift assays indicating M3 is the EaRFX5 binding sites in the EaMHCIa promoter. These results contribute to elucidating the function of EaRFX5 in fish immune response, and provide the first evidence of positive regulation of MHCIa expression by RFX5 in fish.

Grouper ATF1 plays an antiviral role in response to iridovirus and nodavirus infection.

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.

Epinephelus coioides Hsp27 negatively regulates innate immune response and apoptosis induced by Singapore grouper iridovirus (SGIV) infection.

Heat shock proteins (Hsps) are important for maintaining protein homeostasis and cell survival. In this study, Hsp27 of Epinephelus coioides, an economically important marine fish in China and Southeast Asian countries, was characterized. E. coioides Hsp27 contains the consered ACD_HspB1_like domain and three p38 MAPK phosphorylation sites, located at Thr-13, Thr-60 and Ser-167. E. coioides Hsp27 was distributed in both the cytoplasm and nucleus, its mRNA was detected in all 14 tissues examined, and its expression was up-regulated after challenge with Singapore grouper iridovirus (SGIV), an important E. coioides pathogen. Over-expression of E. coioides Hsp27 significantly upregulated the expressions of the key SGIV genes (VP19, LITAF, MCP, and ICP18), downgraded the expressions of the E. coioides immune factors (IRF3, IRF7, ISG15, and TRAF6) and proinflammatory factors (TNF-α, IL-8), downgraded the activation of nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), and substantially inhibited the cell apoptosis induced by SGIV infection. These data illustrated that E. coioides Hsp27 might be involved in SGIV infection by negatively regulating the innate immune response.

Functional analysis of Epinephelus coioides peroxisome proliferative-activated receptor α (PPARα): Involvement in response to viral infection.

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-1ß, 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.

Nuclear factor kappa B/p65 plays a positive role in peroxisome proliferator-activated receptor δ expression in orange-spotted grouper Epinephelus coioides.

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.

Characterization of Kruppel-like factor 6 in Epinephelus coioides: The role in viral infection and the transcriptional regulation on Peroxisome proliferator-activated receptor δ.

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.

Characterization of ssDNA aptamers specifically directed against Trachinotus ovatus NNV (GTONNV)-infected cells with antiviral activities.

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.

PPAR-δ of orange-spotted grouper exerts antiviral activity against fish virus and regulates interferon signaling and inflammatory factors.

Peroxisome proliferator-activated receptor δ (PPAR-δ), also called PPAR-ß or PPAR-ß/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.

Characterization of cathepsin C from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.

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.

Fish SUMO3 functions as a critical antiviral molecule against iridovirus and nodavirus.

Protein SUMOylation (SUMO is small ubiquitin-related modifier) is a dynamic process that is strictly regulated under physiological and pathological conditions. We previously cloned and characterized two SUMO homologue genes (EcSUMO1 and EcSUMO2) from orange-spotted grouper (Epinephelus coioides). In the present study, the SUMO3 homologue from E. coioides (EcSUMO3) was cloned and its possible roles in fish immunity were analyzed. The open reading frame of EcSUMO3 contains 285 base pairs encoding a 94 amino acid protein with a predicted molecular mass of 10.73 kDa. The protein sequence of EcSUMO3 revealed similar domains with mammals, including the UBQ (ubiquitin-like proteins) domain, the hydrophobic surface, the Ulp1-Smt3 interaction sites, a VKTE motif and the C-terminal Gly residues. EcSUMO3 shares 46.83% and 89.58% identity with EcSUMO1 and EcSUMO2, respectively, and it shares 94%, 98%, and 98% identity with SUMO3 from Oreochromis niloticus, Danio rerio, and Homo sapiens, respectively. Quantitative real-time polymerase chain reaction analysis indicated that EcSUMO3 was constitutively expressed in all of the analyzed tissues in healthy grouper. EcSUMO3 expression levels were remarkably (p < 0.01) up-regulated in grouper spleen (GS) cells in response to stimulation with red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV). EcSUMO3 was distributed in both the cytoplasm and nucleus in GS cells. EcSUMO3 enhanced SGIV and RGNNV replication during viral infection in vitro. These results are important for better understanding of the SUMO pathway in fish and provide insights into the regulatory mechanism of viral infection in E. coioides under farmed conditions.

Identification of Beclin-1 from orange-spotted grouper (Epinephelus coioides) involved in viral 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.

Molecular cloning and characterization of grouper Krϋppel-like factor 9 gene: Involvement in the fish immune response to viral infection.

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.

Molecular cloning and characterization of Aos1 and Uba2 from the orange-spotted grouper (Epinephelus coioides).

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are essential regulators of a wide range of cellular processes. Covalent attachment of SUMO requires an enzymatic cascade consisting of a single E1-activating enzyme (Aos1 and Uba2 heterodimer), a single E2-conjugating enzyme (Ubc9), and one of several E3 ligases that facilitate transfer of SUMO from Ubc9 to the substrate. In the present study, the Aos1 and Uba2 homologues (EcAos1 and EcUba2) from the orange-spotted grouper (Epinephelus coioides) were cloned and their possible roles in fish immunity were analyzed. The open reading frame (ORF) of EcAos1 contains 1050 base pairs (bp) encoding a 350 amino acid protein with a predicted molecular mass of 38.97 kDa EcAos1 has a nuclear localization signal (NLS) at residues 193-203. The ORF of EcUba2 contains 1950 bp encoding a 650 amino acid protein with a predicted molecular mass of 71.3 kDa EcUba2 has a NLS at residues 608-630. Quantitative real-time polymerase chain reaction analysis indicated that both EcAos1 and EcUba2 were distributed in all examined tissues. The expression levels of EcAos1 and EcUba2 in the spleen and head kidney of E. coioides were differentially up-regulated when challenged with polyinosine-polycytidylic acid. Green fluorescence of both pEGFP-C1-EcAos1 and pEGFP-C1-EcUba2 was distributed in the nucleus of GS cells. When the NLSs of EcAos1 and EcUba2 were deleted, the cellular localizations all changed. Over-expression of EcAos1 and EcUba2 inhibited red-spotted grouper nervous necrosis virus infection and replication. These results are important for better understanding of the SUMO pathway in fish and provide insights into the regulatory mechanism of viral infection in E. coioides under farmed conditions.