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.

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.

SGIV VP82 inhibits the interferon response by degradation of IRF3 and IRF7.

During virus-host co-evolution, viruses have developed multiple strategies to dampen IFN response and prevent its antiviral activity in host cells. To date, the interactions between host IFN response and the immune evasion strategies exploited by fish iridoviruses still remain largely uncertain. Here, a potential immune evasion protein candidate of Singapore grouper iridovirus (SGIV), VP82 (encoded by SGIV ORF82) was screened and its roles during viral replication were investigated in detail. Firstly, VP82 overexpression dramatically decreased IFN or ISRE promoter activity and the transcription levels of IFN stimulated genes (ISGs) stimulated by grouper cyclic GMP-AMP synthase (EccGAS)/stimulator of interferon genes (EcSTING), TANK-binding kinase 1 (EcTBK1), IFN regulatory factor 3 (EcIRF3)and EcIRF7. Secondly, Co-IP assays indicated that VP82 interacted with EcIRF3 and EcIRF7, but not EcSTING and EcTBK1, which was consistent with the co-localization between VP82 and EcIRF3 or EcIRF7. Furthermore, VP82 promoted the degradation of EcIRF3 and EcIRF7 in a dose-dependent manner via the autophagy pathway. Finally, VP82 overexpression accelerated SGIV replication, evidenced by the increased transcriptions of viral core genes and viral production. Moreover, the antiviral action of EcIRF3 or EcIRF7 was significantly depressed in VP82 overexpressed cells. Together, VP82 was speculated to exert crucial roles for SGIV replication by inhibiting the IFN response via the degradation of IRF3 and IRF7. Our findings provided new insights into understanding the immune evasion strategies utilized by fish iridovirus through IFN regulation.

Dual-specificity phosphatase 1 inhibits Singapore grouper iridovirus replication via regulating apoptosis in Epinephelus coioides.

The dual-specificity phosphatase (DUSP) family plays key roles in the maintenance of cellular homeostasis and apoptosis etc. In this study, the DUSP member DUSP1 of Epinephelus coioides was characterized: the length was 2371 bp including 281 bp 5' UTR, 911 bp 3' UTR, and a 1125 bp open reading frame encoding 374 amino acids. E. coioides DUSP1 has two conserved domains, a ROHD and DSPc along with a p38 MAPK phosphorylation site, localized at Ser308. E. coioides DUSP1 mRNA can be detected in all of the tissues examined, and the subcellular localization showed that DUSP1 was mainly distributed in the nucleus. Singapore grouper iridovirus (SGIV) infection could induce the differential expression of E. coioides DUSP1. Overexpression of DUSP1 could inhibit SGIV-induced cytopathic effect (CPE), the expressions of SGIV key genes, and the viral titers. Overexpression of DUSP1 could also regulate SGIV-induced apoptosis, and the expression of apoptosis-related factor caspase 3. The results would be helpful to further study the role of DUSP1 in viral infection.

Singapore grouper iridovirus VP20 interacts with grouper TBK1 and IRF3 to attenuate the interferon immune response.

Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus, family Iridoviridae, is a highly pathogenic agent and causes heavy economic losses in the global grouper aquaculture. Recent studies demonstrated that SGIV infection attenuated antiviral immune and inflammatory response induced by poly (I:C) in vitro. However, little was known about the potential functions of the immune regulatory proteins encoded by SGIV. Here, we identified the detailed roles of VP20 and clarified the potential mechanism underlying its immune regulatory function during SGIV infection. Our results showed that VP20 was an IE gene, and partially co-localized with Golgi apparatus and lysosomes in grouper cells. Overexpression of VP20 enhanced SGIV replication, demonstrated by the increase in the transcription levels of viral core genes and the protein synthesis of MCP. Reporter gene assays showed that SGIV VP20 overexpression significantly reduced the IFN promoter activity induced by poly (I:C), grouper stimulator of interferon genes (EcSTING) and TANK-binding kinase 1 (EcTBK1). Consistently, the transcription levels of IFN related genes were significantly decreased in VP20 overexpressing cells compared to those in control cells. Co-IP assay and confocal microscopy observations indicated that VP20 co-localized and interacted with EcTBK1 and EcIRF3, but not EcSTING. In addition, VP20 was able to degrade EcIRF3 and attenuate the antiviral action of EcIRF3, while had no effect on EcTBK1. Together, SGIV VP20 was speculated to promote viral replication through attenuating the IFN response mediated by TBK1-IRF3 in vitro. Our findings provided new insights into the immune regulatory function of SGIV encoded unknown proteins.

Fish ELOVL7a is involved in virus replication via lipid metabolic reprogramming.

The elongation of very long chain fatty acids (ELOVL) proteins are key rate-limiting enzymes that catalyze fatty acid synthesis to form long chain fatty acids. ELOVLs also play regulatory roles in the lipid metabolic reprogramming induced by mammalian viruses. However, little is known about the roles of fish ELOVLs during virus infection. Here, a homolog of ELOVL7 was cloned from Epinephelus coioides (EcELOVL7a), and its roles in red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infection were investigated. The transcription level of EcELOVL7a was significantly increased upon RGNNV and SGIV infection or other pathogen-associated molecular patterns stimulation in grouper spleen (GS) cells. Subcellular localization analysis showed that EcELOVL7a encoded an endoplasmic reticulum (ER) related protein. Overexpression of EcELOVL7a promoted the viral production and virus release during SGIV and RGNNV infection. Furthermore, the lipidome profiling showed that EcELOVL7a overexpression reprogrammed cellular lipid components in vitro, evidenced by the increase of glycerophospholipids, sphingolipids and glycerides components. In addition, VLCFAs including FFA (20:2), FFA (20:4), FFA (22:4), FFA (22:5) and FFA (24:0), were enriched in EcELOVL7a overexpressed cells. Consistently, EcELOVL7a overexpression upregulated the transcription level of the key lipid metabolic enzymes, including fatty acid synthase (FASN), phospholipase A 2α (PLA 2α), and cyclooxygenases -2 (COX-2), LPIN1, and diacylglycerol acyltransferase 1α (DGAT1α). Together, our results firstly provided the evidence that fish ELOVL7a played an essential role in SGIV and RGNNV replication by reprogramming lipid metabolism.

Singapore Grouper Iridovirus VP131 Drives Degradation of STING-TBK1 Pathway Proteins and Negatively Regulates Antiviral Innate Immunity.

Iridoviruses are large DNA viruses which cause great economic losses to the aquaculture industry and serious threats to ecological diversity worldwide. Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus, causes high mortality in grouper aquaculture. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. Here, we reported that the protein encoded by SGIV ORF131R (VP131) was localized predominantly within the endoplasmic reticulum (ER). Ectopic expression of GFP-VP131 significantly enhanced SGIV replication, while VP131 knockdown decreased viral infection in vitro, suggesting that VP131 functioned as a proviral factor during SGIV infection. Overexpression of GFP-VP131 inhibited the interferon (IFN)-1 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), TANK-binding kinase 1 (EcTBK1), or melanoma differentiation-associated gene 5 (EcMDA5), whereas such activation induced by mitochondrial antiviral signaling protein (EcMAVS) was not affected. Moreover, VP131 interacted with EcSTING and degraded EcSTING through both the autophagy-lysosome pathway and ubiquitin-proteasome pathway, and targeted for the K63-linked ubiquitination. Of note, we also found that EcSTING significantly accelerated the formation of GFP-VP131 aggregates in co-transfected cells. Finally, GFP-VP131 inhibited EcSTING- or EcTBK1-induced antiviral activity upon red-spotted grouper nervous necrosis virus (RGNNV) infection. Together, our results demonstrated that the SGIV VP131 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion. IMPORTANCE STING has been identified as a critical factor participating in the innate immune response which recruits and phosphorylates TBK1 and IFN regulatory factor 3 (IRF3) to induce IFN production and defend against viral infection. However, viruses also distort the STING-TBK1 pathway to negatively regulate the IFN response and facilitate viral replication. Here, we reported that SGIV VP131 interacted with EcSTING within the ER and degraded EcSTING, leading to the suppression of IFN production and the promotion of SGIV infection. These results for the first time demonstrated that fish iridovirus evaded the host antiviral response via abrogating the STING-TBK1 signaling pathway.

Antiviral role of grouper FoxO1 against RGNNV and SGIV infection.

As a key regulator of the innate immune system, FoxO1 has a variety of activities in biological organisms. In the present study, grouper FoxO1 (EcFoxO1) was cloned and the antiviral activity in red grouper neuron necrosis virus (RGNNV) and Singapore grouper iridescent virus (SGIV) was examined. The open reading frame (ORF) of EcFoxO1 contains 2,034 base pairs that encode a protein of 677 amino acids with a predicted molecular weight of 73.21 kDa. EcFoxO1 was shown to be broadly distributed in healthy grouper tissues, and was up-regulated in vitro in response to stimulation by RGNNV and SGIV. EcFoxO1 has a whole-cell distribution in grouper spleen (GS) cells. EcFoxO1 decreased the replication of RGNNV and SGIV, and activated interferon (IFN) 3, IFN-stimulated response element (ISRE), and nuclear factor-κB (NF-κB) promoter activities. EcFoxO1 could interact with EcIRF3. Together, the results demonstrated that EcFoxO1 might be an important regulator of grouper innate immune response against RGNNV and SGIV infection.

Identification of circRNAs and circRNA-mRNA network of Epinephelus coioides during Singapore grouper iridovirus infection.

Circular RNA (circRNA), one of the important non-coding RNA molecules with a closed-loop structure, plays a key regulatory role in cell processing. In this study, circRNAs of Epinephelus coioides, an important marine cultured fish in China, were isolated and characterized, and the network of circRNAs and mRNA was explored during Singapore grouper iridovirus (SGIV) infection, one of the most important double stranded DNA virus pathogens of marine fish. 10 g of raw data was obtained by high-throughput sequencing, and 2599 circRNAs were classified. During SGIV infection, 123 and 37 circRNAs occurred differential expression in spleen and spleen cells, indicating that circRNAs would be involved in the viral infection. GO annotation and KEGG demonstrated that circRNAs could target E. coioides genes to regulate cell activity and the activation of immune factors. The results provide some insights into the circRNAs mediated immune regulatory network during bony fish virus infection.

Singapore grouper iridovirus VP122 targets grouper STING to evade the interferon immune response.

Singapore grouper iridovirus (SGIV) is a highly pathogenic Iridoviridae that causes hemorrhage and spleen enlargement in grouper. Despite previous genome annotation efforts, many open reading frames (ORFs) in SGIV remain uncharacterized, with largely unknown functions. In this study, we identified the protein encoded by SGIV ORF122, now referred to as VP122. Notably, overexpression of VP122 promoted SGIV replication. Moreover, VP122 exhibited antagonistic effects on the natural antiviral immune response through the cGAS-STING signaling pathway. It specifically inhibited the cGAS-STING-triggered transcription of various immune-related genes, including IFN1, IFN2, ISG15, ISG56, PKR, and TNF-α in GS cells. Additionally, VP122 significantly inhibited the activation of the ISRE promoter mediated by EccGAS and EcSTING but had no effect on EccGAS or EcSTING alone. Immunoprecipitation and Western blotting experiments revealed that VP122 specifically interacts with EcSTING but not EccGAS. Notably, this interaction between VP122 and EcSTING was independent of any specific domain of EcSTING. Furthermore, VP122 inhibited the self-interaction of EcSTING. Interestingly, VP122 did not affect the recruitment of EcTBK1 and EcIRF3 to the EcSTING complex. Collectively, our results demonstrate that SGIV VP122 targets EcSTING to evade the type I interferon immune response, revealing a crucial role for VP122 in modulating the host-virus interaction.

Singapore grouper iridovirus infection counteracts poly I:C induced antiviral immune response in vitro.

Groupers are important mariculture fish in South China and Southeast Asian countries. However, the increasing frequency of infectious disease outbreaks has caused great economic losses in the grouper industry. Among these pathogens, Singapore grouper iridovirus (SGIV) infection causes high mortality in larval and juvenile stages of grouper. However, the mechanism underlying the action of viral manipulation on cellular immune response still remained largely uncertain. Here, using RNA-seq technology, we investigated the regulatory roles of SGIV infection on synthetic RNA duplex poly I:C induced immune response in vitro. Using reporter gene assays, we found that SGIV infection decreased poly I:C induced interferon promoter activation. Transcriptomic analysis showed that the mRNA expression levels of 2238 genes were up-regulated, while 1247 genes were down-regulated in poly I:C transfected grouper spleen (GS) cells. Interestingly, SGIV infection decreased the expression of 1479 up-regulated genes and increased the expression of 297 down-regulated genes in poly I:C transfected cells. The differentially expressed genes (DEGs) down-regulated by SGIV were directly related to immune, inflammation and viral infection, and JUN, STAT1, NFKB1, MAPK14A, TGFB1 and MX were the 6 top hub genes in the down-regulated DEGs' protein-protein interaction (PPI) network. Furthermore, quantitative real-time PCR (qPCR) analysis confirmed that the interferon signaling and inflammatory-related genes, including cGAS, STING, TBK1, MAVS, TNF, IRAK4 and NOD2 were up-regulated by poly I:C stimulation, but all significantly down-regulated after SGIV infection. Thus, we speculated that SGIV infection counteracted poly I:C induced antiviral immune response and this ability helped itself to escape host immune surveillance. Together, our data will contribute greatly to understanding the potential immune evasion mechanism of iridovirus infection in vitro.

Epinephelus coioides PCSK9 affect the infection of SGIV by regulating the innate immune response.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in mammals is a multifunctional protein. In this study, PCSK9 of marine fish Epinephelus coioides was characterized. The full-length cDNA of E. coioides PCSK9 was 2458 bp in length containing 185 bp 5' UTR, 263 bp 3' UTR and 2010 bp open reading frame (ORF) encoding 669 amino acids with the predicted molecular weight of 71 kDa and the theoretical PI of 6.6. Similar to other members of PCSK9 family, E. coioides PCSK9 has three conserved domains: Inhibitor_ I9 super family, Peptidases_ S8_ PCSK9_ Proteinase K_ like, and PCSK9_ C-CRD super family. E. coioides PCSK9 mRNA could be detected in all the tissues examined by real-time quantitative PCR, with the highest expression in the brain, followed by skin, trunk kidney, head kidney, intestine, blood, liver, spleen, gill, muscle and heart. E. coioides PCSK9 was distributed in both the cytoplasm and nucleus. The expression of E. coioides PCSK9 was significantly upregulated during Singapore grouper iridovirus (SGIV) infection. Upregulated PCSK9 could significantly affect the activities of nuclear factor kappaB (NF-κB) promoter, SGIV-induced apoptosis, and the expressions of the key SGIV genes (ICP18, LITAT, MCP, and VP19) and the E. coioides proinflammatory factors (IL-6, IL-1ß, IL-8, and TNF-α). The results illustrated that E. coioides PCSK9 might be involved in the pathogen infection by regulating the innate immune response.

Molecular cloning, inducible expression and function analysis of Epinephelus coioides Sec6 response to SGIV infection.

Exocyst complex component 3 Sec6 of mammals, one of the components of the exocyst complex, participates in numerous cellular functions, such as promoting cell migration and inhibiting apoptosis. In this study, the Sec6 was obtained from Epinephelus coioides, an economically important cultured fish. The full length of E. coioides Sec6 was 2655 bp including a 245 bp 5' UTR, a 154 bp 3' UTR, and a 2256 bp open reading frame (ORF) encoding 751 amino acids, with a molecular mass of 86.76 kDa and a theoretical pI of 5.57. Sec6 mRNA was detected in all the tissues examined, but the expression level is different in these tissues. Using fluorescence microscopy, Sec6 were distributed in both the nucleus and the cytoplasm. After SGIV infection, the expression of E. coioides Sec6 was significantly up-regulated in both trunk kidney and spleen response to Singapore grouper iridovirus (SGIV), an important pathogens of E. coioides. Sec6 could increase the SGIV-induced cytopathic effects (CPE), the expression of the SGIV genes VP19, LITAF, MCP, ICP18 and MCP, and the viral titers. Besides, E. coioides Sec6 significantly downregulated the promoter of NF-κB and AP-1, and inhibited the SGIV-induced apoptosis. The results demonstrated that E. coioides Sec6 might play important roles in SGIV infection.

Fish TRIM21 exhibits antiviral activity against grouper iridovirus and nodavirus infection.

Growing evidences have demonstrated that multiple TRIM (tripartite motif) proteins exert critical roles in host defense against different microbial pathogens. Although mammalian TRIM21 has been reported to function as an important regulatory factor in antiviral immune and inflammatory response, the role of fish TRIM21 against virus infection still remains largely unknown. In the present study, we investigated the characteristics of TRIM21 gene (EcTRIM21) from orange spotted grouper (Epinephelus coioides). The full-length EcTRIM21 cDNA encoded a 557 amino acid peptide with 92.1% and 31.14% identity with giant grouper (Epinephelus lanceolatus) and human (Homo sapiens), respectively. EcTRIM21 contained four conserved domains, including RING, B-Box, PRY and SPRY domain. EcTRIM21 expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, suggesting that EcTRIM21 might be involved in host defense against fish virus infections. Subcellular localization showed that EcTRIM21 were distributed in the cytoplasm in a punctate manner. Overexpression of EcTRIM21 in vitro significantly inhibited RGNNV and SGIV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral core genes. Consistently, knockdown of EcTRIM21 by small interfering RNA (siRNA) promoted the replication of RGNNV and SGIV in vitro. Furthermore, EcTRIM21 overexpression increased both interferon (IFN) and interferon stimulated response element (ISRE) promoter activities. In addition, the transcription levels of IFN signaling related molecules were positively regulated by EcTRIM21 overexpression. Together, our data demonstrated that fish TRIM21 exerted antiviral activity against fish viruses through positive regulation of host interferon response.

Identification and characterization of lncRNAs and the interaction of lncRNA-mRNA in Epinephelus coioides induced with Singapore grouper iridovirus infection.

Singapore grouper iridovirus (SGIV) is a highly pathogenic double-stranded DNA virus, and the fatality rate of SGIV-infected grouper is more than 90%. Up to now, there is no effective methods to control the disease. Long non-coding RNAs (lncRNAs) might play an important role in individual growth and development, immune regulation and other life processes. In this study, lncRNAs were identified in Epinephelus coioides, an important economic aquaculture marine fish in China and Southeast Asia, and the regulatory relationships of lncRNAs and mRNA response to SGIV infection were analyzed. A total of 11,678 lncRNAs were identified and classified from the spleen and GS (grouper spleen) cells. 105 differentially expressed lncRNAs (DElncRNAs) were detected during SGIV infection. The lncRNAs and the regulated mRNAs were analyzed using co-expression network, lncRNA target gene annotation and GO enrichment. At 24 and 48 h after SGIV infection, 118 and 339 lncRNA-mRNA pairs in GS cells were detected, and 728 and 688 differentially expressed lncRNA-mRNA pairs in spleen were obtained, respectively. GO and KEGG were used to predict the DE lncRNAs' target genes, and deduce the DE lncRNAs-affected signaling pathways. In GS cells, lncRNAs might participate in cell part, binding and catalytic activity; and lncRNAs might be involved in immune system process and transcription factor activity in spleen. These data demonstrated that lncRNAs could regulate the expression of immune-related genes response to viral infection, and providing a new insight into understanding the complexity of immune regulatory networks mediated by lncRNAs during viral infection in teleost fish.

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.

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.

The roles of grouper TAK1 in regulating the infection of Singapore grouper iridovirus.

Transforming growth factor-ß activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase family. It is an upstream factor of the IκB kinase, which activates IKKα and IKKß. TAK1 is a key factor in the induction of nuclear factor κB (NF-κB) and plays a crucial role in the activation of inflammatory responses. However, the roles of TAK1 during viral infection in teleost fish are largely unknown. In this study, we cloned a TAK1 homolog (HgTAK1) from the hybrid grouper (Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀). The open reading frame of HgTAK1 consists of 1728 nucleotides encoding 575 amino acids, and the predicted molecular weight is 64.32 kDa HgTAK1 has an S_TKc domain, which consists of a serine/threonine protein kinase and a catalytic domain. Expression pattern analysis showed that HgTAK1 was distributed in all tested tissues, with abundant contents in the heart, head kidney, and blood. Additionally, HgTAK1 was distributed in the cytoplasm of grouper spleen (GS) cells. After Singapore grouper iridovirus (SGIV) infection, the expression of HgTAK1 increased in GS cells. Overexpression of HgTAK1 could promote the replication of SGIV in GS cells and inhibit the activation of NF-κB and IFN stimulated response elements (ISRE) in reporter assay. When co-expressed with IRF3 or HgIRF7 in GS cells, HgTAK1 obviously down-regulated IRF3- or IRF7-mediated the NF-κB and ISRE promoter induction. The interaction between HgTAK1 and IRF3 or IRF7 has been identified by co-immunoprecipitation assay. These findings provide a basis for understanding the innate immune mechanism of the grouper response to viral 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.