Singapore Grouper Iridovirus Replication Research Articles

Singapore grouper iridovirus VP146 modulates the cGAS-STING signaling pathway to escape the interferon immune response

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that has caused significant economic losses to the grouper aquaculture industry. So far, the structure and function of SGIV proteins have been successively reported. In the present paper, the protein of SGIV VP146 was cloned and identified. VP146 was whole-cell distributed in GS cells. VP146 promoted SGIV replication and inhibited the transcription of interferon-related genes as well as pro-inflammatory cytokines in GS cells. In addition, VP146 was involved in the regulation of the cGAS-STING signaling pathway, and decreased cGAS-STING induced the promoter of ISRE and NF-κB. VP146 interacted with the proteins of cGAS, STING, TBK1, and IRF3 from grouper, but did not affect the binding of grouper STING to grouper TBK1 and grouper IRF3. Interestingly, grouper STING was able to affect the intracellular localization of VP146. Four segment structural domains of grouper STING were constructed, and grouper STING-CTT could affect the intracellular localization of VP146. VP146 had no effect on the self-binding of EcSITNG, nor on the binding of EcSTING to EcTBK1 and EcIRF3. Together, the results demonstrated that SGIV VP146 modulated the cGAS-STING signaling pathway to escape the interferon immune response.

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.

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.

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.

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.

Antiviral Effect and Mechanism of Edaravone against Grouper Iridovirus Infection.

Singapore grouper iridovirus (SGIV) is a virus with high fatality rate in the grouper culture industry. The outbreak of SGIV is often accompanied by a large number of grouper deaths, which has a great impact on the economy. Therefore, it is of great significance to find effective drugs against SGIV. It has been reported that edaravone is a broad-spectrum antiviral drug, most widely used clinically in recent years, but no report has been found exploring the effect of edaravone on SGIV infections. In this study, we evaluated the antiviral effect of edaravone against SGIV, and the anti-SGIV mechanism of edaravone was also explored. It was found that the safe concentration of edaravone on grouper spleen (GS) cells was 50 µg/mL, and it possessed antiviral activity against SGIV infection in a dose-dependent manner. Furthermore, edaravone could significantly disrupt SGIV particles and interference with SGIV binding to host cells, as well as SGIV replication in host cells. However, edaravone was not effective during the SGIV invasion into host cells. This study was the first time that it was determined that edaravone could exert antiviral effects in response to SGIV infection by directly interfering with the processes of SGIV infecting cells, aiming to provide a theoretical basis for the control of grouper virus disease.

Lcalmnb2: Asian seabass gene involved in resisting SGIV

Diseases cause significant economic loss in aquaculture. Molecular breeding is an effective way to improve long-lasting disease resistance. DNA markers associated with disease resistance are essential in molecular breeding. The Singapore grouper iridovirus (SGIV) causes severe mortality in hatcheries of Asian seabass (Lates calcarifer). To identify DNA markers associated with SGIV resistance and understand more about functions of candidate genes in SGIV resistance, the lmnb2 gene from L. calcarifer was characterized. The gene contained 13 exons and 12 introns. Its open reading frame (ORF) of 1731 bp encoded a protein with 576 amino acids. A SNP, identified in the first intron of the gene, was significantly associated with SGIV resistance. Lcalmnb2 was ubiquitously expressed in organs of the healthy Asian seabass. In vitro studies revealed that Lcalmnb2 promoted cell proliferation and migration. Overexpression of the gene repressed the replication of SGIV in Asian seabass cells, while knockdown of the gene prompted SGIV replication. In virus-infected cells, SGIV utilized Lamin B2 for host DNA transport during viral replication, leading to the induction of nuclear abnormalities. This study sheds new light on the involvement of Lcalmnb2 in resisting SGIV infection. The identified SNP may serve as a useful DNA marker for selecting SGIV-resistant Asian seabass individuals in future breeding programs.

Taurochenodeoxycholic acid exerts anti-viral activities upon SGIV infection via anti-inflammatory response

Taurine chenodeoxycholic acid (TCDCA), a combination of taurine and chenodeoxycholic acid, plays anti-inflammatory roles in many diseases. However, little is known about its antiviral activity in aquaculture. Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus, is an important viral pathogen in grouper aquaculture. Here, the effects of TCDCA on SGIV replication in vitro and in vivo were investigated. TCDCA treatment inhibited SGIV infection in grouper spleen (GS) cells, evidenced by the delayed CPE progression and decreased viral gene transcription, protein expression and viral progeny production. TCDCA treatment suppressed the mRNA level of pro-inflammatory factors and NF-κB promoter activities in SGIV-infected cells compared with mock-infected cells. Inhibition of NF-κB activity enhanced the inhibitory effects of TCDCA on SGIV replication, indicating that TCDCA inhibited SGIV replication in vitro via regulating host inflammatory response. Furthermore, TCDCA treatment protected grouper from SGIV infection, evidenced by the decreased mortality, the down-regulated transcription levels of viral genes and the improved pathological symptoms. In addition, the expression levels of SGIV induced pro-inflammation factors were decreased, while those of the antioxidant genes were increased after the supplement with TCDCA. Thus, our findings will not only contribute greatly to understanding the mechanism of SGIV pathogenesis, but also demonstrate that TCDCA might be a potential antiviral agent against SGIV infection.

Grouper Atg14 promotes Singapore grouper iridovirus (SGIV) replication by inhibiting the host innate immune response

As one of the important members of the autophagy-related protein family, Atg14 plays a key role in the formation and maturation of autophagosomes. However, little is known about the potential roles of fish Atg14 and its roles in virus infection. In the present study, the homolog of Atg14 (EcAtg14) from the orange-spotted grouper (Epinephelus coioides) was cloned and characterized. The open reading frame (ORF) of EcAtg14 consists of 1530 nucleotides, encoding 509 amino acids, with a predicted molecular weight of 56.9 kDa. EcAtg14 was distributed in all tested tissues, with higher expression in liver, blood and spleen. The expression of EcAtg14 was increased in grouper spleen (GS) cells after Singapore grouper iridovirus (SGIV) infection. EcAtg14 was distributed in the cytoplasm of GS cells. Overexpression of EcAtg14 promoted SGIV replication in GS cells and inhibited IFN3, ISRE and NF-κB promoter activities. Co-immunoprecipitation results showed that there was an interaction between EcAtg14 and EcBeclin. EcAtg14 also promoted the synthesis of LC3-II in GS cells. These findings provide a basis for understanding the innate immune mechanism of grouper against viral infection.

A novel fish CRADD inhibits Singapore grouper iridovirus (SGIV) infection by mediating apoptosis and modulating SGIV CARD (VP48)-caspase-2 interaction

The bipartite linker protein CRADD is a molecule that contains death domain (DD)- and caspase recruitment domain (CARD), and remarkably assists in inducing apoptosis and activating caspase-2. This study describes a novel fish CRADD (EcCRADD) isolated from the orange-spotted grouper (Epinephelus coioides), meanwhile demonstrating its inhibitory effect in viral replication. Our findings suggest that EcCRADD can form long-chain filamentous structures that may be involved in apoptosis triggered by nuclear fragmentation. EcCRADD engages the pro-apoptotic protein EcRIP1 (receptor interacting protein 1) through the DD domain and activates caspase-2 through the CARD domain. This bridging allows EcCRADD to promote Singapore grouper iridovirus (SGIV)-induced apoptosis and synergistically inhibits SGIV replication in vitro. SGIV contains a CARD protein encoded by ORF048L (VP48). In contrast to EcCRADD, the overexpression of VP48 significantly promotes SGIV replication and inhibits SGIV-induced apoptosis. Furthermore, we found that VP48 may utilize caspase-2 as a scaffolding molecule to activate NF-κB and promote cell survival. However, EcCRADD could activate p53 in the presence of caspase-2 and inhibit this VP48-induced NF-κB activity in a dose-dependent manner. Lastly, EcCRADD inhibits the nuclear expression of VP48 via caspase-2. EcCRADD competes with VP48 to bind with caspase-2. Our study provides insights into host–virus interactions in fish, specifically that EcCRADD inhibits virus replication via the caspase-2-activated p53 pathway and interferes with the VP48-activated NF-κB signaling pathway.

Fish SCD1 promotes SGIV infection via modulating the formation of lipid droplets and TBK1/MDA5-activated IFN signal pathway

Stearoyl CoA desaturase (SCD1), the rate-limiting enzyme for monounsaturated fatty acid production, involved in host metabolic regulation. Recent reports indicated that SCD1 played a crucial role in the process of viral infection. To investigate the function of SCD1 in teleost fish and the role it played in fish virus infection, EcSCD1 was cloned and characterized in the orange-spotted grouper, Epinephelus coioides. The open reading frame (ORF) of EcSCD1 consisted of 1005 nucleotides encoding a protein containing 334 amino acids with a predicted molecular weight of 38.33 kDa, which shared 74.05% and 60.48% identity with zebrafish and human, respectively. EcSCD1 possessed four transmembrane domains and three conserved His-rich regions. In healthy groupers, EcSCD1 was primarily expressed in the liver and brain. Subcellular localization assay revealed that EcSCD1 was mainly distributed in the cytoplasm with uniform distribution or dot-like aggregation forms, and EcSCD1 was co-localized with the endoplasmic reticulum (ER). Singapore grouper iridovirus (SGIV) as well as poly (dA: dT) stimulation induced the expression of EcSCD1. On the other hand, EcSCD1 overexpression increased SGIV infection and SCD1 enzymatic product oleic acid (OA) recovered SGIV infection suppressed by SCD1 inhibitor. In addition, EcSCD1 overexpression facilitated the formation of lipid droplets (LDs), which was crucial for SGIV replication. Furthermore, EcSCD1 overexpression down-regulated the expression of interferon signaling molecules and the activities of interferon (IFN)1/3 and interferon stimulated response element (ISRE) promotors. Moreover, EcSCD1 overexpression restricted TANK binding kinase 1 (TBK1)−/melanoma differentiation-associated protein 5 (MDA5)-, but not mitochondrial antiviral signaling protein (MAVS)-evoked interferon immune responses. Overall, our findings offered novel insights into the regulatory mechanism of EcSCD1 during virus infection.

Antiviral immunity of grouper MAP kinase phosphatase 1 to Singapore grouper iridovirus infection

Singapore grouper iridovirus (SGIV), with various mechanisms for evading and modulating host, has inflicted heavy economic losses in the grouper aquaculture. MAP kinase phosphatase 1 (MKP-1) regulates mitogen-activated protein kinases (MAPKs) to mediate the innate immune response. Here, we cloned EcMKP-1, an MKP-1 homolog from the orange-spotted grouper Epinephelus coioides, and investigated its role in the infection of SGIV. In juvenile grouper, EcMKP-1 was highly upregulated and peaked at different times after injection with lipopolysaccharide, polyriboinosinic polyribocytidylic acid and SGIV. EcMKP-1 expression in heterologous fathead minnow cells was able to suppress SGIV infection and replication. Furthermore, EcMKP-1 was a negative regulator of c-Jun N-terminal kinase (JNK) phosphorylation early in SGIV infection. EcMKP-1 decreased the apoptotic percentage and caspase-3 activity during the late stage of SGIV replication. Our results demonstrate critical functions of EcMKP-1 in antiviral immunity, JNK dephosphorylation and anti-apoptosis during SGIV infection.

Grouper cGAS is a negative regulator of STING-mediated interferon response.

Cyclic GMP-AMP synthase (cGAS) is one of the classical pattern recognition receptors that recognizes mainly intracytoplasmic DNA. cGAS induces type I IFN responses to the cGAS-STING signaling pathway. To investigate the roles of cGAS-STING signaling pathway in grouper, a cGAS homolog (named EccGAS) was cloned and identified from orange-spotted grouper (Epinephelus coioides). The open reading frame (ORF) of EccGAS is 1695 bp, encodes 575 amino acids, and contains a Mab-21 typical structural domain. EccGAS is homologous to Sebastes umbrosus and humans at 71.8% and 41.49%, respectively. EccGAS mRNA is abundant in the blood, skin, and gills. It is uniformly distributed in the cytoplasm and colocalized in the endoplasmic reticulum and mitochondria. Silencing of EccGAS inhibited the replication of Singapore grouper iridovirus (SGIV) in grouper spleen (GS) cells and enhanced the expression of interferon-related factors. Furthermore, EccGAS inhibited EcSTING-mediated interferon response and interacted with EcSTING, EcTAK1, EcTBK1, and EcIRF3. These results suggest that EccGAS may be a negative regulator of the cGAS-STING signaling pathway of fish.

Characterization and functional analysis of GSK3β from Epinephelus coioides in Singapore grouper iridovirus infection

Glycogen synthase kinase 3β (GSK3β), a serine/threonine protein kinase, is a crucial regulator of several signaling pathways and plays a vital role in cell proliferation, growth, apoptosis, and immune responses. However, the role of GSK3β during viral infection in teleosts remains largely unknown. In the present study, a GSK3β homologue from Epinephelus coioides (EcGSK3β) was cloned and characterized. The open reading frame of EcGSK3β consists of 1323 bp, encoding a 440 amino acid protein, with a predicted molecular mass of 48.23 kDa. Similar to its mammalian counterpart, EcGSK3β contains an S_TKc domain. EcGSK3β shares 99.77% homology with the giant grouper (Epinephelus lanceolatus). Quantitative real-time PCR analysis indicated that EcGSK3β mRNA was broadly expressed in all tested tissues, with abundant expression in the skin, blood, and intestines. Additionally, the expression of EcGSK3β increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that EcGSK3β is mainly distributed in the cytoplasm. EcGSK3β overexpression promoted SGIV replication during viral infection in vitro. In contrast, silencing of EcGSK3β inhibited SGIV replication. EcGSK3β significantly downregulated the activities of interferon-β, interferon-sensitive response element, and NF-κB. Taken together, these findings are important for a better understanding of the function of GSK3β in fish and reveal its involvement in the host response to viral immune challenge.

Grouper TRIM23 exerts antiviral activity against iridovirus and nodavirus.

TRIM (tripartite motif) proteins have been demonstrated to exert critical roles in host defense against different microbial pathogens. Among them, TRIM23 acts as an important regulatory factor in antiviral immune and inflammatory responses, but the roles of fish TRIM23 against virus infection still remain largely unknown. Here, we investigated the characteristics of TRIM23 homolog from orange spotted grouper (Epinephelus coioides) (EcTRIM23). EcTRIM23 encoded a 580 amino acid peptide, which shared 93.1%, 89.73% and 86.36% identity with golden perch (Perca flavescens), zebrafish (Danio rerio) and human (Homo sapiens), respectively. The transcription levels of EcTRIM23 were significantly up-regulated in response to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTRIM23 overexpression in vitro significantly inhibited RGNNV and SGIV replication, evidenced by the delayed cytopathic effect (CPE) progression and the decreased expression of viral core genes. EcTRIM23 significantly increased the expression levels of interferon (IFN) related signaling molecules and pro-inflammatory cytokines, as well as the promoter activities of IFN and NF-κB, suggesting that EcTRIM23 exerted antiviral function by positively regulating host IFN response. Exogenous EcTRIM23 exhibited either diffuse or aggregated localization in grouper cells. After co-transfection, TANK binding kinase 1 (TBK1), TNF receptor associated factor (TRAF) 3 and TRAF4, TRAF5 and TRAF6 were found to interact with EcTRIM23 in grouper cells. Moreover, these proteins could be recruited and co-localized with EcTRIM23 in vitro. Together, our results demonstrated that fish TRIM23 exerted antiviral activity against fish viruses by interacting with multiple host proteins to regulate immune responses.

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.

Research on the indirect antiviral function of medicinal plant ingredient quercetin against grouper iridovirus infection

Grouper iridovirus is a devastating pathogen that belongs to the genus Ranavirus. Based on the previous results that natural ingredient quercetin isolated from Illicium verum Hook. f. could effectively inhibit Singapore grouper iridovirus (SGIV) replication, suggesting that quercetin could serve as potential antiviral agent against grouper iridovirus. To know about whether quercetin has indirect antiviral activity against SGIV, this study made the investigation in vitro and in vivo, and the potential mechanism was also explored. Pretreating the cells with quercetin (12.5 μg/mL) significantly inhibited the replication of SGIV, similar results were also confirmed in vivo. Importantly, quercetin pretreatment could induce the expression of genes involved in type I interferon (IFN) system (IFN, STAT1, PKR, MxI and ISG15) and TLR9. It suggested that quercetin exerted the indirect antiviral activity against SGIV infection through promoting the recognition of SGIV and activating the IFN pathway to establish the antiviral status of host cell. Taken together, our results shedded light on the indirect antiviral function of natural ingredient quercetin, and clearly demonstrated that natural ingredient quercetin will be an excellent potential agent against SGIV infection in grouper aquaculture.

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

Transforming growth factor-β 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 IKKβ. 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.

Singapore Grouper Iridovirus Induces Glucose Metabolism in Infected Cells by Activation of Mammalian Target of Rapamycin Signaling.

Singapore grouper iridovirus (SGIV), a member of the Iridoviridae family, is an important marine cultured fish pathogen worldwide. Our previous studies have demonstrated that lipid metabolism was essential for SGIV entry and replication, but the roles of glucose metabolism during SGIV infection still remains largely unknown. In this study, we found that the transcription levels of key enzymes involved in glycolysis were regulated in varying degrees during SGIV infection based on the transcriptomic analysis. Quantitative PCR and western blot analysis also indicated that the expression of both glucose transporters (GLUT1 and GLUT2) and the enzymes of glucose metabolism (hexokinase 2, HK2 and pyruvate dehydrogenase complex, PDHX) were upregulated during SGIV infection in vivo or in vitro, suggesting that glycolysis might be involved in SGIV infection. Exogenous glucose supplementation promoted the expression of viral genes and infectious virion production, while glutamine had no effect on SGIV infection, indicating that glucose was required for SGIV replication. Consistently, pharmacological inhibition of glycolysis dramatically reduced the protein synthesis of SGIV major capsid protein (MCP) and infectious virion production, and promotion of glycolysis significantly increased SGIV infection. Furthermore, knockdown of HK2, PDHX, or GLUT1 by siRNA decreased the transcription and protein synthesis of SGIV MCP and suppressed viral replication, indicating that those enzymes exerted essential roles in SGIV replication. In addition, inhibition of mTOR activity in SGIV-infected cells effectively reduced the expression of glycolysis key enzymes, including HK2, PDHX, GLUT1, and GLUT2, and finally inhibited SGIV replication, suggesting that mTOR was involved in SGIV-induced glycolysis. Thus, our results not only provided new insights into the mechanism of how SGIV infection affects host cell glycolysis, but also contributed to further understanding of the iridovirus pathogenesis.

Identification and functional characterization of Cystatin B in orange-spotted grouper, Epinephelus coioides

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.