Grouper Iridovirus Research Articles

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.

Curcumin inhibits Singapore grouper iridovirus infection through multiple antiviral mechanisms

Singapore grouper iridovirus (SGIV) is one of the main pathogens that causes high mortality and substantial economic losses in the grouper aquaculture industry. Curcumin is a polyphenolic compound extracted from the traditional Chinese medicinal material, turmeric. In this study, we investigated the antiviral properties of curcumin against SGIV based on quantitative real-time PCR (qRT-PCR), western blotting, and viral titer assessment with in vitro models, and found that different concentrations of curcumin inhibited SGIV infection. Furthermore, curcumin significantly impaired attachment of the virion. Moreover, the qRT-PCR results showed that cellular immune and inflammatory responses related to the NF-κB signaling pathway were regulated by curcumin. Examinations of the expression of antioxidant enzymes indicated that the antioxidant capacity played a critical role in preventing SGIV infection, which may depend on the Nrf2/Keap1 signaling pathway. Importantly, curcumin also had a strong antiviral activity in vivo according to the increased survival rates of groupers and inhibition of SGIV gene expression. This study demonstrated that curcumin could inhibit SGIV infection both in vitro through multiple antiviral mechanisms and reduce the mortality of SGIV-infected groupers in vivo. Overall, our findings show that SGIV is sensitive to curcumin and its antiviral activity makes it a potential candidate drug for treating SGIV infection.

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.

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.

Characterization of pIgR and its association with resistance to iridovirus in Asian seabass

The improvement of disease resistance is vital in aquaculture as diseases are one of the serious threats to successful aquaculture. Understanding of molecular mechanisms underlying disease resistance is important in improving resistance against diseases. The pIgR (polymeric immunoglobulin receptor) gene plays an important role in the mucosal immunity of fish. However, not much is known about its roles in inhibiting pathogens in aquaculture species. Here, the pIgR gene from Asian seabass (Lates calcarifer) was characterized, and its functions on the iridovirus infection were examined. LcapIgR consisted of a 2683 bp ORF, a 69 bp 5’ UTR and a 1597 bp 3’ UTR. qRT-PCR revealed that LcapIgR was expressed in all 11 organs examined, being the highest in the heart, followed by intestine, kidney, and eye. Upon a SGIV (Singapore grouper iridovirus) challenge of the fish, the expression of LcapIgR in kidney increased significantly. Overexpression of LcapIgR in an Asian seabass cell line reduced iridovirus entry into cells, and significantly lowered the transcription and replication of the gene encoding for the iridovirus major capsid protein. These results suggest that LcapIgR play an important role in repressing the pathogenic activity of iridovirus. In addition, two SNPs were identified in the 3’ UTR of LcapIgR. Genotyping of both SNPs in survival and dead Asian seabass after challenging with SGIV revealed that both SNPs were possibly associated with resistance to SGIV. Therefore, both SNPs may play a useful role in the selection of fish that are resistant against iridovirus at the fingerling stage.

Establishment of a new intestinal cell line from the grouper (Epinephelus coioides) for aquatic virus pathogenesis

A new cell line (ECGI-21) was developed from the intestine of grouper (Epinephelus coioides). The cell line was routinely grown in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum (FBS) at 28 °C. ECGI-21 cells had an epithelioid morphology. At present, the cell line has been stably passed over 80 generations and has been successfully cryopreserved. The origin of grouper was confirmed by the sequence of 18S ribosomal RNA (18S rRNA). Chromosome analysis indicated that the modal chromosome number was 48. The green fluorescence signals were detected when pEGFP-N3 was transfected into ECGI-21 cells. Moreover, the ECGI-21 cells were susceptible to Singapore grouper iridovirus (SGIV) and Red-spotted grouper nervous necrosis virus (RGNNV), which was demonstrated by the presence of a significant cytopathic effect (CPE), increased expression of viral proteins after viral infection. Meantime, abundant viral particles were observed in the cells infected with SGIV and RGNNV by electron microscopy. Furthermore, virus replication significantly increased the level of interferon (IFN) related signaling molecules and pro-inflammatory cytokine expression in cells. In summary, ECGI-21 cell line was established and become the first stable intestinal cell line from marine fish. It provides a useful tool for studying problems of gastrointestinal health, viral pathogenesis and virus-host interaction in vitro.

Comparative transcriptomic analysis reveals different host cell responses to Singapore grouper iridovirus and red-spotted grouper nervous necrosis virus

Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) are important pathogens that cause high mortality and heavy economic losses in grouper aquaculture. Interestingly, SGIV infection in grouper cells induces paraptosis-like cell death, while RGNNV infection induces autophagy and necrosis characterized morphologically by vacuolation of lysosome. Here, a comparative transcriptomic analysis was carried out to identify the different molecular events during SGIV and RGNNV infection in grouper spleen (EAGS) cells. The functional enrichment analysis of DEGs suggested that several signaling pathways were involved in CPE progression and host immune response against SGIV or RGNNV. Most of DEGs featured in the KEGG “lysosome pathway” were up-regulated in RGNNV-infected cells, indicating that RGNNV induced lysosomal vacuolization and autophagy might be due to the disturbance of lysosomal function. More than 100 DEGs in cytoskeleton pathway and mitogen-activated protein kinase (MAPK) signal pathway were identified during SGIV infection, providing additional evidence for the roles of cytoskeleton remodeling in cell rounding during CPE progression and MAPK signaling in SGIV induced cell death. Of note, consistent with changes at the transcriptional levels, the post-translational modifications of MAPK signaling-related proteins were also detected during RGNNV infection, and the inhibitors of extracellular signal-regulated kinase (ERK) and p38 MAPK significantly suppressed viral replication and virus induced vacuoles formation. Moreover, the majority of DEGs in interferon and inflammation signaling were obviously up-regulated during RGNNV infection, but down-regulated during SGIV infection, suggesting that SGIV and RGNNV differently manipulated host immune response in vitro. In addition, purine and pyrimidine metabolism pathways were also differently regulated in SGIV and RGNNV-infection cells. Taken together, our data will provide new insights into understanding the potential mechanisms underlying different host cell responses against fish DNA and RNA virus.

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.

Aptamer-Based High-Throughput Screening Model for Efficient Selection and Evaluation of Natural Ingredients against SGIV Infection

Singapore grouper iridovirus (SGIV) causes high economic losses in mariculture. Effective drugs for managing SGIV infection are urgently required. Medicinal plant resources are rich in China. Medicinal plants have a long history and significant curative effects in the treatment of many diseases. Reverse-transcription quantitative real-time PCR is the most commonly used method for detecting virus infection and assessing antiviral efficacy with high accuracy. However, their applications are limited due to high reagent costs and complex time-consuming operations. Aptamers have been applied in some biosensors to achieve the accurate detection of pathogens or diseases through signal amplification. This study aimed to establish an aptamer-based high-throughput screening (AHTS) model for the efficient selection and evaluation of medicinal plants components against SGIV infection. Q2-AHTS is an expeditious, rapid method for selecting medicinal plant drugs against SGIV, which was characterized as being dram, high-speed, sensitive, and accurate. AHTS strategy reduced work intensity and experimental costs and shortened the whole screening cycle for effective ingredients. AHTS should be suitable for the rapid selection of effective components against other viruses, thus further promoting the development of high-throughput screening technology.

Effective protection against Ranavirus-type grouper iridovirus and Vibrio harveyi by priming a bivalent GrouperVAC-Irido-R-Vh followed by boosting a monovalent V. harveyi vaccine

Natural outbreaks of Ranavirus-type grouper iridovirus (GIV-R) and/or Vibrio harveyi result in massive mortalities and severe economic losses in grouper industry. However, effective vaccines against both pathogens have not yet been applied commercially. In this study, a water-in-oil formation of formalin-killed bivalent vaccine (GrouperVAC-Irido-R-Vh) against both GIV-R and V. harveyi and a monovalent vaccine (GrouperVAC-Vh) against V. harveyi were developed and evaluated both under laboratory and field trial tests. The result showed that the single intraperitoneally-injected grouper juveniles (N = 35) with bivalent GrouperVAC-Irido-R-Vh under laboratory condition were protected against GIV-R and V. harveyi at 21 days post vaccination (dpv) with relative percent survivals (RPS) of >90% and 80%, respectively. In a field trial test (N = 7500), the effective protections of single intraperitoneally-injected fish were also observed in vaccinated grouper juveniles at 90 dpv, with RPSs of 87.5% and 70.4% against GIV-R and V. harveyi challenge, respectively, indicating that the bivalent GrouperVAC-Irido-R-Vh conferred effective protections against both GIV-R and V. harveyi infection for at least 3 months. Moreover, based on primary injection of GrouperVAC-Irido-R-Vh, a booster immunization of the single component of GrouperVAC-Vh (N = 4500) can prolong conferring effective protection against vibriosis caused by V. harveyi from 3 months to 9 months with an RPS of 88.2%. Given that the large-sized grouper is much less sensitive to GIV-R but remains highly susceptible to V. harveyi, we propose that the optimal immunization programs against GIV-R and V. harveyi are primary immunization with the bivalent GrouperVAC-Irido-R-Vh vaccine followed by boosting a monovalent GrouperVAC-Vh vaccine.

Antiviral Activities of Green Tea Components against Grouper Iridovirus Infection In Vitro and In Vivo.

(1) Background: Singapore grouper iridovirus (SGIV) can cause extensive fish deaths. Therefore, developing treatments to combat virulent SGIV is of great economic importance to address this challenge to the grouper aquaculture industry. Green tea is an important medicinal and edible plant throughout the world. In this study, we evaluated the use of green tea components against SGIV infection. (2) Methods: The safe working concentrations of green tea components were identified by cell viability detection and light microscopy. Additionally, the antiviral activity of each green tea component against SGIV infection was determined with light microscopy, an aptamer (Q5c)-based fluorescent molecular probe, and reverse transcription quantitative PCR. (3) Results: The safe working concentrations of green tea components were green tea aqueous extract (GTAE) ≤ 100 μg/mL, green tea polyphenols (TP) ≤ 10 μg/mL, epigallocatechin-3-gallate (EGCG) ≤ 12 μg/mL, (-)-epigallocatechin (EGC) ≤ 10 μg/mL, (-)-epicatechin gallate (EGC) ≤ 5 μg/mL, and (-)-epicatechin (EC) ≤ 50 μg/mL. The relative antiviral activities of the green tea components determined in terms of MCP gene expression were TP > EGCG > GTAE > ECG > EGC > EC, with inhibition rates of 99.34%, 98.31%, 98.23%, 88.62%, 73.80%, and 44.31%, respectively. The antiviral effect of aptamer-Q5c was consistent with the results of qPCR. Also, TP had an excellent antiviral effect in vitro, wherein the mortality of fish in only the SGIV-injection group and TP + SGIV-injection group were 100% and 11.67%, respectively. (4) Conclusions: In conclusion, our results suggest that green tea components have effective antiviral properties against SGIV and may be candidate agents for the effective treatment and control of SGIV infections in grouper aquaculture.

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-1β, 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.

Antiviral effect and mechanism of metformin against grouper iridovirus infection

AbstractSingapore grouper iridovirus (SGIV) infection is associated with high mortality in mariculture, and effective treatments are urgently required. Given the wide‐ranging applications of metformin, we aimed to assess the effects of metformin against SGIV. The safe and effective concentration of metformin in grouper spleen cells was determined as 250 μg/mL. We detected the inhibitory activity of metformin against SGIV infection in grouper spleen cells using an aptamer‐based fluorescent molecular probe and reverse transcription‐quantitative real‐time polymerase chain reaction. Metformin effectively inhibited SGIV infection in a dose‐dependent manner and showed an excellent inhibitory effect against SGIV in vitro (70%). We further investigated metformin's possible antiviral mechanism and showed that metformin did not damage SGIV invading host cells, but could damage SGIV particles (59.57%) and interfere with SGIV binding (23.25%) and replication (29.48%) in host cells. Metformin mainly inhibited SGIV by damaging SGIV particles. Overall, these results suggested that metformin had direct and host‐mediated antiviral effects against SGIV and thus had great potential for the development of effective drugs to control SGIV infection in aquaculture.

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.

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.

Antiviral activities of Glycyrrhiza uralensis components against Singapore grouper iridovirus

AbstractSingapore grouper iridovirus (SGIV) is a serious pathogen that negatively impacts grouper aquaculture. Because of its 90% fatality rate, effective medications that control SGIV infection are urgently needed. Glycyrrhiza uralensis is a well‐known medicinal plant that contains active ingredients with antiviral, antibacterial, and other biological activities. In this study, we evaluated the application of G. uralensis components against SGIV infection. The safe working concentrations of G. uralensis components, which were identified by light microscope observation and CCK‐8 detection, were as follows: aqueous extract (GUF) ≤ 10 mg/ml, glycyrrhizin ≤ 100 μg/ml, glycyrrhetinic acid ≤ 50 μg/ml, liquiritin ≤ 25 μg/ml, isoliquiritigenin ≤ 10 μg/ml, and liquiritigenin ≤ 10 μg/ml. The ingredients did not have significant effects against SGIV infection, whereas GUF showed significant anti‐SGIV infection activity in a concentration‐dependent manner, and it also destroyed the structure of virus particles. Therefore, GUF might impact the binding of virus particles to cell receptors and the stage of virus replication in host cells. These results show that G. uralensis aqueous extracts have good anti‐SGIV effects and great potential for treating SGIV infection.

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.

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.