Singapore Grouper Iridovirus Infection Research Articles

Ubiquitin-Proteasome System Is Required for Efficient Replication of Singapore Grouper Iridovirus.

The ubiquitin–proteasome system (UPS) serves as the major intracellular pathway for protein degradation and plays crucial roles in several cellular processes. However, little is known about the potential actions of the UPS during fish virus infection. In this study, we elucidated the possible roles of UPS in the life cycle of Singapore grouper iridovirus (SGIV); a large DNA virus that usually causes serious systemic diseases with high mortality in groupers. Data from transcriptomic analysis of differentially expressed genes illustrated that expression of 65 genes within the UPS pathway, including ubiquitin encoding, ubiquitination, deubiquitination, and proteasome, were up- or down-regulated during SGIV infection. Using different proteasome inhibitors, inhibition of the proteasome decreased SGIV replication in vitro, accompanied by inhibition of virus assembly site formation, and viral gene transcription and protein transportation. Over-expression of ubiquitin partly rescued the inhibitory effect of ubiquitin inhibitor on SGIV replication, suggesting that UPS was required for fish iridovirus infection in vitro. Viral or host proteins regulated by proteasome inhibition during SGIV infection were investigated with two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sixty-two differentially expressed proteins, including 15 viral and 47 host proteins, were identified after SGIV infection. The host proteins were involved in ubiquitin-mediated protein degradation, metabolism, cytoskeleton, macromolecular biosynthesis, and signal transduction. Among them, 11 proteins were negatively regulated upon MG132 treatment during SGIV infection. This is believed to be the first study to provide evidence that UPS was essential for fish virus infection and replication.

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

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

Transcriptomics analysis reveals candidate genes and pathways for susceptibility or resistance to Singapore grouper iridovirus in orange-spotted grouper (Epinephelus coioides)

In this study, the transcriptional response of grouper to Singapore grouper iridovirus (SGIV) stimulation was characterized using RNA sequencing. Transcriptome sequencing of three test groups in the grouper was performed using the Illumina MiSeq platform. The three test groups were a control group, which was injected with PBS buffer; a high-susceptible (HS) group, which died shortly after the SGIV injection; and a high-resistance (HR) group, which survived the SGIV injection. In total, 38,253 unigenes were generated. When the HS group was compared with the control group, 885 unigenes were upregulated and 487 unigenes were downregulated. When the HR and control groups were compared, 1114 unigenes were upregulated and 420 were downregulated, and when the HR and HS groups were compared, 1010 unigenes were upregulated and 375 were downregulated. In the KEGG analysis, two immune-related pathways, the p53 and peroxisome proliferator-activated receptor pathways, were detected with highly significant enrichment. In addition, 7465 microsatellites and 22,1569 candidate single nucleotide polymorphisms were identified from our transcriptome data. The results suggested several pathways that are associated with traits of disease susceptibility or disease resistance, and provided extensive information about novel gene sequences, gene expression profiles, and genetic markers. This may contribute to vaccine research and a breeding program against SGIV infection in grouper.

The CXC chemokines and CXC chemokine receptors in orange-spotted grouper (Epinephelus coioides) and their expression after Singapore grouper iridovirus infection

Chemokines comprise a group of small molecular weight (6–14 kDa) cytokines; chemokine receptors are a superfamily of seven transmembrane domain G-coupled receptors. Both chemokines and their receptors have important roles in immune surveillance, inflammation, and development. Recently, 9 CXC chemokine ligands (CXCLs) and 8 CXC chemokine receptors (CXCRs) were identified and cloned from orange-spotted grouper (Epinephelus coioides) and annotated by phylogenetic and syntenic analyses. We detected mRNA transcripts for CXCLs and CXCRs in healthy tissues of E. coioides. Our data show that CXCL genes are highly expressed in the spleen, kidney and liver and that CXCR genes are ubiquitously expressed, rather than being expressed only in immune organs. Analysis of gene expression after Singapore grouper iridovirus infection indicated that CXCL and CXCR genes are regulated in a gene-specific manner. CXCL8 and CXCL12a were significantly upregulated in the spleen, kidney and liver of resistant fish, indicating potential roles in immunity against the pathogen. Additionally, CXCR4a was upregulated in all three organs in resistant fish, suggesting that CXCL8 or CXCL12a may participate in the immune response via interaction with CXCR4a. In addition, the new orange-spotted grouper receptor CXCR1b was found to be upregulated in the spleen and kidney of resistant fish, indicating that this receptor plays an important role in immune responses to viral infection. These results are valuable for comparative immunological studies and provide insight into the roles of these genes in viral infection.

Molecular cloning and characterization of FADD from the orange-spotted grouper (Epinephelus coioides)

Fas-associated protein with death domain (FADD) is the key adaptor protein that transmits apoptotic signals mediated by the main death receptors. Besides being an essential instrument in cell death, FADD is also implicated in proliferation, cell cycle progression, tumor development, inflammation, innate immunity, and autophagy. In the present study, a FADD homologue (EcFADD) from the orange-spotted grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcFADD contains 808 base pairs (bp), including a 573 bp open reading frame that encodes a 190 amino acid protein with a predicted molecular mass of 21.81 kDa. Quantitative real-time polymerase chain reaction analysis indicated that EcFADD was distributed in all examined tissues. The expression of EcFADD in the spleen of E. coioides was differentially up-regulated when challenged with Singapore grouper iridovirus (SGIV) or polyinosine-polycytidylic acid(poly[I:C]). EcFADD was abundantly distributed in both the cytoplasm and nucleus in grouper spleen (GS) and fathead minnow (FHM) epithelial cells. Over-expression of EcFADD inhibited SGIV infection and replication and SGIV-induced apoptosis. To achieve antiviral and anti-apoptosis activities, FADD promoted the activation of interferon-stimulated response element (ISRE) and type I interferon (IFN) genes in the antiviral IFN signaling pathway and inhibited activation of apoptosis-related transcription factors p53. Our results not only characterize FADD but also reveal new immune functions and the molecular mechanisms by which FADD responds to virus infection and virus-induced apoptosis.

Probing and characterizing the high specific sequences of ssDNA aptamer against SGIV-infected cells

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

Identification of Inhibitory Compounds Against Singapore Grouper Iridovirus Infection by Cell Viability-Based Screening Assay and Droplet Digital PCR.

Singapore grouper iridovirus (SGIV) is one of the major causative agents of fish diseases and has caused significant economic losses in the aquaculture industry. There is currently no commercial vaccine or effective antiviral treatment against SGIV infection. Annually, an increasing number of small molecule compounds from various sources have been produced, and many are proved to be potential inhibitors against viruses. Here, a high-throughput in vitro cell viability-based screening assay was developed to identify antiviral compounds against SGIV using the luminescent-based CellTiter-Glo reagent in cultured grouper spleen cells by quantificational measurement of the cytopathic effects induced by SGIV infection. This assay was utilized to screen for potential SGIV inhibitors from five customized compounds which had been reported to be capable of inhibiting other viruses and 30 compounds isolated from various marine organisms, and three of them [ribavirin, harringtonine, and 2-hydroxytetradecanoic acid (2-HOM)] were identified to be effective on inhibiting SGIV infection, which was further confirmed with droplet digital PCR (ddPCR). In addition, the ddPCR results revealed that ribavirin and 2-HOM inhibited SGIV replication and entry in a dose-dependent manner, and harringtonine could reduce SGIV replication rather than entry at the working concentration without significant toxicity. These findings provided an easy and reliable cell viability-based screening assay to identify compounds with anti-SGIV effect and a way of studying the anti-SGIV mechanism of compounds.

Fish miR-146a promotes Singapore grouper iridovirus infection by regulating cell apoptosis and NF-κB activation.

miR-146a was reported to participate in various pathophysiological conditions in mammals, such as inflammation and immune responses, oncogenesis and cell damage. However, its function in low vertebrates has not been well elucidated. In this study, we characterized the expression profiles and functions of miR-146a in fish cells during iridovirus infection. We found that the reported fish miR-146a genes encoded an identical mature sequence, which shared high similarity with its mammalian orthologues, suggesting a putative functional conservation of miR-146a between fish and other vertebrates. Using a well-established infection model of Singapore grouper iridovirus (SGIV) in fathead minnow cells, we found that SGIV infection induced the expression of miR-146a to a dramatic extent. More importantly, we found that miR-146a promoted SGIV propagation, as demonstrated by higher expression of viral genes and increased virus titres in miR-146a-overexpressing cells. Mechanistically, we found that miR-146a overexpression suppressed, while miR-146a knockdown promoted, NF-κB activation and SGIV-induced cell apoptosis, two major cellular events involved in SGIV infection. Our study suggested that the induction of miR-146a by SGIV infection may function through a feed-forward mechanism to promote viral infection by restraining anti-viral cellular responses.

Fish TRIM32 functions as a critical antiviral molecule against iridovirus and nodavirus

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

Singapore grouper iridovirus protein VP088 is essential for viral infectivity.

Viral infection is a great challenge in healthcare and agriculture. The Singapore grouper iridovirus (SGIV) is highly infectious to numerous marine fishes and increasingly threatens mariculture and wildlife conservation. SGIV intervention is not available because little is known about key players and their precise roles in SGVI infection. Here we report the precise role of VP088 as a key player in SGIV infection. VP088 was verified as an envelope protein encoded by late gene orf088. We show that SGIV could be neutralized with an antibody against VP088. Depletion or deletion of VP088 significantly suppresses SGIV infection without altering viral gene expression and host responses. By precisely quantifying the genome copy numbers of host cells and virions, we reveal that VP088 deletion dramatically reduces SGIV infectivity through inhibiting virus entry without altering viral pathogenicity, genome stability and replication and progeny virus release. These results pinpoint that VP088 is a key player in SGIV entry and represents an ideal target for SGIV intervention.

Development and characterization of aptamer-based enzyme-linked apta-sorbent assay for the detection of Singapore grouper iridovirus infection

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

Subcellular redistribution and sequential recruitment of macromolecular components during SGIV assembly.

Virus infection consists of entry, synthesis of macromolecular components, virus assembly and release. Understanding of the mechanisms underlying each event is necessary for the intervention of virus infection in human healthcare and agriculture. Here we report the visualization of Singapore grouper iridovirus (SGIV) assembly in the medaka haploid embryonic stem (ES) cell line HX1. SGIV is a highly infectious DNA virus that causes a massive loss in marine aquaculture. Ectopic expression of VP88GFP, a fusion between green fluorescent protein and the envelope protein VP088, did not compromise the ES cell properties and susceptibility to SGIV infection. Although VP88GFP disperses evenly in the cytoplasm of non-infected cells, it undergoes aggregation and redistribution in SGIV-infected cells. Real-time visualization revealed multiple key stages of VP88GFP redistribution and the dynamics of viral assembly site (VAS). Specifically, VP88GFP entry into and condensation in the VAS occurred within a 6-h duration, a similar duration was observed also for the release of VP88GFP-containing SGIV out of the cell. Taken together, VP088 is an excellent marker for visualizing the SGIV infection process. Our results provide new insight into macromolecular component recruitment and SGIV assembly.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0292-3) contains supplementary material, which is available to authorized users.

C-Jun N-terminal kinases 3 (JNK3) from orange-spotted grouper, Epinephelus coioides, inhibiting the replication of Singapore grouper iridovirus (SGIV) and SGIV-induced apoptosis

C-Jun N-terminal kinases (JNKs), a subgroup of serine-threonine protein kinases that activated by phosphorylation, are involve in physiological and pathophysiological processes. JNK3 is one of JNK proteins involved in JNK3 signaling transduction. In the present study, two JNK3 isoforms, Ec-JNK3 X1 and Ec-JNK3 X2, were cloned from orange-spotted grouper, Epinephelus coioides. Both Ec-JNK3 X1 and Ec-JNK3 X2 were mainly expressed in liver, gill, skin, brain and muscle of juvenile grouper. The relative expression of Ec-JNK3 X2 mRNA was much higher in muscle and gill than that of Ec-JNK3 X1. Isoform-specific immune response to challenges was revealed by the expression profiles in vivo. Immunofluorescence staining indicated that JNK3 was localized in the cytoplasm of grouper spleen (GS) cells and shown immune response to SGIV infection in vitro. Over-expressing Ec-JNK3 X1 and/or Ec-JNK3 X2 inhibited the SGIV infection and replication and the SGIV-induced apoptosis. To achieve the antiviral and anti-apoptosis activities, JNK3 promoted the activation of genes ISRE and type I IFN in the antiviral IFN signaling pathway, and inhibited the activation of transcription factors NF-κB and p53 relating to apoptosis, respectively. Ec-JNK3 X2 showed stronger activities in antivirus and anti-apoptosis than that of Ec-JNK3 X1. Our results not only define the characterization of JNK3 but also reveal new immune functions and the molecular mechanisms of JNK3 on iridoviruses infection and the virus-induced apoptosis.

RING domain is essential for the antiviral activity of TRIM25 from orange spotted grouper.

Tripartite motif-containing 25 (TRIM25) has been demonstrated to exert crucial roles in the regulation of innate immune signaling. However, the roles of fish TRIM25 in antiviral immune response still remained uncertain. Here, a novel fish TRIM25 gene from orange spotted grouper (EcTRIM25) was cloned and its roles in grouper virus infection were elucidated. EcTRIM25 encoded a 734-aa protein which shared 68% identity to large yellow croaker (Larimichthys crocea). Amino acid alignment showed that EcTRIM25 contained three conserved domains, including a RING-finger domain, a B box/coiled-coil domain and a SPRY domain. In healthy grouper, the transcript of EcTRIM25 was predominantly detected in skin, spleen and intestine. After stimulation with Singapore grouper iridovirus (SGIV) or poly I:C, the relative expression of EcTRIM25 in grouper spleen was significantly increased at the early stage of injection. Subcellular localization analysis showed that EcTRIM25 distributed throughout the cytoplasm in grouper cells. Notably, the deletion RING domain affected its accurate localization and displayed microtubule like structures or bright aggregates in GS cells. After incubation with SGIV or red spotted grouper nervous necrosis virus (RGNNV), overexpression of full length of EcTRIM25 invitro significantly decreased the viral gene transcription of SGIV and RGNNV. Consistently, the deletion of RING domain obviously affected the inhibitory effect of EcTRIM25. Furthermore, overexpression of EcTRIM25 significantly increased the expression level of interferon related signaling molecules, including interferon regulatory factor (IRF) 3, interferon-induced 35-kDa protein (IFP35), MXI, IRF7 and myeloid differentiation factor 88 (MyD88), suggesting that the positive regulation of interferon immune response by EcTRIM25 might affected RGNNV replication directly. Meanwhile, the expression levels of pro-inflammation cytokines were differently regulated by the ectopic expression of EcTRIM25. We proposed that the regulation of IRF7, MyD88 and pro-inflammation cytokines might contribute more important roles in SGIV infection. In addition, the RING domain of EcTRIM25 also played critical roles in the regulation of interferon immune and inflammation response. Together, our results will provide new evidences that the RING domain was essential for the antiviral action of fish TRIM25 against iridovirus and nodavirus infection.

Establishment of a new cell line from the snout tissue of golden pompano Trachinotus ovatus, and its application in virus susceptibility.

A new marine-fish cell line, designated GPS, was established from the snout tissue of golden pompano Trachinotus ovatus. GPS cells multiplied well in Leibovitz's L-15 containing 10% foetal bovine serum (FBS) at 28° C and the cells have been subcultured for >60 passages. Polymerase chain reaction (PCR) amplification of 16S ribosomal (r)RNA confirmed the origin of this cell line from T. ovatus. Chromosome analysis showed that GPS cells exhibited chromosomal aneuploidy with a modal chromosome number of 54. Bright green fluorescence signal was observed in enhanced green fluorescent protein (EGFP)-N3 transfected cells, indicating that GPS cells could be used to investigate gene functions in vitro. The GPS cells were highly susceptible to Singapore grouper iridovirus (SGIV), which was demonstrated by the presence of severe cytopathic effect (CPE) and increased viral titres. Real-time quantitative PCR and Western blot analysis showed that the viral gene transcription and protein synthesis occurred during SGIV infection in GPS cells. Thus, this study described the characteristic of a new cell line from the snout tissue of T. ovatus that could be used as a tool for propagation of iridovirus and genetic manipulation to investigate host-pathogen interactions.

Fish TRIM8 exerts antiviral roles through regulation of the proinflammatory factors and interferon signaling.

The tripartite motif (TRIM)-containing proteins usually exert important regulatory roles during multiple biological processes. TRIM8 has been demonstrated to be a RING domain-containing E3 ubiquitin ligase which plays critical roles in inflammation and cancer. In this study, a TRIM8 homolog from grouper, Epinephelus coioides (EcTRIM8) was cloned, and its effects on fish virus replication were investigated. The full-length EcTRIM8 cDNA encoded a polypeptide of 568 amino acids with 92% identity to TRIM8 homolog from large yellow croaker (Larimichthys crocea). Sequence alignment analysis indicated that EcTRIM8 contained conserved RING finger, B-box and coiled-coil domain. Expression patterns analysis showed that EcTRIM8 was predominant in kidney, gill, fin, liver, spleen and brain. After challenging with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the EcTRIM8 transcript was significantly increased at the early stage of injection. Under fluorescence microscopy, we observed different distribution patterns of EcTRIM8 in grouper spleen (GS) cells, including punctate fluorescence evenly situated throughout the cytoplasm and bright aggregates. The ectopic expression of EcTRIM8 in vitro significantly inhibited the replication of SGIV and red spotted grouper nervous necrosis virus (RGNNV), evidenced by the obvious reduction in the severity of cytopathic effect (CPE) and the significant decrease in viral gene transcription and protein synthesis. Moreover, the transcription of the proinflammatory factors and interferon related immune factors were differently regulated by EcTRIM8 during SGIV or RGNNV infection. In addition, overexpression of EcTRIM8 significantly increased the transcription of interferon regulator factor 3 (IRF3) and IRF7, and enhanced IRF3 or IRF7 induced interferon-stimulated response element (ISRE) promoter activity. Together, our results firstly demonstrated that fish TRIM8 could exert antiviral function through the regulation of the expression of proinflammatory cytokines and interferon related transcription factors in response to fish viruses.

MHC class IIα polymorphisms and their association with resistance/susceptibility to Singapore grouper iridovirus (SGIV) in orange-spotted grouper, Epinephelus coioides

The major histocompatibility complex (MHC) is a crucial component of the immune systems of vertebrate animals and is associated with their resistance/susceptibility to pathogenic diseases. In this study, the association between MHC IIα gene polymorphisms and disease resistance/susceptibility was analyzed in the orange-spotted grouper (Epinephelus coioides), by challenging it with Singapore grouper iridovirus (SGIV). Thirty-seven alleles showing high levels of polymorphism (24.7% mutation sites) were isolated from 80 individuals. In the peptide-binding region, the ratio of nonsynonymous (dN) substitutions to synonymous (dS) substitutions was 1.427 (>1), suggesting that the loci are evolving under positive balancing selection. Eight alleles were selected to estimate their distributions in high-resistance (HR) and high-susceptibility (HS) groups of fish. The EPCO-DAA*0101 allele was significantly associated with susceptibility to SGIV infection (P<0.05) and the EPCO-DAA*0104, EPCO-DAA*0601, EPCO-DAA*1101, and EPCO-DAA*1201 alleles were significantly associated with resistance to SGIV (P<0.05 for each). To confirm these correlations, an additional independent challenge experiment was performed in the Wenchang population of the orange-spotted grouper. The frequency distribution showed that the EPCO-DAA*0101 allele was significantly more frequent in the Wenchang HS (WC-HS) group than in the WC-HR group (P<0.05), whereas the EPCO-DAA*1101 allele was significantly more frequent in the WC-HR group than in the WC-HS group (P<0.05), consistent with the first challenge. However, the frequency distributions of the EPCO-DAA*0104, EPCO-DAA*0601, and EPCO-DAA*1201 alleles did not differ significantly between the WC-HR and WC-HS groups (P>0.05). These results indicate that the EPCO-DAA*0101 allele confers susceptibility to SGIV infection, whereas the EPCO-DAA*1101 allele confers resistance to it. These disease-resistance-related MHC markers could be useful for the molecular-marker-assisted selective breeding of the orange-spotted grouper. Statement of relevanceMolecular marker-assisted selective breeding program.

Antiviral function of grouper MDA5 against iridovirus and nodavirus.

Melanoma differentiation-associated gene 5 (MDA5) is a critical member of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family which can recognize viral RNA and enhances antiviral response in host cells. In this study, a MDA5 homolog from orange spotted grouper (Epinephelus coioides) (EcMDA5) was cloned, and its roles on grouper virus infection were characterized. The full-length EcMDA5 cDNA encoded a polypeptide of 982 amino acids with 74% identity with MDA5 homolog from rock bream (Oplegnathus fasciatus). Amino acid alignment analysis indicated that EcMDA5 contained three functional domains: two caspase activation and recruitment domain (CARDs), a DEAD box helicase-like (DExDc) domain, a helicase superfamily C-terminal domain (HELICc), and a C-terminal regulatory domain (RD). Upon challenge with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the transcript of EcMDA5 was significantly up-regulated especially at the early stage post-injection. Under fluorescence microscopy, we observed that EcMDA5 mostly localized in the cytoplasm of grouper spleen (GS) cells. Interestingly, during virus infection, the distribution pattern of EcMDA5 was significantly altered in SGIV infected cells, but not in red spotted grouper nervous necrosis virus (RGNNV) infected cells, suggested that EcMDA5 might interact with viral proteins during SGIV infection. The ectopic expression of EcMDA5 invitro obviously delayed virus infection induced cytopathic effect (CPE) progression and significantly inhibited viral gene transcription of RGNNV and SGIV. Moreover, overexpression of EcMDA5 not only significantly increased interferon (IFN) and IFN-stimulated response element (ISRE) promoter activities in a dose dependent manner, but also enhanced the expression of IRF3, IRF7 and TRAF6. In addition, the transcription level of the proinflammatory factors, including TNF-α, IL-6 and IL-8 were differently altered by EcMDA5 overexpression during SGIV or RGNNV infection, suggesting that the regulation on proinflammatory cytokines by EcMDA5 were also important for RGNNV infection. Together, our results demonstrated for the first time that the inhibitory effect of fish MDA5 on iridovirus replication might be mainly through the regulation of proinflammatory cytokines.

JNK1 Derived from Orange-Spotted Grouper, Epinephelus coioides, Involving in the Evasion and Infection of Singapore Grouper Iridovirus (SGIV).

c-Jun N-terminal kinase (JNK) regulates cellular responses to various extracellular stimuli, environmental stresses, pathogen infections, and apoptotic agents. Here, a JNK1, Ec-JNK1, was identified from orange-spotted grouper, Epinephelus coioides. Ec-JNK1 has been found involving in the immune response to pathogen challenges in vivo, and the infection of Singapore grouper iridovirus (SGIV) and SGIV-induced apoptosis in vitro. SGIV infection activated Ec-JNK1, of which phosphorylation of motif TPY is crucial for its activity. Over-expressing Ec-JNK1 phosphorylated transcription factors c-Jun and promoted the infection and replication of SGIV, while partial inhibition of the phosphorylation of Ec-JNK1 showed the opposite effects by over-expressing the dominant-negative EcJNK1-Δ183-185 mutant. Interestingly, SGIV enhanced the viral infectivity by activating Ec-JNK1 which in turn drastically inhibited the antiviral responses of type 1 IFN, indicating that Ec-JNK1 could be involved in blocking IFN signaling during SGIV infection. In addition, Ec-JNK1 enhanced the activation of AP-1, p53, and NF-κB, and resulted in increasing the levels of SGIV-induced cell death. The caspase 3-dependent activation correlated with the phosphorylation of Ec-JNK1 and contributed to SGIV-induced apoptosis. Taken together, SGIV modulated the phosphorylation of Ec-JNK1 to inactivate the antiviral signaling, enhance the SGIV-induced apoptosis and activate transcription factors for efficient infection and replication. The “positive cooperativity” molecular mechanism mediated by Ec-JNK1 contributes to the successful evasion and infection of iridovirus pathogenesis.

Molecular clone and characterization of c-Jun N-terminal kinases 2 from orange-spotted grouper, Epinephelus coioides.

c-Jun N-terminal kinase 2 (JNK2) is a multifunctional mitogen-activated protein kinases involving in cell differentiation and proliferation, apoptosis, immune response and inflammatory conditions. In this study, we reported a new JNK2 (Ec-JNK2) derived from orange-spotted grouper, Epinephelus coioides. The full-length cDNA of Ec-JNK2 was 1920bp in size, containing a 174bp 5'-untranslated region (UTR), 483bp 3'-UTR, and a 1263bp open reading frame (ORF), which encoded a putative protein of 420 amino acids. The deduced protein sequence of Ec-JNK2 contained a conserved Thr-Pro-Tyr (TPY) motif in the domain of serine/threonine protein kinase (S-TKc). Ec-JNK2 has been found to involve in the immune response to pathogen challenges in vivo, and the infection of Singapore grouper iridovirus (SGIV) in vitro. Immunofluorescence staining showed that Ec-JNK2 was localized in the cytoplasm of grouper spleen (GS) cells, and moved to the nucleus after infecting with SGIV. Ec-JNK2 distributed in all immune-related tissues examined. After challenging with lipopolysaccharide (LPS), SGIV and polyriboinosinic polyribocytidylic acid (poly I:C), the mRNA expression of Ec-JNK2 was significantly (P<0.01) up-regulated in juvenile orange-spotted grouper. Over-expressing Ec-JNK2 in fathead minnow (FHM) cells increased the SGIV infection and replication, while over-expressing the dominant-negative Ec-JNK2Δ181-183 mutant decreased it. These results indicated that Ec-JNK2 could be an important molecule in the successful infection and evasion of SGIV.