Qiwei Qin

Grouper MAVS functions as a crucial antiviral molecule against nervous necrosis virus infection.

ABSTRACT Mitochondrial antiviral signaling protein (MAVS), also known as IPS‐1, VISA, and Cardif, has been well studied for its crucial roles in the mammalian interferon immune response. To better understand the actions of MAVS in fish immune response, a MAVS homolog from orange spotted grouper (Epinephelus coioides) (EcMAVS) was cloned and characterized in this study. EcMAVS encoded a 563‐amino acid peptide which showed 64% and 20% identity to rock bream (Oplegnathus fasciatus) and human (Homo sapiens), respectively. Sequence alignment analysis showed that EcMAVS shared a conserved CARD domain at N terminal, a central proline‐rich region and a TM domain at C terminal. Phylogenetic analysis indicated that EcMAVS showed the nearest relationship to rock bream, followed by other fishes, birds and mammals. In healthy grouper, the transcript of EcMAVS was predominantly detected in gill, intestine and skin. In vitro, the expression level of EcMAVS was significantly increased during red‐spotted grouper nervous necrosis virus (RGNNV) infection, but only slightly increased at the late stage of Singapore grouper iridovirus (SGIV) infection, suggested the EcMAVS might exert various roles in response to different viruses. Subcellular localization analysis showed that the fluorescence in EcMAVS transfected cells primarily co‐localized with mitochondria. Overexpression of EcMAVS in grouper cells significantly inhibited the replication of RGNNV, demonstrated by the delay of CPE progression and the decrease of viral gene transcription. Differently, the replication of SGIV was almost not affected by the ectopic expression of EcMAVS. Furthermore, our results also showed that EcMAVS overexpression significantly increased the expression of interferon related cytokines, and activated both IRF3‐ and IRF7‐mediated interferon promoter activities. Taken together, our results demonstrated that grouper MAVS exerted antiviral function against nodavirus infection via up‐regulating the interferon immune response. HighlightsEcMAVS showed 64% and 20% identity to rock bream and human, respectively.EcMAVS localized in mitochondrion.EcMAVS overexpression significantly inhibited RGNNV replication.EcMAVS overexpression enhanced both IRF3‐ and IRF7‐mediated interferon response.

Fish DDX3X exerts antiviral function against grouper nervous necrosis virus infection

Abstract Human DEAD box ATP‐dependent RNA helicase DDX3X has been demonstrated to exert crucial functions in carcinogenesis and antiviral immune response. However, to our knowledge, few information focused on the functions of fish DDX3X. In this study, we cloned and characterized a DDX3X homolog from orange spotted grouper (Epinephelus coioides) (EcDDX3X). EcDDX3X encoded a 733‐amino acid protein which shared 97% and 76% identity to spiny damselfish (Acanthochromis polyacanthus) and human (Homo sapiens), respectively. Amino acid alignment analysis showed that EcDDX3X contained conserved DExDc and Helic C domains. The transcription levels of EcDDX3X were significantly increased in poly I:C transfected cells and red‐spotted grouper nervous necrosis virus (RGNNV) infected cells. Under fluorescence microscopy, the green fluorescence was observed evenly in the cytoplasm in EcDDX3X transfected cells. The ectopic expression of EcDDX3X significantly inhibited the replication of RGNNV, evidenced by the decreased numbers of the vacuoles evoked by RGNNV infection, and the reduced transcription levels of RGNNV coat protein (CP) and RNA‐dependent RNA polymerase (RdRp) genes. In contrast, the replication of Singapore grouper iridovirus (SGIV) in grouper spleen (GS) cells was not significantly affected by EcDDX3X overexpression. Further studies showed that overexpression of EcDDX3X in vitro significantly increased the expression levels of several interferon associated cytokines or effectors. Moreover, the regulatory effect of EcDDX3X on interferon immune response was dependent on its N terminal region, but not the DExDc and Helic C domain. In addition, we also found that overexpression of EcDDX3X significantly increased the interferon promoter activity, and the activation of interferon immune response was regulated by both IRF3 and IRF7. Together, our results firstly showed that fish DDX3X exerted crucial roles in antiviral immunity against RNA virus infection via upregulating interferon antiviral responses. HighlightsEcDDX3X was cloned and characterized.EcDDX3X encoded a cytoplasmic protein.EcDDX3X overexpression significantly inhibited the replication of RGNNV.EcDDX3Xoverexpression enhanced IRF3‐ and IRF7‐mediated interferon response.

Rapid and sensitive detection of redspotted grouper nervous necrosis virus (RGNNV) infection by aptamer-coat protein-aptamer sandwich enzyme-linked apta-sorbent assay (ELASA)

Redspotted grouper nervous necrosis virus (RGNNV) is one of the most devastating pathogens in the aquaculture of the grouper, Epinephlus sp., worldwide. The early and rapid diagnosis of RGNNV is important for the prevention of RGNNV infection. In this study, an aptamer (A10)-based sandwich enzyme-linked apta-sorbent assay (ELASA) was developed for RGNNV diagnosis. This sandwich ELASA showed high specificity for the RGNNV coat protein (CP) and virions in virus-infected cells and tissues. At the optimized working concentration of 200 nM of aptamer, the ELASA could detect RGNNV in the lysates of as few as 4xa0×xa0103 RGNNV-infected GB cells. Incubation for 10 min was sufficient to produce accurate results. The sandwich ELASA was most stable at incubation temperatures of 4-25°C, but could still distinguish RGNNV-infected samples from the controls at 37°C. It could detect RGNNV infection in brain lysates diluted 1/10, with results consistent with those of reverse transcription PCR, although with 10% less sensitivity. The main equipment required includes dissection tools, a water bath, Pierce™ Streptavidin Coated Plates and a microplate reader. The sandwich ELASA has great potential utility for the rapid and sensitive diagnosis of RGNNV in its early stages by fish farmers.

Expression and functional characterization of TRIF in orange-spotted grouper (Epinephelus coioides)

Abstract Antiviral immune responses are triggered by the innate immune recognition of viral infection. Toll/interleukin‐1 receptor (TIR) domain containing adapter inducing interferon‐&bgr; (TRIF) is an adapter in responding to activation of Toll‐like receptors, which provides early clearance of viral pathogens. Our study focuses on the functional characterization of grouper TRIF (EcTRIF) based on the comparison of its sequence and functional evolution from grouper fish to mammals. The results show that the open reading frame of EcTRIF encoded a protein of 580 amino acids. Real‐time PCR analysis indicates that EcTRIF was constitutively expressed in all the analyzed tissues in healthy grouper. EcTRIF was significantly induced in spleen post‐LPS and poly (I:C) stimulation. Fluorescence microscopy shows that EcTRIF is colocalized with a Golgi apparatus marker, implying its unique subcellular localization in the Golgi apparatus. Luciferase reporter assays confirmed that EcTRIF was able to activate the IFN and NF‐&kgr;B promoter. Overexpression of EcTRIF in grouper brain cells inhibited the replication of red‐spotted grouper nervous necrosis virus (RGNNV). These results indicate that EcTRIF plays an important role in modulating antiviral innate immune responses. Our results have applications in functional studies on TRIF in teleost fish and immune evolution. HighlightsEcTRIF were significantly induced in spleen post LPS and poly(I:C) stimulation.EcTRIF is colocalized with a Golgi apparatus marker, implying its unique subcellular localization in Golgi apparatus.EcTRIF is able to active the IFN and NF‐&kgr;B promoter.EcTRIF can inhibit the replication of RGNNV in vitro.

Singapore grouper iridovirus (SGIV) TNFR homolog VP51 functions as a virulence factor via modulating host inflammation response.

Virus encoded tumor necrosis factor receptor (TNFR) homologues are usually involved in immune evasion by regulating host immune response or cell death. Singapore grouper iridovirus (SGIV) is a novel ranavirus which causes great economic losses in aquaculture industry. Previous studies demonstrated that SGIV VP51, a TNFR-like protein regulated apoptotic process in VP51 overexpression cells. Here, we developed a VP51-deleted recombinant virus Δ51-SGIV by replacing VP51 with puroR-GFP. Deletion of VP51 resulted in the decrease of SGIV virulence, evidenced by the reduced replication in vitro and the decreased cumulative mortalities in Δ51-SGIV challenged grouper compared to WT-SGIV. Moreover, VP51 deletion significantly increased virus induced apoptosis, and reduced the expression of pro-inflammatory cytokines in vitro. In addition, the expression of several pro-inflammatory genes were decreased in Δ51-SGIV infected grouper compared to WT-SGIV. Thus, we speculate that SGIV VP51 functions as a critical virulence factor via regulating host cell apoptosis and inflammation response.

Data for: 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 16.43u202fnM. 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 aptamers truncated methods could be applied in the research of identifying aptamers 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.

Establishment of a new fish cell line from the caudal fin of golden pompano Trachinotus ovatus and its susceptibility to iridovirus: new caudal fin cell line of t. ovatus

A new cell line derived from the caudal fin of golden pompano Trachinotus ovatus (TOCF) was successfully established and characterized. TOCF cells grew well at 28° C in L-15 medium supplemented with 10% foetal bovine serum (FBS). The cell line has been subcultured in more than 100 passages. Molecular characterization of 18S ribosomal (r)RNA and cytochrome oxidase subunit 1 (COI) confirmed that the TOCF cells were derived indeed from T. ovatus. TOCF cells have a modal chromosome number of 54. It was further showed that TOCF cells were transfected successfully with pEGFP-N3 and pDsRED-N1 plasmid, suggesting that TOCF cells could be used to research gene functions in vitro. Viral susceptibility tests showed that TOCF cells were susceptible to Singapore grouper iridovirus (SGIV), observed by the occurrence of the cytopathic effect (CPE) with the formation of inclusion bodies. In addition, the expression of major capsid protein (MCP) gene of SGIV changed during virus infection in TOCF cells. Thus, our present results described the characteristic of a TOCF cell line that could be a valuable tool for genetic manipulation, as well as isolation and propagation of iridovirus studies.

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

ABSTRACT 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 21.81kDa. 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. HIGHLIGHTSEcFADD in the spleen of E. coioides was differentially up‐regulated when challenged with SGIV or poly[I:C].EcFADD was abundantly distributed in both the cytoplasm and nucleus in FHM and GS cells.Over‐expression of EcFADD inhibited SGIV infection and replication and SGIV‐induced apoptosis.FADD promoted the activation of ISRE and IFN genes in the antiviral IFN signaling pathway and inhibited activation of p53.

Fish TRIM35 negatively regulates the interferon signaling pathway in response to grouper nodavirus infection

Tripartite motif-containing protein 35 (TRIM35) has been demonstrated to exert critical roles in cancer, cell death and other multiple cell processes. However, the precisely roles of TRIM35 during virus infection still remained largely unknown. In the current study, we cloned a TRIM35 gene from orange spotted grouper (EcTRIM35) and uncovered its roles in response to nodavirus infection. EcTRIM35 encoded a 456-aa protein which showed 65% and 32% identity to large yellow croaker (Larimichthys crocea) and human (Homo sapiens), respectively. Structure prediction and amino acid alignment analysis indicated that EcTRIM35 contained three conserved domains, including RING domain, B-BOX and SPRY domain. In healthy grouper, the high expression level of EcTRIM35 could be detected in liver, spleen and intestine. After infection with red-spotted grouper nervous necrosis (RGNNV) and Singapore grouper iridovirus (SGIV) in GS cells, the transcript of EcTRIM35 was significantly up-regulated with the infection time increased. Under fluorescence microscopy, the bright fluorescence aggregates were observed in EcTRIM35 transfected cells, but the fluorescence distribution was obviously altered in the EcTRIM35-ΔRING transfected cells. After incubation with RGNNV, the overexpression of EcTRIM35 inxa0vitro significantly enhanced the viral replication, evidenced by the enhancement of cytopathic effect (CPE) severity and the up-regulation of the viral gene transcription. Moreover, the ectopic expression of EcTRIM35 significantly decreased the expression of interferon signaling molecules or effectors. Further studies elucidated that EcTRIM35 overexpression significantly weakened the MAVS-, MITA- or TBK1-induced interferon immune response, but showed no effects on MDA5-induced immune response. Thus, our results will shed new lights on the roles of fish TRIM35 in innate immune response against grouper virus infection.

Grouper STAT1a is involved in antiviral immune response against iridovirus and nodavirus infection

ABSTRACT Signal Transducer and Activator of Transcription 1 (STAT1) has been demonstrated to function as a critical mediator in multiple cell processes, such as cell proliferation, cell death, and innate immune response. Interestingly, two orthologues of human STAT1, including STAT1a and STAT1b genes have been identified in different fish. However, the detailed roles of fish STAT1a in virus replication still remained largely uncertain. Here, we cloned a STAT1a from orange‐spotted grouper Epinephelus coioides (EcSTAT1a) and characterized its roles during fish virus infection. EcSTAT1a encoded a 751‐aa peptide which shared 97% and 93% identity to STAT1 from mandarin fish (Siniperca chuatsi) and Malabar grouper (Epinephelus malabaricus), respectively. Amino acid alignment analysis showed that EcSTAT1a contained a STAT‐int domain, a STAT‐alpha domain, a STAT‐bind domain (DNA binding domain), a SH2 domain and a STAT1‐TAZ2 bind domain. In examined tissues from healthy grouper, the expression of EcSTAT1a was predominant in intestine, gill and liver. In grouper cells, the relative expression levels of EcSTAT1a was significantly increased during red‐spotted grouper nervous necrosis virus (RGNNV) or Singapore grouper iridovirus (SGIV) infection. Under fluorescence microscopy, we found that EcSTAT1a mainly localized in the cytoplasm. The ectopic expression of EcSTAT1a in vitro significantly delayed the cytopathic effect (CPE) progression evoked by RGNNV and SGIV. Further studies showed that the expression levels of viral genes, including SGIV major capsid protein (MCP), VP19, ICP‐18, LITAF and RGNNV coat protein (CP), RNA‐dependent RNA polymerase (RdRp) were all significantly reduced in EcSTAT1a overexpressing cells compared to the control vector transfected cells, suggested that EcSTAT1a exerted antiviral activity against iridovirus and nodavirus. Furthermore, overexpression of EcSTAT1a significantly increased the expression of interferon related cytokines or effectors and pro‐inflammatory factors. Together, our results elucidated that EcSTAT1a might function as a critical antiviral factor by regulating the host interferon immune and inflammation response. HighlightsEcSTAT1a showed the highest identity to mandarin fish.EcSTAT1a was predominant in intestine, gill and liver.EcSTAT1a mainly localized in the cytoplasm.Overexpression of EcSTAT1a inhibited RGNNV and SGIV replication.EcSTAT1a significantly enhanced interferon immune and inflammatory response.