Publications (70)198.98 Total impact
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Article: Molecular characterization and function of a p38 MAPK gene from Litopenaeus vannamei.
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ABSTRACT: p38 mitogen-activated protein kinases (MAPKs) are broadly expressed from yeasts to mammals, and are involved in the regulation of cells responsible to various extracellular stimuli. In this study, a p38 MAPK gene (designated as Lvp38) from Litopenaeus vannamei, was cloned and characterized. It contained the conserved structures of a Thr-Gly-Tyr (TGY) motif and a substrate-binding site, Ala-Thr-Arg-Trp (ATRW). The tissue distribution patterns showed that Lvp38 was widely expressed in all examined tissues, with the highest expression in hemocytes, nerves, and intestines. Quantitative real-time PCR revealed that Lvp38 was upregulated in gills and hemocytes after infection with the Gram-negative Vibrio alginolyticus and the Gram-positive Staphylococcus aureus. Reporter gene assays indicated that Lvp38 activated the expression of antimicrobial peptides (AMPs) of Drosophila and shrimp. Knockdown of Lvp38 by RNA interference (RNAi) resulted in a higher mortality of L. vannamei under V. alginolyticus and S. aureus infection, as well as a reduction in the expression of three shrimp AMP genes, namely, PEN4, crustin, and ALF2. Taken together, our data indicated that Lvp38 played a role in defending against bacterial infections.Fish & Shellfish Immunology 03/2013; · 3.32 Impact Factor -
Article: Litopenaeus vannamei Toll-interacting protein (LvTollip) is a potential negative regulator of the shrimp Toll pathway involved in the regulation of the shrimp antimicrobial peptide gene penaeidin-4 (PEN4).
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ABSTRACT: The Toll-like receptor (TLR)-nuclear factor (NF)-κB signaling pathway is evolutionarily conserved from insects to mammals as a regulator of the expression of immune-related genes. In mammals, TLR-NF-κB signaling is tightly controlled because excessive activation of this pathway can result in severe damage to the host. The mammalian Toll-interacting protein (Tollip) has an important function in the negative regulation of this pathway, but no reports about invertebrate Tollip have been published to date. In this study, we cloned Litopenaeus vannamei Tollip (LvTollip) and investigated its function in the regulation of the NF-κB pathway-controlled antimicrobial peptide genes (AMPs). The LvTollip full-length cDNA is 1231 bp long and contains an open reading frame of 813 bp that encodes a 270-amino acid protein. LvTollip shares significant similarities to mammalian Tollips, which contain a centrally localized protein kinase C conserved region 2 (C2) domain and a C-terminal CUE domain. After challenges with the white spot syndrome virus (WSSV) or Vibrio alginolyticus, the expression levels of LvTollip were altered in the gill, hemocyte, hepatopancreatic, intestinal, and muscle tissues. In Drosophila S2 cells, LvTollip localized in the membrane and the cytoplasm and significantly inhibited the promoter activities of the NF-κB pathway-controlled AMP penaeidin-4 (PEN4). In LvTollip-knockdown shrimp, the expression level of AMP PEN4 was increased. However, the mortality rates of LvTollip-knockdown shrimp in response to WSSV or V. alginolyticus infections were not significantly different from those of the control group. Our results suggested that LvTollip might be involved in the negative regulation of PEN4 and that LvTollip expression was responsive to microbial infections.Developmental and comparative immunology 03/2013; · 3.29 Impact Factor -
Article: Mandarin fish caveolin-1 interaction with major capsid protein of infectious spleen and kidney necrosis virus and its role in early stages of infection.
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ABSTRACT: Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. ISKNV is one of the major agents that cause mortality and economic losses to the freshwater fish culture industry in Asian countries particularly that of mandarin fish (Siniperca chuatsi). In the present study, we report that the interaction of mandarin fish caveolin-1 (mCav-1) with the ISKNV major capsid protein (ISKNV MCP) was detected by using a virus overlay assay and confirmed by pull-down assay and co-immunoprecipitation. This interaction was independent of the classic caveolin-1 scaffolding domain (CSD), which is responsible for interacting with several signaling proteins and receptors. Confocal immunofluorescence microscopy showed that ISKNV MCP co-localized with mCav-1 in the perinuclear region of virus-infected mandarin fish fry (MFF-1) cells, which appeared as soon as four hours post-infection. The subcellular fractionation analysis showed that ISKNV MCP was associated with caveolae in the early stages of viral infection. The RNA interference silencing of mCav-1 did not change the virus-cell binding, but efficiently inhibited the entry of virions into the cell. Taken together, these results suggested that mCav-1 plays an important role in the early stages of ISKNV infection.Journal of Virology 01/2013; · 5.40 Impact Factor -
Article: Identification and Function of Leucine-Rich Repeat Flightless-I-Interacting Protein 2 (LRRFIP2) in Litopenaeus vannamei.
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ABSTRACT: Leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2) is a myeloid differentiation factor 88-interacting protein with a positive regulatory function in toll-like receptor signaling. In this study, seven LRRFIP2 protein variants (LvLRRFIP2A-G) were identified in All the seven LvLRRFIP2 protein variants encode proteins with a DUF2051 domain. LvLRRFIP2s were upregulated in hemocytes after challenged with lipopolysaccharide, poly I:C, CpG-ODN2006, , , and white spot syndrome virus (WSSV). Dual-luciferase reporter assays in Schneider 2 cells revealed that LvLRRFIP2 activates the promoters of and shrimp AMP genes. The knockdown of LvLRRFIP2 by RNA interference resulted in higher cumulative mortality of upon but not and WSSV infections. The expression of AMP genes were reduced by dsLvLRRFIP2 interference. These results indicate that LvLRRFIP2 has an important function in antibacterials via the regulation of AMP gene expression.PLoS ONE 01/2013; 8(2):e57456. · 4.09 Impact Factor -
Article: The potential role of microfilaments in host cells for infection with infectious spleen and kidney necrosis virus infection.
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ABSTRACT: Infectious spleen and kidney necrosis virus (ISKNV) belongs to the genus Megalocytivirus from the family Iridoviridae. Megalocytivirus causes severe economic losses to tropical freshwater and marine culture industry in Asian countries and is devastating to the mandarin fish farm industry in China particularly. We investigated the involvement of microfilaments in the early and late stages of ISKNV infection in MFF-1 cells by selectively perturbing their architecture using well-characterized inhibitors of actin dynamics. The effect of disruption of actin cytoskeleton on ISKNV infection was evaluated by indirect immunofluorescence analysis or real-time quantitative PCR. The depolymerization of the actin filaments with cytochalasin D, cytochalasin B, or latrunculin A reduced ISKNV infection. Furthermore, depolymerization of filamentous actin by inhibitors did not inhibit binding of the virus but affected virus internalization in the early stages of infection. In addition, the depolymerization of actin filaments reduced total ISKNV production in the late stages of ISKNV. This study demonstrated that ISKNV required an intact actin network during infection. The findings will help us to better understand how iridoviruses exploit the cytoskeleton to facilitate their infection and subsequent disease.Virology Journal 01/2013; 10:77. · 2.34 Impact Factor -
Article: Litopenaeus vannamei Sterile-Alpha and Armadillo Motif Containing Protein (LvSARM) Is Involved in Regulation of Penaeidins and antilipopolysaccharide factors.
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ABSTRACT: The Toll-like receptor (TLR)-mediated NF-κB pathway is tightly controlled because overactivation may result in severe damage to the host, such as in the case of chronic inflammatory diseases and cancer. In mammals, sterile-alpha and armadillo motif-containing protein (SARM) plays an important role in negatively regulating this pathway. While Caenorhabditis elegans SARM is crucial for an efficient immune response against bacterial and fungal infections, it is still unknown whether Drosophila SARM participates in immune responses. Here, Litopenaeus vannamei SARM (LvSARM) was cloned and functionally characterized. LvSARM shared signature domains with and exhibited significant similarities to mammalian SARM. Real-time quantitative PCR analysis indicated that the expression of LvSARM was responsive to Vibrio alginolyticus and white spot syndrome virus (WSSV) infections in the hemocyte, gill, hepatopancreas and intestine. In Drosophila S2 cells, LvSARM was widely distributed in the cytoplasm and could significantly inhibit the promoters of the NF-κB pathway-controlled antimicrobial peptide genes (AMPs). Silencing of LvSARM using dsRNA-mediated RNA interference increased the expression levels of Penaeidins and antilipopolysaccharide factors, which are L.vannamei AMPs, and increased the mortality rate after V. alginolyticus infection. Taken together, our results reveal that LvSARM may be a novel component of the shrimp Toll pathway that negatively regulates shrimp AMPs, particularly Penaeidins and antilipopolysaccharide factors.PLoS ONE 01/2013; 8(2):e52088. · 4.09 Impact Factor -
Article: Activating Transcription Factor 4 and X Box Binding Protein 1 of Litopenaeus vannamei Transcriptional Regulated White Spot Syndrome Virus Genes Wsv023 and Wsv083.
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ABSTRACT: In response to endoplasmic reticulum (ER) stress, the signaling pathway termed unfolded protein response (UPR) is activated. To investigate the role of UPR in Litopenaeus vannamei immunity, the activating transcription factor 4 (designated as LvATF4) which belonged to a branch of the UPR, the [protein kinase RNA (PKR)-like ER kinase, (PERK)]-[eukaryotic initiation factor 2 subunit alpha (eIF2α)] pathway, was identified and characterized. The full-length cDNA of LvATF4 was 1972 bp long, with an open reading frame of 1299 bp long that encoded a 432 amino acid protein. LvATF4 was highly expressed in gills, intestines and stomach. For the white spot syndrome virus (WSSV) challenge, LvATF4 was upregulated in the gills after 3 hpi and increased by 1.9-fold (96 hpi) compared to the mock-treated group. The LvATF4 knock-down by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Reporter gene assays show that LvATF4 could upregulate the expression of the WSSV gene wsv023 based on the activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element (ATF/CRE). Another transcription factor of L. vannamei, X box binding protein 1 (designated as LvXBP1), has a significant function in [inositol-requiring enzyme-1(IRE1) - (XBP1)] pathway. This transcription factor upregulated the expression of the WSSV gene wsv083 based on the UPR element (UPRE). These results suggest that in L. vannamei UPR signaling pathway transcription factors are important for WSSV and might facilitate WSSV infection.PLoS ONE 01/2013; 8(4):e62603. · 4.09 Impact Factor -
Article: Pathogenicity and complete genome sequence analysis of the mud crab dicistrovirus-1.
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ABSTRACT: A virus with a particle diameter of approximately 30nm and no envelope was purified from diseased mud crab, Scylla paramamosain and it was demonstrated to be pathogenic to mud crab. The complete nucleotide sequence analysis indicated that its genome was a single molecule of linear positive-sense ssRNA with a length of 10,415 nucleotides, excluding the 3'poly (A) tail. It consisted of two open reading frames (ORF) separated by an intergenic region (IGR) and flanked by a 5'untranslated region (5'-UTR) and a 3'untranslated region (3'-UTR). The 5'-ORF encode five putative non-structural proteins, including BIR (Baculovirus Inhibitor of Apoptosis Protein Repeat), helicase, VPg (the genome-linked viral protein), 3C-like protease and RdRP (RNA-dependent RNA polymerase), while the 3'-ORF encode the structural protein precursors. This genome organization was consistent with the typical organization of dicistrovirus and the virus was designated as mud crab dicistrovirus-1 (MCDV-1). The results of the phylogenetic analysis of the putative structural protein precursor suggest that MCDV-1 has a closer genetic relationship with Taura syndrome virus (TSV) than do other dicistroviruses and that MCDV-1 is a new member of the family Dicistroviridae and assigned into the genus Aparavirus.Virus Research 10/2012; · 2.94 Impact Factor -
Article: Identification and characterization of Inositol-requiring enzyme-1 and X-box binding protein 1, two proteins involved in the unfolded protein response of Litopenaeus vannamei.
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ABSTRACT: The inositol-requiring enzyme-1 (IRE1)-X-box binding protein 1 (IRE1-XBP1) pathway is the key branch of the unfolded protein response (UPR). To investigate the role of the IRE1-XBP1 pathway in reducing environmental stress and increasing anti-viral immunity in Litopenaeus vannamei, homologues of IRE1 (designated as LvIRE1) and XBP1 (designated as LvXBP1) were identified and characterized. The full-length cDNA of LvIRE1 is 4908bp long, with an open reading frame (ORF) that encodies a putative 1174 amino acid protein. The full-length cDNA of LvXBP1 is 1746bp long. It contains two ORFs that encode putative 278 amino acid and 157 amino acid proteins, respectively. LvXBP1 mRNA has the predicted IRE1 splicing motifs CNG'CNGN located within the loop regions of two short hairpins. Sequencing of the splicing fragment induced by endoplasmic reticulum (ER)-stress showed a 3bp or 4bp frame shift from the predicted sites. The spliced form LvXBP1 (LvXBP1s) contained an ORF encodes a putative 463 amino acid protein. The reporter gene assays indicated that LvXBP1s activates the promoter of L. vannamei immunoglobulin heavy chain binding protein (LvBip), an important UPR effector. RT-PCR showed that LvXBP1 was spliced during the experiments. For heat shock treatment, the total LvXBP1 expression was increased and peaked at about 36h, whereas the percentages of the two isoforms were relatively stable. For the WSSV challenge, LvXBP1 was upregulated during the experiment and the percentage of the spliced form continuously declined after 18h of infection. Knock-down of LvXBP1 by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Furthermore, the expression profiles of LvIRE1 and LvXBP1 in the gills, hemocytes, intestines, and hepatopancreas of the WSSV-challenged shrimp were detected using real-time RT-PCR. Taken together, these results confirm that the IRE1-XBP1 pathway is important for L. vannamei environmental stress resistance, suggest that L. vannamei IRE1-XBP1 may activated by WSSV and be annexed to serve the virus.Developmental and comparative immunology 04/2012; 38(1):66-77. · 3.29 Impact Factor -
Article: The viral TRAF protein (ORF111L) from infectious spleen and kidney necrosis virus interacts with TRADD and induces caspase 8-mediated apoptosis.
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ABSTRACT: Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus of the Iridoviridae family. It causes a serious and potentially pandemic disease in wild and cultured fishes. ISKNV infection induces evident apoptosis in mandarin fish (Siniperca chuatsi) and zebrafish (Danio renio). However, the mechanism is still unknown. After a genome-wide bioinformatics analysis of ISKNV-encoded proteins, the ISKNV open reading frame 111L (ORF111L) shows a high similarity to the tumour necrosis factor receptor-associated factor (TRAF) encoded by fish, mice and mammals, which is essential for apoptotic signal transduction. Moreover, ORF111L was verified to directly interact with the zebrafish TNF receptor type 1 associated death domain protein (TRADD). A recombinant plasmid containing the DNA sequence of ORF111L was constructed and microinjected into zebrafish embryos at the 1-2 cell stage to investigate its biological function in vivo. ORF111L overexpression in the embryos resulted in increased apoptosis. ORF111L-induced apoptosis was clearly associated with significant caspase 8 upregulation and activation. The knockdown of zebrafish caspase 8 expression effectively blocked the apoptosis induced by ORF111L overexpression. Significantly, ORF111L overexpression resulted in much stronger effect on caspase 8 and caspase 3 upregulation compared to zebrafish TRAF2. This is the first report of a viral protein similar to TRAF that interacts with TRADD and induces caspase 8-mediated apoptosis, which may provide novel insights into the pathogenesis of ISKNV infection.PLoS ONE 01/2012; 7(5):e37001. · 4.09 Impact Factor -
Article: Identification and Function of Myeloid Differentiation Factor 88 (MyD88) in Litopenaeus vannamei.
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ABSTRACT: Myeloid differentiation factor 88 (MyD88) is a universal and essential signaling protein in Toll-like receptor/interleukin-1 receptor-induced activation of nuclear factor-kappa B. In this study, two MyD88 protein variants (LvMyD88 and LvMyD88-1) were identified in Litopenaeus vannamei. The LvMyD88 cDNA is 1,848 bp in length and contains an open reading frame (ORF) of 1,428 bp, whereas the LvMyD88-1 cDNA is 1,719 bp in length and has an ORF of 1,299 bp. Both variants encode proteins with death and Toll/interleukin-1 receptor domains and share 91% sequence identity. In healthy L. vannamei, the LvMyD88 genes were highly expressed in hemocytes but at a low level in the hepatopancreas. The LvMyD88s expression was induced in hemocytes after challenge with lipopolysaccharide, CpG-ODN2006, Vibrio parahaemolyticus, Staphyloccocus aureus, and white spot syndrome virus, but not by poly I∶C. Overexpression of LvMyD88 and LvMyD88-1 in Drosophila Schneider 2 cells led to activation of antimicrobial peptide genes and wsv069 (ie1), wsv303, and wsv371. These results suggested that LvMyD88 may play a role in antibacterial and antiviral response in L. vannamei. To our knowledge, this is the first report on MyD88 in shrimp and a variant of MyD88 gene in invertebrates.PLoS ONE 01/2012; 7(10):e47038. · 4.09 Impact Factor -
Article: A novel viral SOCS from infectious spleen and kidney necrosis virus: interacts with Jak1 and inhibits IFN-α induced Stat1/3 activation.
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ABSTRACT: Interferon (IFN)-induced Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway is important in controlling immune responses and is negatively response-regulated by the suppressor of cytokine signaling (SOCS) proteins. However, several viruses have developed various strategies to inhibit this pathway to circumvent the anti-viral immunity of the host. The infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus in the family Iridoviridae and a causative agent of epizootics in fish. ISKNV ORF103R encodes a predicted viral SOCS (vSOCS) with high homology to the vertebrate SOCS1, but lacks a SOCS-box domain. Interestingly, vSOCS only exists in the genus Megalocytivirus. ISKNV-vSOCS can block the IFN-α-induced Jak/Stat pathway in HepG2 cells. Over-expression of ISKNV-vSOCS inhibited the activities of IFN-stimulated response element (ISRE) promoter; however, the inhibitions by ISKNV-vSOCS were dose-dependent. ISKNV-vSOCS interacted with Jak1 protein and inhibited its tyrosine kinase activity in vitro. ISKNV-vSOCS also impaired the phosphorylation of Stat1 and Stat3 proteins and suppressed their activations. The point mutations (F18D, S66A, S85A, and R64K) of ISKNV-vSOCS significantly impaired the inhibition of IFN-α-induced ISRE-promoter activation. In conclusion, vSOCS inhibits IFN-α-induced Stat1/Stat3 signaling, suggesting that Megalocytivirus has developed a novel strategy to evade IFN anti-viral immunity via vSOCS protein.PLoS ONE 01/2012; 7(7):e41092. · 4.09 Impact Factor -
Article: Infectious spleen and kidney necrosis virus (a fish iridovirus) enters Mandarin fish fry cells via caveola-dependent endocytosis.
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ABSTRACT: Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. Megalocytiviruses have been implicated in more than 50 fish species infections and currently threaten the aquaculture industry, causing great economic losses in China, Japan, and Southeast Asia. However, the cellular entry mechanisms of megalocytiviruses remain largely uncharacterized. In this study, the main internalization mechanism of ISKNV was investigated by using mandarin fish fry (MFF-1) cells. The progression of ISKNV infection is slow, and infection is not inhibited when the cells are treated with ammonium chloride (NH(4)Cl), chloroquine, sucrose, and chlorpromazine, which are inhibitors of clathrin-dependent endocytosis. The depletion of cellular cholesterol by methyl-β-cyclodextrin results in the significant inhibition of ISKNV infection; however, the infection is resumed with cholesterol replenishment. Inhibitors of caveolin-1-involved signaling events, including phorbol 12-myristate 13-acetate (PMA), genistein, and wortmannin, impair ISKNV entry into MFF-1 cells. Moreover, ISKNV entry is dependent on dynamin and the microtubule cytoskeleton. Cofraction analysis of ISKNV and caveolin-1 showed that ISKNV colocates with caveolin-1 during virus infection. These results indicate that ISKNV entry into MFF-1 cells proceeds via classical caveola-mediated endocytosis and is dependent on the microtubules that serve as tracks along which motile cavicles may move via a caveola-caveosome-endoplasmic reticulum (ER) pathway. As a fish iridovirus, ISKNV entry into MFF-1 cells is different from the clathrin-mediated endocytosis of frog virus 3 entry into mammalian cells (BHK-21) at 28°C, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection.Journal of Virology 12/2011; 86(5):2621-31. · 5.40 Impact Factor -
Article: Isolation and characterization of cDNAs encoding Ars2 and Pasha homologues, two components of the RNA interference pathway in Litopenaeus vannamei.
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ABSTRACT: The RNA interference (RNAi) is an evolutionarily conserved protective mechanism in eukaryotes against parasitic foreign nucleic acids. Previous studies demonstrated that the RNAi mechanism is important for shrimp antiviral immunity. Here, we report the identification and functional analysis of two key components of the shrimp RNAi activity: Litopenaeus vannamei arsenite resistance gene 2 (LvArs2) and partner of drosha (LvPasha). The full-length cDNA of LvArs2 was 3470 bp, including a 5' untranslated region (UTR) of 167 bp, a 3' UTR of 639 bp, and an open reading frame (ORF) of 2664 bp that encoded 887 amino acid residues with an estimated molecular mass of 102.5 kDa. The full-length cDNA of LvPasha was 2654 bp, including a 5' UTR of 99 bp, a 3' UTR of 560 bp, and an ORF of 1995 bp that encoded 664 amino acid residues with an estimated molecular mass of 74.2 kDa. Co-immunoprecipitation demonstrated that LvArs2 interacted with L. vannamei Dicer2 (LvDcr2) and LvPasha in Drosophila Schneider 2 (S2) cells, suggesting that LvArs2 may be involved in regulation of the miRNA/siRNA pathways in L.vannamei. Subcellular localization assays demonstrated both LvArs2 and LvPasha proteins mainly presented in the nucleus. After Poly(C-G) stimulation, the expression of LvArs2 was suppressed and expression of LvPasha was enhanced in shrimp gills. These results suggest that LvArs2 and LvPasha may participate in the defense against RNA viruses in crustacea.Fish & Shellfish Immunology 12/2011; 32(2):373-80. · 3.32 Impact Factor -
Article: Sequence analysis of 12 genome segments of mud crab reovirus (MCRV).
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ABSTRACT: Mud crab reovirus (MCRV) is the causative agent of a serious disease with high mortality in cultured mud crab (Scylla serrata). This study sequenced and analyzed 12 genome segments of MCRV. The 12 genome segments had a total length of 24.464 kb, showing a total G+C content of 41.29% and predicted 15 ORFs. Sequence analysis showed that the majority of MCRV genes shared low homology with the counterpart genes of other reoviruses, e.g., the amino acid identity of RNA-dependent RNA polymerase (RdRp) was lower than 13.0% compared to the RdRp sequences of other reoviruses. Nucleotide and amino acid sequences of RdRp and capping enzyme suggested MCRV as a single group. Further genome-based phylogenetical analysis of conserved termini and reovirus polymerase motif indicates that this MCRV belongs to a new genus of the Reoviridae family, tentatively named as Crabreovirus.Virology 11/2011; 422(2):185-94. · 3.35 Impact Factor -
Article: Antigenic identification of virion structural proteins from infectious spleen and kidney necrosis virus.
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ABSTRACT: Infectious spleen and kidney necrosis virus (ISKNV), belonging to the genus Megalocytivirus in the family Iridoviridae, is one of the major agents causing mortality and economic losses to the freshwater fish culture industry in Asian countries. Currently, little information regarding the antigenic properties of Megalocytivirus (especially ISKNV) is available. Our previous study using four different workflows with systematic and comprehensive proteomic approaches led to the identification of 38 ISKNV virion-associated proteins (J. Virol. 2869-2877, 2011). Thus, in this report, the antigenicity of 31 structural proteins from ISKNV virion was investigated. A one-dimensional gel electrophoresis immunoblot profile coupled with MALDI-TOF-TOF MS/MS was applied to identify six immunogenic viral proteins, namely, ORFs major capsid protein (006L), 054L, 055L, 101L, 117L, and 125L. Then, the antigenicity of 31 structural proteins was characterized by Western blot by using pooled sera from mandarin fish that survived ISKNV infection. Of the 31 viral proteins, 22 were recognized by the fish ISKNV antiserum. Furthermore, this antiserum neutralizes MFF-1 cells ISKNV infection. To our knowledge, this study is the first report on the immunogenicity of viral proteins and characterization of the proteome of megalocytivirus infective agents. Our findings are expected to promote the development of effective vaccine candidates.Fish & Shellfish Immunology 08/2011; 31(6):919-24. · 3.32 Impact Factor -
Article: Molecular cloning, characterization and expression analysis of two novel Tolls (LvToll2 and LvToll3) and three putative Spätzle-like Toll ligands (LvSpz1-3) from Litopenaeus vannamei.
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ABSTRACT: Toll-like receptor-mediated NF-κB pathways are essential for inducing immune related-gene expression in the defense against bacterial, fungal and viral infections in insects and mammals. Although a Toll receptor (LvToll1) was cloned in Litopenaeus vannamei, relatively little is known about other types of Toll-like receptors and their endogenous cytokine-like ligand, Spätzle. Here, we report two novel Toll-like receptors (LvToll2 and LvToll3) and three Spätzle-like proteins (LvSpz1-3) from L. vannamei. LvToll2 has 1009 residues with an extracellular domain containing 18 leucine-rich repeats (LRRs) and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain of 139 residues. LvToll3 is 1244 residues long with an extracellular domain containing 23 LRRs and a cytoplasmic TIR domain of 138 residues. The Spätzle-like proteins LvSpz1, LvSpz2 and LvSpz3 are 237, 245 and 275 residues in length, respectively, and all of them have a putative C-terminal cystine-knot domain. In Drosophila Schneider 2 (S2) cells, LvToll1 and LvToll3 were localized to the membrane and cytoplasm, and LvToll2 was confined to the cytoplasm. In Drosophila S2 cells, LvToll2 could significantly activate the promoters of NF-κB-pathway-controlled antimicrobial peptide genes, whereas LvToll1 and LvToll3 had no effect on them. LvSpz1 exerted some degree of inhibition on the promoter activities of Drosophila Attacin A and L. vannamei Penaeidin4. LvSpz3 also inhibited the Drosophila Attacin A promoter, but LvSpz2 could only slightly activate it. LvToll1, LvToll2 and LvToll3 were constitutive expressed in various tissues, while LvSpz1, LvSpz2 and LvSpz3 exhibited tissue-specific expression in the epithelium, eyestalk, intestine, gill and muscle. In the gill, after Vibrio alginolyticus challenge, LvToll1 was upregulated, but LvToll2 and LvToll3 showed no obvious changes. LvSpz1 and LvSpz3 were also strongly induced by V. alginolyticus challenge, but LvSpz2 only showed a slight downregulation. In the gill, after white spot syndrome virus (WSSV) challenge, LvToll1, LvToll2, LvToll3, LvSpz1 and LvSpz3 were upregulated, but LvSpz2 showed no obvious change, except for a slight downregulation at 12h post-injection of WSSV. These findings might be valuable in understanding the innate immune signal pathways of shrimp and enabling future studies on the host-pathogen interactions in V. alginolyticus and WSSV infections.Developmental and comparative immunology 07/2011; 36(2):359-71. · 3.29 Impact Factor -
Article: Entry of tiger frog virus (an Iridovirus) into HepG2 cells via a pH-dependent, atypical, caveola-mediated endocytosis pathway.
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ABSTRACT: Tiger frog virus (TFV), in the genus Ranavirus of the family Iridoviridae, causes high mortality of cultured tiger frog tadpoles in China. To explore the cellular entry mechanism of TFV, HepG2 cells were treated with drugs that inhibit the main endocytic pathways. We observed that TFV entry was inhibited by NH(4)Cl, chloroquine, and bafilomycin, which can all elevate the pH of acidic organelles. In contrast, TFV entry was not influenced by chlorpromazine or overexpression of a dominant-negative form of Esp15, which inhibit the assembly of clathrin-coated pits. These results suggested that TFV entry was not associated with clathrin-mediated endocytosis, but was related to the pH of acidic organelles. Subsequently, we found that endocytosis of TFV was dependent on membrane cholesterol and was inhibited by the caveolin-1 scaffolding domain peptide. Dynamin and actin were also required for TFV entry. In addition, TFV virions colocalized with the cholera toxin subunit B, indicating that TFV enters as caveola-internalized cargo into the Golgi complex. Taken together, our results demonstrated that TFV entry occurs by caveola-mediated endocytosis with a pH-dependent step. This atypical caveola-mediated endocytosis is different from the clathrin-mediated endocytosis of frog virus 3 (FV3) by BHK cells, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection in lower vertebrates.Journal of Virology 07/2011; 85(13):6416-26. · 5.40 Impact Factor -
Article: Molecular cloning, characterization and expression analysis of the tumor necrosis factor (TNF) superfamily gene, TNF receptor superfamily gene and lipopolysaccharide-induced TNF-α factor (LITAF) gene from Litopenaeus vannamei.
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ABSTRACT: In vertebrates, the tumor necrosis factor (TNF)-receptor (TNFR) system participates in diverse physiological and pathological events, such as inflammation and protective immune responses to microbial infections. There are few reports about the role of the invertebrate TNF-TNFR system in immune responses. Here, we isolated and characterized the TNF superfamily (LvTNFSF) gene, TNFR superfamily (LvTNFRSF) gene and lipopolysaccharide-induced TNF-α factor (LvLITAF) gene from Litopenaeus vannamei. LvTNFSF consists of 472 amino acids with a conserved C-terminal TNF domain and has 89.8% identity with the Marsupenaeus japonicus TNF superfamily gene. LvTNFRSF consists of 296 amino acids with a conserved TNFR domain and has 18.0% identity with Chlamys farreri TNFR, 14.6% identity with Drosophila melanogaster Wengen and 14.6% identity with Homo sapiens TNFR1. LvLITAF consists of 124 amino acids with the LITAF domain and shows 62.6% identity with D. melanogaster LITAF and 32.3% identity with H. sapiens LITAF. The promoter region of LvTNFSF was cloned and used to construct a luciferase reporter. In Drosophila S2 cells, the promoter of LvTNFSF can be activated by LvLITAF, L. vannamei NF-κB family proteins (LvRelish and LvDorsal) and LvSTAT. Unlike its mammalian counterparts, LvTNFRSF could not activate the NF-κB pathway in Drosophila S2 cells. Using real-time quantitative PCR, we obtained expression profiles of LvTNFSF, LvTNFRSF and LvLITAF in the gill, intestine and hepatopancreas of L. vannamei after challenge with Gram-negative Vibrio alginolyticus, Gram-positive Staphylococcus aureus, the fungus Candida albicans and white spot syndrome virus (WSSV). Taken together, our results reveal that LvTNFSF, LvTNFRSF and LvLITAF may be involved in shrimp immune responses to pathogenic infections.Developmental and comparative immunology 06/2011; 36(1):39-50. · 3.29 Impact Factor -
Article: The viral ankyrin repeat protein (ORF124L) from infectious spleen and kidney necrosis virus attenuates nuclear factor-κB activation and interacts with IκB kinase β.
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ABSTRACT: The ankyrin (ANK) repeat is one of the most common protein-protein interaction motifs, found predominantly in eukaryotes and bacteria, but the functions of the ANK repeat are rarely researched in animal viruses, with the exception of poxviruses. Infectious spleen and kidney necrosis virus (ISKNV) is a typical member of the genus Megalocytivirus in the family Iridoviridae and is a causative agent of epizootics in fish. The genome of ISKNV contains four putative viral ANK (vANK) repeat proteins and their functions remain largely unknown. In the present study, it was found that ORF124L, a vANK repeat protein in ISKNV, encodes a protein of 274 aa with three ANK repeats. Transcription of ORF124L was detected at 12 h post-infection (p.i.) and reached a peak at 40 h p.i. ORF124L was found to localize to both the nucleus and the cytoplasm in mandarin fish fry cells. ISKNV ORF124L interacted with the mandarin fish IκB kinase β protein (scIKKβ), and attenuated tumour necrosis factor alpha (TNF-α)- or phorbol myristate acetate (PMA)-induced activity of a nuclear factor κB (NF-κB)-luciferase reporter but did not interfere with the activity of an activator protein 1 (AP-1)-luciferase reporter. Phosphorylation of IκBα and nuclear translocation of NF-κB were also impaired by ISKNV ORF124L. In summary, ORF124L was identified as a vANK repeat protein and its role in inhibition of TNF-α-induced NF-κB signalling was investigated through interaction with the mandarin fish IKKβ. This work may help to improve our understanding of the function of fish iridovirus ANK repeat proteins.Journal of General Virology 04/2011; 92(Pt 7):1561-70. · 3.36 Impact Factor
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Institutions
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2005–2012
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Sun Yat-Sen University
- School of Life Sciences
Guangzhou, Guangdong Sheng, China
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2002–2008
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The University of Hong Kong
- School of Biological Sciences
Hong Kong, Hong Kong
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2003–2007
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Zhongshan University
Zhongshan, Guangdong Sheng, China
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