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Find video protocols related to scientific articles indexed in Pubmed.
Entry of a Novel Marine DNA Virus, Singapore Grouper Iridovirus, into Host Cells Occurs via Clathrin-Mediated Endocytosis and Macropinocytosis in a pH-Dependent Manner.
J. Virol.
PUBLISHED: 08-27-2014
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Iridoviruses are nucleocytoplasmic DNA viruses which cause great economic losses in the aquaculture industry but also show significant threat to global biodiversity. However, a lack of host cells has resulted in poor progress in clarifying iridovirus behavior. We investigated the crucial events during virus entry using a combination of single-virus tracking and biochemical assays, based on the established virus-cell infection model for Singapore grouper iridovirus (SGIV). SGIV infection in host cells was strongly inhibited when cells were pretreated with drugs blocking clathrin-mediated endocytosis, including sucrose and chlorpromazine. Inhibition of key regulators of macropinocytosis, including Na(+)/H(+) exchanger, Rac1 GTPase, p21-activated kinase 1 (PAK1), protein kinase C (PKC), and myosin II, significantly reduced SGIV uptake. Cy5-labeled SGIV particles were observed to colocalize with clathrin and macropinosomes. In contrast, disruption of cellular cholesterol by methyl-?-cyclodextrin and nystatin had no effect on virus infection, suggesting that SGIV entered grouper cells via the clathrin-mediated endocytic pathway and macropinocytosis but not via caveola-dependent endocytosis. Furthermore, inhibitors of endosome acidification such as chloroquine and bafilomycin A1 blocked virus infection, indicating that SGIV entered cells in a pH-dependent manner. In addition, SGIV particles were observed to be transported along both microtubules and actin filaments, and intracellular SGIV motility was remarkably impaired by depolymerization of microtubules or actin filaments. The results of this study for the first time demonstrate that not only the clathrin-dependent pathway but also macropinocytosis are involved in fish DNA enveloped virus entry, thus providing a convenient tactic for exploring the life cycle of DNA viruses.
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Involvement of fish signal transducer and activator of transcription 3 (STAT3) in SGIV replication and virus induced paraptosis.
Fish Shellfish Immunol.
PUBLISHED: 07-25-2014
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Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor which plays crucial roles in immune regulation, inflammation, cell proliferation, transformation, and other physiological processes of the organism. In this study, a novel STAT3 gene from orange spotted grouper (Ec-STAT3) was cloned and characterized. Bioinformatic analysis revealed that full-length of Ec-STAT3 was 3105-bp long and contained a 280-bp 5'UTR, a 470-bp 3'UTR, and a 2355-bp open reading frame (ORF) that encoded a 784-amino acid peptide. The deduced protein of Ec-STAT3 showed 98% identity to that of turbot (Scophthalmus maximus). Amino acid alignment showed that Ec-STAT3 contained four conserved domains, including a protein interaction domain, a coiled coil domain, a DNA binding domain, and an SH2 domain. Quantitative real-time PCR analysis showed that the highest expression level was detected in the liver, followed by skin and spleen. After injection with Singapore grouper iridovirus (SGIV), the transcript of Ec-STAT3 in spleen was increased significantly. To further explore the function of Ec-STAT3, we investigated the roles of Ec-STAT3 in SGIV infection in vitro. Immune fluorescence analysis indicated that SGIV infection altered the distribution of phosphorylated Ec-STAT3 in nucleus, and a small part of phosphorylated Ec-STAT3 was associated with virus assembly sites, suggesting that Ec-STAT3 might be important for SGIV infection. Using STAT3 specific inhibitor, S3I-201, we found that inhibition of Ec-STAT3 activation decreased the SGIV replication significantly. Moreover, inhibition of Ec-STAT3 activation obviously altered SGIV infection induced cell cycle arrest and the expression of pro-survival genes, including Bcl-2, Bcl-xL and Bax inhibitor. Together, our results firstly demonstrated the critical roles of fish STAT3 in DNA virus replication and virus induced paraptosis, but also provided new insights into the mechanism of iridovirus pathogenesis.
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Involvement of the PI3K and ERK signaling pathways in largemouth bass virus-induced apoptosis and viral replication.
Fish Shellfish Immunol.
PUBLISHED: 04-24-2014
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Increased reports demonstrated that largemouth Bass, Micropterus salmoides in natural and artificial environments were always suffered from an emerging iridovirus disease, largemouth Bass virus (LMBV). However, the underlying mechanism of LMBV pathogenesis remained largely unknown. Here, we investigated the cell signaling events involved in virus induced cell death and viral replication in vitro. We found that LMBV infection in epithelioma papulosum cyprini (EPC) cells induced typical apoptosis, evidenced by the appearance of apoptotic bodies, cytochrome c release, mitochondrial membrane permeabilization (MMP) destruction and reactive oxygen species (ROS) generation. Two initiators of apoptosis, caspase-8 and caspase-9, and the executioner of apoptosis, caspase-3, were all significantly activated with the infection time, suggested that not only mitochondrion-mediated, but also death receptor-mediated apoptosis were involved in LMBV infection. Reporter gene assay showed that the promoter activity of transcription factors including p53, NF-?B, AP-1 and cAMP response element-binding protein (CREB) were decreased during LMBV infection. After treatment with different signaling pathway inhibitors, virus production were significantly suppressed by the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway and extracellular-signal-regulated kinases (ERK) signaling pathway. Furthermore, LMBV infection induced apoptosis was enhanced by PI3K inhibitor LY294002, but decreased by addition of ERK inhibitor UO126. Therefore, we speculated that apoptosis was sophisticatedly regulated by a series of cell signaling events for efficient virus propagation. Taken together, our results provided new insights into the molecular mechanism of ranavirus infection.
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Molecular cloning and characterization of two types of I?B? orthologues in orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 02-19-2014
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Inhibitors of kappa B (I?Bs) are the members of primary regulators of NF-?B, which can inhibit NF-?B activity by blocking the NF-?B in an inactive state in the cytoplasm. In this study, two types of I?B? orthologues (EcI?B?A and EcI?B?B) from orange-spotted grouper, Epinephelus coioides, were cloned and characterized. EcI?B?A and EcI?B?B encoded putative proteins containing 308 and 318 amino acids, which shared 59% and 53% identity to I?B?A and I?B?B of Danio rerio, respectively. Amino acid sequence alignment showed that both EcI?B?A and EcI?B?B contained a conserved degradation motif DSGLDS in the N-terminal region and a PEST sequence in the C-terminal region. In addition, EcI?B?A and EcI?B?B contained 5 and 6 ankyrin repeats, respectively. The genomic DNA of EcI?B?A and EcI?B?B consisted of 6 exons and 5 introns. Both of their transcripts were widely distributed in different tissues, and the expression levels were different in response to various stimuli, including lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Dual-luciferase reporter assay suggested that both EcI?B?A and EcI?B?B were able to inhibit Ecc-Rel and Ecp65 induced NF-?B promoter activity in grouper spleen (GS) cells. Subcellular localization analysis showed that EcI?B?B was present predominantly in the cytoplasm, while EcI?B?A was distributed throughout both the nucleus and the cytoplasm. Furthermore, overexpression of EcI?B?A and EcI?B?B in GS cells inhibited the viral gene transcriptions of MCP, ORF019 and ORF162 of SGIV. Taken together, our findings suggested that both EcI?B?A and EcI?B?B were involved in grouper innate immunity against virus.
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Singapore grouper iridovirus-encoded semaphorin homologue (SGIV-sema) contributes to viral replication, cytoskeleton reorganization and inhibition of cellular immune responses.
J. Gen. Virol.
PUBLISHED: 02-17-2014
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Semaphorins are a large, phylogenetically conserved family of proteins that are involved in a wide range of biological processes including axonal steering, organogenesis, neoplastic transformation, as well as immune responses. In this study, a novel semaphorin homologue gene belonging to the Singapore grouper iridovirus (SGIV), ORF155R (termed SGIV-sema), was cloned and characterized. The coding region of SGIV-sema is 1728 bp in length, encoding a predicted protein with 575 aa. SGIV-sema contains a ~370 aa N-terminal Sema domain, a conserved plexin-semaphorin-integrin (PSI) domain, and an immunoglobulin (Ig)-like domain near the C terminus. SGIV-sema is an early gene product during viral infection and predominantly distributed in the cytoplasm with a speckled and clubbed pattern of appearance. Functionally, SGIV-sema could promote viral replication during SGIV infection in vitro, with no effect on the proliferation of host cells. Intriguingly, ectopically expressed SGIV-sema could alter the cytoskeletal structure of fish cells, characterized by a circumferential ring of microtubules near the nucleus and a disrupted microfilament organization. Furthermore, SGIV-sema was able to attenuate the cellular immune response, as demonstrated by decreased expression of inflammation/immune-related genes such as IL-8, IL-15, TNF-? and mediator of IRF3 activation (MITA), in SGIV-sema-expressing cells before and after SGIV infection. Ultimately, our study identified a novel, functional SGIV gene that could regulate cytoskeletal structure, immune responses and facilitate viral replication.
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Molecular cloning and characterization of a new G-type lysozyme gene (Ec-lysG) in orange-spotted grouper, Epinephelus coioides.
Dev. Comp. Immunol.
PUBLISHED: 01-22-2014
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Lysozyme acts as an innate immunity molecule against pathogen infection. In this study, a new G-type lysozyme gene with a typical G-type lysozyme domain (designated as Ec-lysG) was cloned and characterized from the orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysG cDNA contains 1419 bp and encodes a 256-residue protein containing a 25-residue signal peptide at the N-terminus. BLAST analysis reveals Ec-lysG shares 64% identity with Siniperca chuatsi, but 63% to another reported G-type lysozyme from orange-spotted grouper (OSG-lysG). The genomic DNA of Ec-lysG contains four exons and three introns, with a total length of 2062 bp. An amino acid sequence alignment showed that Ec-lysG shares the fundamental structural features of G-type lysozyme, including the catalytic residues, substrate binding sites, and soluble lytic transglycosylase domain. Quantitative PCR showed that Ec-lysG transcript is most abundant in the head kidney, and less abundant in the heart. The expression of Ec-lysG was differentially upregulated in the head kidney after stimulation with lipopolysaccharide, Vibrio alginolyticus, and Singapore grouper iridovirus (SGIV). A subcellular localization analysis showed that Ec-lysG is distributed predominantly in the cytoplasm. Recombinant Ec-lysG (rEc-lysG) has optimal activity at pH 7.5 and 35°C. rEc-lysG showed lytic activities against Gram-positive bacterium Streptococcus iniae, Staphylococcus aureus, and Micrococcus lysodeikticus, and the Gram-negative bacterium V. alginolyticus. Scanning electron microscopy (SEM) showed that rEc-lysG acts on M. lysodeikticus cell walls. The overexpression of Ec-lysG in grouper cells did not significantly delay the occurrence of the cytopathic effect (CPE) induced by SGIV, and did not inhibit viral gene transcription. In conclusion, Ec-lysG might be a potent antibacterial protein, with a role in innate immunity.
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Isolation and characterization of a new class of DNA aptamers specific binding to Singapore grouper iridovirus (SGIV) with antiviral activities.
Virus Res.
PUBLISHED: 01-13-2014
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The Singapore grouper iridovirus (SGIV), a member of the genus Ranavirus, is a major viral pathogen that has caused heavy economic losses to the grouper aquaculture industry in China and Southeast Asia. No efficient method of controlling SGIV outbreaks is currently available. Systematic evolution of ligands by exponential enrichment (SELEX) is now widely used for the in vitro selection of artificial ssDNA or RNA ligands, known as aptamers, which bind to targets through their stable three-dimensional structures. In our current study, we generated ssDNA aptamers against the SGIV, and evaluated their ability to block SGIV infection in cultured fish cells and cultured fish in vivo. The anti-SGIV DNA aptamers, LMB-761, LMB-764, LMB-748, LMB-439, LMB-755, and LMB-767, were selected from a pool of oligonucleotides randomly generated using a SELEX iterative method. The analysis of the secondary structure of the aptamers revealed that they all formed similar stem-loop structures. Electrophoretic mobility shift assays showed that the aptamers bound SGIV specifically, as evidenced by a lack cross-reactivity with the soft shell turtle iridovirus. The aptamers produced no cytotoxic effects in cultured grouper spleen cells (GS). Assessment of cytopathic effects (CPE) and viral titer assays showed that LMB-761, LMB-764, LMB-748, LMB-755, and LMB-767 significantly inhibited SGIV infection in GS cells. The in vivo experiments showed that LMB-761 and LMB-764 reduced SGIV-related mortality, and no negative effects were observed in orange-spotted grouper, Epinephelus coioides, indicating that these DNA aptamers may be suitable antiviral candidates for controlling SGIV infections in fish reared in marine aquaculture facilities.
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Isolation and characterization of tumor necrosis factor receptor-associated factor 6 (TRAF6) from grouper, Epinephelus tauvina.
Fish Shellfish Immunol.
PUBLISHED: 01-06-2014
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Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. In the present study, a TRAF6 (named as Et-TRAF6) was identified from the marine fish grouper, Epinephelus tauvina by RACE PCR. The full-length cDNA of Et-TRAF6 comprised 1949 bp with a 1713 bp open reading frame (ORF) that encodes a putative protein of 570 amino acids. Similar to most TRAF6s, Et-TRAF6 includes one N-terminal RING domain (78aa-116aa), two zinc fingers of TRAF-type (159aa-210aa and 212aa-269aa), one coiled-coil region (370aa-394aa), and one conserved C-terminal meprin and TRAF homology (MATH) domain (401aa-526aa). Quantitative real-time PCR analysis revealed that Et-TRAF6 mRNA is expressed in all tested tissues, with the predominant expression in the stomach and intestine. The expression of Et-TRAF6 was up-regulated in the liver after challenge with Lipoteichoic acid (LTA), Peptidoglycan (PGN), Zymosan, polyinosine-polycytidylic acid [Poly(I:C)] and Polydeoxyadenylic acid · Polythymidylic acid sodium salt [Poly(dA:dT)]. The expression of Et-TRAF6 was also up-regulated in the liver after infection with Vibrio alginolyticus, Singapore grouper iridovirus (SGIV) and grouper nervous necrosis virus (GNNV). Recombinant Et-TRAF6 (rEt-TRAF6) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Et-TRAF6 serum preparation. Intracellular localization revealed that Et-TRAF6 is distributed in both cytoplasm and nucleus, and predominantly in the cytoplasm. These results together indicated that Et-TRAF6 might be involved in immune responses toward bacterial and virus challenges.
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An insulin-like growth factor homologue of Singapore grouper iridovirus modulates cell proliferation, apoptosis and enhances viral replication.
J. Gen. Virol.
PUBLISHED: 09-23-2013
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Insulin-like growth factors (IGFs) play crucial roles in regulating cell differentiation, proliferation and apoptosis. In this study, a novel IGF homologue gene (IGF-like) encoded by Singapore grouper iridovirus (SGIV) ORF062R (termed SGIV-IGF), was cloned and characterized. The coding region of SGIV-IGF is 771 bp in length, with a variable number of tandem repeats (VNTR) locus at the 3-end. We cloned one isoform of this novel gene, 582 bp in length, containing the predicted IGF domain and 3.6 copy numbers of the 27 bp repeat unit. SGIV-IGF was an early transcribed gene during viral infection, and SGIV-IGF was distributed predominantly in the cytoplasm with a diffused granular appearance. Intriguingly, overexpression of SGIV-IGF was able to promote the growth of grouper embryonic cells (GP cells) by promoting G1/S phase transition, which was at least partially dependent on its 3-end VNTR locus. Furthermore, viral titre assay and real-time quantitative PCR (RT-qPCR) analysis proved that SGIV-IGF could promote SGIV replication in grouper cells. In addition, overexpression of SGIV-IGF mildly facilitated apoptosis in SGIV-infected non-host fathead minnow (FHM) cells. Together, our study demonstrated a novel functional gene of SGIV which may regulate viral replication and cellular processes through multiple mechanisms that appear to be cell type-dependent.
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Characterization of cathepsin B gene from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.
Fish Shellfish Immunol.
PUBLISHED: 09-19-2013
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The lysosomal cysteine protease cathepsin B of papain family is a key regulator and signaling molecule that involves in various biological processes, such as the regulation of apoptosis and activation of virus. In the present study, cathepsin B gene (Ec-CB) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-CB cDNA was composed of 1918 bp and encoded a polypeptide of 330 amino acids with higher identities to cathepsin B of teleosts and mammalians. Ec-CB possessed typical cathepsin B structural features including an N-terminal signal peptide, the propeptide region and the cysteine protease domain which were conserved in other cathepsin B sequences. Phylogenetic analysis revealed that Ec-CB was most closely related to Lutjanus argentimaculatus. RT-PCR analysis showed that Ec-CB transcript was expressed in all the examined tissues which abundant in spleen, kidney and gill. After challenged with Singapore grouper iridovirus (SGIV) stimulation, the mRNA expression of cathepsin B in E. coioides was up-regulated at 24 h post-infection. Subcellular localization analysis revealed that Ec-CB was distributed predominantly in the cytoplasm. When the fish cells (GS or FHM) were treated with the cathepsin B specific inhibitor CA-074Me, the occurrence of CPE induced by SGIV was delayed, and the viral gene transcription was significantly inhibited. Additionally, SGIV-induced typical apoptosis was also inhibited by CA-074Me in FHM cells. Taken together, our results demonstrated that the Ec-CB might play a functional role in SGIV infection.
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Characterization of an envelope gene VP19 from Singapore grouper iridovirus.
Virol. J.
PUBLISHED: 09-17-2013
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Viral envelope proteins are always proposed to exert important function during virus infection and replication. Vertebrate iridoviruses are enveloped large DNA virus, which can cause great economic losses in aquaculture and ecological destruction. Although numerous iridovirus envelope proteins have been identified using bioinformatics and proteomic methods, their roles in virus infection remained largely unknown.
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Identification and characterization of Rab7 from orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 07-19-2013
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Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. During the virus-host co-evolution, host Rab7 was also exploited by virus to complete their life cycle. To date, however, the roles of fish Rab7 in virus infection remained largely unknown. Here, we cloned and characterized a Rab7 gene from grouper, Epinephelus coioides (Ec-Rab7). The full-length Ec-Rab7 cDNA was composed of 1182 bp and encoded a polypeptide of 207 amino acids which shared 99% identity with that from Anoplopoma fimbria or Oreochromis niloticus. Ec-Rab7 contained five conserved domains of Rab GTPase family including GTP-binding or GTPase regions as well as an effector site. RT-PCR analysis revealed that Ec-Rab7 ubiquitously expressed in all detected tissues and its transcript in spleen was up-regulated after challenge with Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-Rab7 was distributed in the cytoplasm as spots and mostly colocalized with lysosomes. Notably, the ectopic expressed Ec-Rab7 partly aggregated into the viral factories in cells infected by SGIV. Furthermore, overexpression of Ec-Rab7 accelerated the occurrence of cytopathic effect (CPE) induced by SGIV infection and promoted viral gene transcription. In addition, far western blotting assay revealed that Ec-Rab7 might interact with viral proteins, including SGIV VP69 and VP101. Taken together, our data suggested that Ec-Rab7 might be potentially involved in SGIV replication.
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Medaka haploid embryonic stem cells are susceptible to Singapore grouper iridovirus as well as to other viruses of aquaculture fish species.
J. Gen. Virol.
PUBLISHED: 07-04-2013
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Viral infection is a challenge in high-density aquaculture, as it leads to various diseases and causes massive or even complete loss. The identification and disruption of host factors that viruses utilize for infection offer a novel approach to generate viral-resistant seed stocks for cost-efficient and sustainable aquaculture. Genetic screening in haploid cell cultures represents an ideal tool for host factor identification. We have recently generated haploid embryonic stem (ES) cells in the laboratory fish medaka. Here, we report that HX1, one of the three established medaka haploid ES cell lines, was susceptible to the viruses tested and is thus suitable for genetic screening to identify host factors. HX1 cells displayed a cytopathic effect and massive death upon inoculation with three highly infectious and notifiable fish viruses, namely Singapore grouper iridovirus (SGIV), spring viremia of carp virus (SVCV) and red-spotted grouper nervous necrosis virus (RGNNV). Reverse transcription-PCR and Western blot analyses revealed the expression of virus genes. SGIV infection in HX1 cells elicited a host immune response and apoptosis. Viral replication kinetics were determined from a virus growth curve, and electron microscopy revealed propagation, assembly and release of infectious SGIV particles in HX1 cells. Our results demonstrate that medaka haploid ES cells are susceptible to SGIV, as well as to SVCV and RGNNV, offering a unique opportunity for the identification of host factors by genetic screening.
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Identification and characterization of a tumor necrosis factor receptor like protein encoded by Singapore grouper iridovirus.
Virus Res.
PUBLISHED: 06-23-2013
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Virus encoded tumor necrosis factor receptors (TNFRs) have been demonstrated to facilitate virus to escape from apoptosis or other host immune response for viral replication. Singapore grouper iridovirus (SGIV), a large DNA virus which belongs to genus Ranavirus, is a major pathogen resulting in heavy economic losses to grouper aquaculture. Here, SGIV ORF096 (VP96) encoding a putative homolog of TNFR was identified and characterized. Multiple sequence alignment indicated that SGIV-VP96 contained two extracellular cysteine-rich domains (CRDs) with conserved four or six cysteine residues, but lacked the transmembrane domain at the C-terminus. SGIV-VP96 was identified as an early (E) gene and localized in the cytoplasm in transfected or infected cells. Overexpression of SGIV-VP96 in vitro enhanced cell proliferation, and improved cell survival against SGIV infection. Furthermore, virus infection induced apoptosis and caspase-3 activity were inhibited in SGIV-VP96 expressing FHM cells compared to the control cells. Taken together, our results suggested that SGIV might utilize virus encoded TNFR like genes to modulate the host apoptotic response for effective virus replication.
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Characterization of LPS-induced TNF? factor (LITAF) from orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 06-13-2013
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Lipopolysaccharide-induced TNF? factor (LITAF) is an important transcription factor that mediates cell apoptosis and inflammatory response. In the present study, we cloned and characterized a LITAF gene from orange-spotted grouper (Epinephelus coioides) (Ec-LITAF). Ec-LITAF encoded a predicted 142 amino acid protein which shared 74% identity to sablefish (Anoplopoma fimbria) LITAF homolog. Multiple amino acid alignment showed that Ec-LITAF contained a typical LITAF domain with two CXXC motifs. Phylogenetic analysis indicated that Ec-LITAF was closely related to that of sablefish. Ec-LITAF mRNA was widely expressed in different tissues and its expression level in spleen was up-regulated after Singapore grouper iridovirus (SGIV) infection. Subcellular localization analysis revealed that the distribution of Ec-LITAF showed diffuse and aggregated patterns in cytoplasm. Interestingly, the distribution of Ec-LITAF overlayed with a viral LITAF homolog (vLITAF) encoded by SGIV. Overexpression of Ec-LITAF in vitro up-regulated the expression of tumor necrosis factors (TNF1 and TNF2) and TNF receptors (TNFR1 and TNFR2), and the expression of itself initiated apoptosis in fish cells. In addition, overexpression of Ec-LITAF not only accelerated SGIV infection induced CPE and cell death, but also increased viral gene transcription. Taken together, our data suggested that Ec-LITAF might play crucial roles during SGIV replication.
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Genome Sequence of Klebsiella pneumoniae HSL4, a New Strain Isolated from Mangrove Sediment for Biosynthesis of 1,3-Propanediol.
Genome Announc
PUBLISHED: 05-11-2013
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Klebsiella pneumoniae HSL4 is a 1,3-propanediol-producing bacterium strain isolated from mangrove sediment. We present here a 5,221,448-bp assembly of its genome sequence. Genome analysis revealed that it contains 10 coding sequences (CDSs) responsible for glycerol fermentation to 1,3-propanediol, 19 CDSs encoding glycerol utilization, and 140 CDSs related to its virulence.
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Identification and characterization of TRP14, a thioredoxin-related protein of 14 kDa from orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 05-07-2013
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Thioredoxin (abbreviated as Trx) is an important ubiquitous disulfide reductase, which can protect organisms against various oxidative stresses. In the present study, a thioredoxin-related protein of 14 kDa (named as Ec-TRP14) was identified from the marine fish grouper, Epinephelus coioides by RACE PCR. The full-length cDNA of Ec-TRP14 was comprised of 1066 bp with a 372 bp open reading frame that encodes a putative protein of 123 amino acids. Similar to most TRP14s, Ec-TRP14 contained the conserved motif C-P-D-C. Ec-TRP14 mRNA is predominately expressed in liver, brain and muscle. The expression of Ec-TRP14 was up-regulated in the liver of grouper challenged with SGIV. Ec-TRP14 was recombined and expressed in Escherichia coli BL21 (DE3), and the rEc-Ec-TRP14 fusion protein was demonstrated to possess the antioxidant activity. The grouper spleen (GS) cells were treated with a high concentration of rEc-TRP14 (8.3 ?g/ml), which significantly enhanced cells viability under damage caused by viral infection. These results together indicated that Ec-TRP14 could function as an important antioxidant in a physiological context, and might be involved in the responses to viral challenge.
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Molecular cloning, expression and functional analysis of ISG15 in orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 01-20-2013
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Interferon-stimulated gene 15 (ISG15) is an ubiquitin homolog that is significantly induced by type I interferons or viral infections. Groupers, Epinephelus spp. being maricultured in China and Southeast Asian countries, always suffer from virus infection, including iridovirus and nodavirus. To date, the roles of grouper genes, especially interferon related genes in virus infection remained largely unknown. Here, the ISG15 homolog (EcISG15) was cloned from grouper Epinephelus coioides and its immune response to Singapore grouper iridovirus (SGIV) and grouper nervous necrosis virus (GNNV) was investigated. The full-length EcISG15 cDNA was composed of 948 bp and encoded a polypeptide of 155 amino acids with 37-68% identity with the known ISG15 homologs from other fish species. Amino acid alignment analysis indicated that EcISG15 contained two ubiquitin-like (UBL) domains and an Ub-conjugation domain (LRGG). Expressional analysis showed that EcISG15 was dramatically induced by GNNV infection, poly I:C or poly dA-dT treatment, but no obvious changes were observed during SGIV infection. Immunofluorescence assay showed that EcISG15 localized mainly in the cytoplasm of grouper cells in response to poly I:C stimulation or GNNV infection, but not in mock or SGIV infected cells. Western blot analysis indicated that the ISGylation was absent in SGIV-infected cells, but significantly enhanced in GNNV-infected or poly I:C transfected cells, suggesting that EcISG15 might play different roles in SGIV and GNNV infection. Furthermore, overexpression of EcISG15 in vitro inhibited the transcription of GNNV genes significantly. Taken together, the results indicated that fish ISG15 might exert important roles against RNA virus infection.
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miR-homoHSV of Singapore Grouper Iridovirus (SGIV) Inhibits Expression of the SGIV Pro-apoptotic Factor LITAF and Attenuates Cell Death.
PLoS ONE
PUBLISHED: 01-01-2013
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Growing evidence demonstrates that various large DNA viruses could encode microRNAs (miRNAs) that regulate host and viral genes to achieve immune evasion. In this study, we report that miR-homoHSV, an miRNA encoded by Singapore grouper iridovirus (SGIV), can attenuate SGIV-induced cell death. Mechanistically, SGIV miR-homoHSV targets SGIV ORF136R, a viral gene that encodes the pro-apoptotic lipopolysaccharide-induced TNF-? (LITAF)-like factor. miR-homoHSV suppressed exogenous and endogenous SGIV LITAF expression, and thus inhibited SGIV LITAF-induced apoptosis. Meanwhile, miR-homoHSV expression was able to attenuate cell death induced by viral infection, presumably facilitating viral replication through the down-regulation of the pro-apoptotic gene SGIV LITAF. Together, our data suggest miR-homoHSV may serve as a feedback regulator of cell death during viral infection. The findings of this study provide a better understanding of SGIV replication and pathogenesis.
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Mangroviflexus xiamenensis gen. nov., sp. nov., a member of the family Marinilabiliaceae isolated from mangrove sediment.
Int. J. Syst. Evol. Microbiol.
PUBLISHED: 10-07-2011
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A Gram-negative, obligately anaerobic, non-spore-forming, long rod-shaped bacterium strain P2(T) was isolated from the offshore mangrove sediment of the South China Sea. Growth was observed at between 22 and 39 °C, with an optimum at 35 °C. The pH range for growth was 5.0-8.5, with an optimum around pH 7.0-7.5. Salt tolerance was determined between 0.2 and 3.5% (w/v), optimum at 0.5-1.0%. Catalase and oxidase activities were negative. Strain P2(T) utilized cysteine, lactate, pyruvate, yeast extract or H(2)/CO(2)+acetate as electron donors, and sulfate or sulfite as electron acceptors. Metabolism was strictly fermentative. The main organic fermentation products were propionate, acetate and succinate. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain P2(T) formed a distinct evolutionary lineage within the family Marinilabiliaceae. Strain P2(T) was most closely related to members of the genera Alkaliflexus (92.0% 16S rRNA gene sequence similarity), Marinilabilia (91.7%) and Anaerophaga (89.9%) of the family Marinilabiliaceae. The DNA G+C content of the novel strain was 44.2 ± 1.0 mol%. The dominant fatty acids of strain P2(T) were iso-C(15:0) (33.5%), anteiso-C(15:0) (18.9%), C(16:0) (5.4%), C(16:0) 3-OH (7.7%) and iso-C(17:0) 3-OH (13.3%). The respiratory quinone was menaquinone 7 (100% of total quinone) and the major polar lipid was phosphatidylethanolamine. Strain P2(T) was distinguishable from members of phylogenetically related genera by differences in several phenotypic properties. On the basis of phylogenetic, phenotypic and physiological evidence, a novel genus, Mangroviflexus, is proposed to harbour strain P2(T) (?=?CGMCC 1.5167(T)?=?DSM 24214(T)) which is described as the type strain of a novel species, Mangroviflexus xiamenensis gen. nov., sp. nov.
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Desulfobaculum xiamenensis gen. nov., sp. nov., a member of the family Desulfovibrionaceae isolated from marine mangrove sediment.
Int. J. Syst. Evol. Microbiol.
PUBLISHED: 08-26-2011
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A taxonomic study was carried out on strain P1(T), which was isolated from mangrove sediment samples collected from Qinglan Port (Hainan, China). Cells were curved rods, that were motile, with a single polar flagellum. The strain was non-spore-forming with a cell size of 0.6×1.5-2.2 µm. Catalase and oxidase activities were not detected. Growth was observed in the temperature range 22-44 °C (optimum, 35-40 °C) and pH range 5.5-8.5 (optimum, pH 7.0). NaCl was required for growth and tolerated at up to 3.5% (w/v) (optimum, 0.5%). Strain P1(T) utilized hydrogen, succinate, L-malate, citrate, oxalate, DL-lactate, pyruvate, or cysteine as electron donors, and sulfate or sulfite as electron acceptors. Fermentation products from pyruvate were acetate, H(2) and CO(2). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain P1(T) formed a distinct evolutionary lineage within the family Desulfovibrionaceae. Strain P1(T) was most closely related to members of the genera Desulfovibrio (92.0-94.3% 16S rRNA gene sequence similarity), Desulfocurvus (91.1%), Bilophila (87.9%) and Lawsonia (86.0%) of the family Desulfovibrionaceae. The DNA G+C content of strain P1(T) was 64.5 mol% and the major cellular fatty acids were iso-C(15:0) (18.8%), anteiso-C(15:0) (5.0%), C(16:0) (14.2%) and iso-C(17:1)?9c (24.4%). The predominant menaquinone was MK-7 (97%). Major polar lipids were phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. Strain P1(T) was distinguishable from members of phylogenetically related genera by differences in several phenotypic properties. On the basis of the phenotypic and phylogenetic data, strain P1(T) represents a novel species of a new genus, for which the name Desulfobaculum xiamenensis gen. nov., sp. nov. is proposed. The type strain of Desulfobaculum xiamenensis is P1(T) (=CGMCC 1.5166(T)=DSM 24233(T)).
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Characterization of p38 MAPKs from orange-spotted grouper, Epinephelus coioides involved in SGIV infection.
Fish Shellfish Immunol.
PUBLISHED: 07-30-2011
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p38 mitogen-activated protein kinases (MAPKs) are broadly expressed signaling molecules that involves in the regulation of cellular responsible for various extracellular stimuli. In this study, three p38 MAPK genes (Ec-p38a, p38b and p38?) were cloned from grouper, Epinephelus coioides and their characteristics were investigated in vitro. Although Ec-p38a, p38b and p38? showed high homologies to other fish p38a MPAK, p38b MAPK and p38? MAPK, respectively, they all contained the conserved structures of Thr-Gly-Tyr (TGY) motif and substrate binding site Ala-Thr-Arg-Trp (ATRW). Phylogenetic analysis indicated that Ec-p38a, p38b and p38? are more closely related to those from fish than mammals. The tissue distribution patterns of Ec-p38a, p38b and p38? were different, and Ec-p38? was up-regulated most obviously in head kidney after Singapore grouper iridovirus (SGIV) infection. Overexpression of Ec-p38? in FHM cells delayed the occurrence of CPE induced by SGIV infection. Further analysis indicated that overexpression of Ec-p38? inhibited viral gene transcription and protein synthesis, as well as SGIV induced typical apoptosis in fish cells. Taken together, our data indicated that Ec-p38? played a crucial role in regulating apoptosis and virus replication during iridovirus infection.
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Transcriptome analysis of orange-spotted grouper (Epinephelus coioides) spleen in response to Singapore grouper iridovirus.
BMC Genomics
PUBLISHED: 07-12-2011
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Orange-spotted grouper (Epinephelus coioides) is an economically important marine fish cultured in China and Southeast Asian countries. The emergence of infectious viral diseases, including iridovirus and betanodavirus, have severely affected food products based on this species, causing heavy economic losses. Limited available information on the genomics of E. coioides has hampered the understanding of the molecular mechanisms that underlie host-virus interactions. In this study, we used a 454 pyrosequencing method to investigate differentially-expressed genes in the spleen of the E. coioides infected with Singapore grouper iridovirus (SGIV).
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Singapore grouper iridovirus, a large DNA virus, induces nonapoptotic cell death by a cell type dependent fashion and evokes ERK signaling.
Apoptosis
PUBLISHED: 06-10-2011
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Virus induced cell death, including apoptosis and nonapoptotic cell death, plays a critical role in the pathogenesis of viral diseases. Singapore grouper iridovirus (SGIV), a novel iridovirus of genus Ranavirus, causes high mortality and heavy economic losses in grouper aquaculture. Here, using fluorescence microscopy, electron microscopy and biochemical assays, we found that SGIV infection in host (grouper spleen, EAGS) cells evoked nonapoptotic programmed cell death (PCD), characterized by appearance of cytoplasmic vacuoles and distended endoplasmic reticulum, in the absence of DNA fragmentation, apoptotic bodies and caspase activation. In contrast, SGIV induced typical apoptosis in non-host (fathead minnow, FHM) cells, as evidenced by caspase activation and DNA fragmentation, suggesting that SGIV infection induced nonapoptotic cell death by a cell type dependent fashion. Furthermore, viral replication was essential for SGIV induced nonapoptotic cell death, but not for apoptosis. Notably, the disruption of mitochondrial transmembrane potential (??m) and externalization of phosphatidylserine (PS) were not detected in EAGS cells but in FHM cells after SGIV infection. Moreover, the extracellular signal-regulated kinase (ERK) signaling was involved in SGIV infection induced nonapoptotic cell death and viral replication. This is a first demonstration of ERK-mediated nonapoptotic cell death induced by a DNA virus. These findings contribute to understanding the mechanisms of iridovirus pathogenesis.
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Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication.
Virus Res.
PUBLISHED: 04-17-2011
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Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances.
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Involvement of the mitogen-activated protein kinase pathway in soft-shelled turtle iridovirus-induced apoptosis.
Apoptosis
PUBLISHED: 03-29-2011
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Iridoviruses are large DNA viruses that infect invertebrates and poikilothermic vertebrates, and result in significant economic losses in aquaculture production, and drastic declines in amphibian populations. Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in farm-raised soft-shelled turtles (Trionyx sinensis). In the present study, the mechanisms of STIV-induced cell death and the roles of the mitogen-activated protein kinase (MAPK) signaling pathway were investigated. STIV infection evoked typical apoptosis in fish cells, as demonstrated by the formation of apoptotic bodies, positive terminal deoxynucleotidyl transferase-mediated nicked-end labeling, and caspase-3 activation. The translocation of cytochrome c from mitochondria to cytoplasm, and caspase-9 activation suggested that a mitochondria-mediated pathway was involved in STIV-induced apoptosis. Moreover, MAPK pathways, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK signaling were activated during STIV infection. Using specific inhibitors, we found that MAPK signaling molecules, including ERK, JNK and p38 MAPK, were important for virus release, whereas, only ERK and p38 MAPK were involved in STIV-induced apoptosis by modulating caspase-3 activity. Taken together, our findings shed light on the roles of the MAPK signaling pathway in iridovirus-induced apoptosis and virus replication, which provides new insights into understanding iridovirus-host interaction.
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A new leukocyte cell-derived chemotaxin-2 from marine fish grouper, Epinephelus coioides: molecular cloning and expression analysis.
Fish Shellfish Immunol.
PUBLISHED: 03-16-2011
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Leukocyte cell-derived chemotaxin-2 (LECT2) is a multifunctional protein involved in cell growth, differentiation and autoimmunity. In this study, a new leukocyte cell-derived chemotaxin-2 (EcLECT2) gene was cloned from grouper, Epinephelus coioides, by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA sequence of EcLECT2 was 595 bp in size, containing a 5-untranslated region (UTR) of 44 bp and a 3-UTR of 83 bp. The deduced protein sequence of the open reading frame (465 bp) showed highest similarity (81%) to the LECT2 of the fresh-water fish Larimichthys crocea. An abundant transcription of the determined EcLECT2 mRNA has been detected in liver and skin of grouper, E. coioides. Furthermore, the expression of EcLECT2 was differentially up-regulated in liver after infection with Staphyloccocus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV), while the expression was down-regulated after stimulation with Concanavalin A (Con A). Recombinant mature EcLECT2 (rEcLECT2) was successfully expressed in Escherichia coli BL21 (DE3), and the antiserum against EcLECT2 was obtained for further investigations. EcLECT2 may be an important molecule in the innate immunity of grouper.
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Roles of stress-activated protein kinases in the replication of Singapore grouper iridovirus and regulation of the inflammatory responses in grouper cells.
J. Gen. Virol.
PUBLISHED: 03-14-2011
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Stress-activated protein kinases (SAPKs), including p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), are usually activated in response to different environmental stimuli, including virus infection. In the present study, the roles of SAPKs during Singapore grouper iridovirus (SGIV) infection were investigated in fish cells. The results showed that increased phosphorylation of JNK1/2 and p38 MAPK occurred during active replication of SGIV in grouper cell cultures. Moreover, downstream effectors (c-Jun, MAPK-activated protein kinase 2, p53, activator protein 1, Myc and nuclear factor of activated T cells) were activated after SGIV infection, suggesting that SGIV replication activated the JNK and p38 MAPK signalling pathways. Notably, using specific inhibitors, it was found that viral gene transcripts, protein expression and viral titres were not affected by inhibition of p38 MAPK but were suppressed significantly by inhibiting JNK1/2 activation. In addition, transcription of grouper immune genes including interferon regulatory factor 1, interleukin-8 and tumour necrosis factor alpha (TNF-?) were regulated by JNK, whilst only TNF-? was regulated by p38 MAPK. It is proposed that the JNK pathway is important for SGIV replication and modulates the inflammatory responses during virus infection.
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Proteomic analysis of Singapore grouper iridovirus envelope proteins and characterization of a novel envelope protein VP088.
Proteomics
PUBLISHED: 02-23-2011
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Singapore grouper iridovirus (SGIV) is an enveloped virus causing heavy economic losses to marine fish culture. The envelope fractions of SGIV were separated from the purified virions by Triton X-100 treatment, and subjected to 1-DE-MALDI-TOF/TOF-MS/MS and LC-MALDI-TOF/TOF-MS/MS analysis. A total of 19 virus-encoded envelope proteins were identified in this study and 73.7% (13/17) of them were predicted to be membrane proteins. Three viral envelope proteins were uniquely identified by 1-DE-MALDI, whereas another ten proteins were identified only by LC-MALDI, with six proteins identified by both workflows. VP088 was chosen as a representative of proteomic identification and characterized further. VP088 was predicted to be a viral transmembrane envelope protein which contains two RGD (Arg-Gly-Asp) motifs, three transmembrane domains, and five N-glycosylation sites. VP088 gene transcript was first detected at 12?h p.i. and reached the peak at 48?h p.i. Combined with the drug inhibition assay, VP088 gene was identified as a late (L) gene. Recombinant VP088 (rVP088) was expressed in Escherichia coli, and the specific antiserum against rVP088 was raised. VP088 was proved to be a viral envelope protein by Western blot and immunoelectron microscopy (IEM). Furthermore, rVP088 can bind to a 94?kDa host cell membrane protein, suggesting that VP088 might function as an attaching protein. Neutralization assay also suggested that VP088 is involved in SGIV infection. This study will lead to a better understanding of molecular mechanisms of the iridoviral pathogenesis and virus-host interactions.
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Characterization of large yellow croaker (Pseudosciaena crocea) ?-actin promoter supports ?-actin gene as an internal control for gene expression modulation and its potential application in transgenic studies in fish.
Fish Shellfish Immunol.
PUBLISHED: 01-31-2011
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As a housekeeping gene, ?-actin is one of the most commonly used reference gene and its promoter is widely used in transgenic studies in mammals and fish. In this study, we used genomic walker technology to clone the ?-actin gene (Lyc?-actin) promoter sequence from large yellow croaker, an economically important marine fish in China. The Lyc?-actin promoter region spans 3350 nucleotides (nt) and contains several transcription factor binding sites and a conserved enhancer motif (ATGGTAATAA) in the first intron. A promoter activity assay showed that this promoter region can drive enhanced green fluorescent protein (EGFP) gene expression in the fish cell line, EPC. Luciferase activity analysis demonstrated that the activity of the Lyc?-actin promoter is not affected by poly(I:C) or lipopolysaccharide (LPS) stimulation. Absolute real-time PCR analysis of various tissues revealed that Lyc?-actin expression levels are not significantly altered by poly(I:C) or inactivated trivalent bacterial vaccine (P > 0.05). These results suggest that ?-actin can be used as a suitable internal control for gene expression modulation in response to immune stimulations in large yellow croaker. In vivo transgenic experiments showed that the Lyc?-actin promoter region can drive efficient EGFP expression in large yellow croaker fries or fertilized zebrafish eggs, supporting its potential application in transgenic studies in fish.
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Identification and functional characterization of an interferon regulatory factor 7-like (IRF7-like) gene from orange-spotted grouper, Epinephelus coioides.
Dev. Comp. Immunol.
PUBLISHED: 01-26-2011
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Interferon regulatory factor (IRF) 7 plays a crucial role in modulating cellular responses to viral infection and cytokines, including interferons (IFNs). In the present study, a novel IRF7 gene (designated as EcIRF7) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length EcIRF7 cDNA is composed of 2089 bp and encodes a polypeptide of 433 amino acids with 81% identity to IRF7 of Siniperca chuatsi, and the genomic DNA of EcIRF7 consists of 9 exons and 8 introns, with a length of approximately 5629 bp. EcIRF7 contains three conserved domains including a DNA-binding domain (DBD), an IRF associated domain (IAD) and a serine-rich domain, all of which are highly conserved across species. Recombinant EcIRF7 was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-EcIRF7 serum preparation. Realtime quantitative PCR (RT-qPCR) analysis revealed a broad expression of EcIRF7, with a relative strong expression in spleen, kidney, skin and intestine. The expression of EcIRF7 was differentially up-regulated after stimulation with Vibrio vulnificus, Staphylococcus aureus and Singapore grouper iridovirus (SGIV). EcIRF7 showed similar intracellular localization pattern to those of mammalian and chicken, and translocated into nucleus after SGIV infection. Further more, EcIRF7 was proved to be capable of activating zebrafish type I IFN promoter and inhibiting the replication of SGIV in grouper spleen (GS) cells. These results suggest that EcIRF7 is potentially involved in grouper immune responses to invasion of viral and bacterial pathogens.
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Identification of a novel marine fish virus, Singapore grouper iridovirus-encoded microRNAs expressed in grouper cells by Solexa sequencing.
PLoS ONE
PUBLISHED: 01-25-2011
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MicroRNAs (miRNAs) are ubiquitous non-coding RNAs that regulate gene expression at the post-transcriptional level. An increasing number of studies has revealed that viruses can also encode miRNAs, which are proposed to be involved in viral replication and persistence, cell-mediated antiviral immune response, angiogenesis, and cell cycle regulation. Singapore grouper iridovirus (SGIV) is a pathogenic iridovirus that has severely affected grouper aquaculture in China and Southeast Asia. Comprehensive knowledge about the related miRNAs during SGIV infection is helpful for understanding the infection and the pathogenic mechanisms.
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Molecular characterization of a Toll-like receptor 22 homologue in large yellow croaker (Pseudosciaena crocea) and promoter activity analysis of its 5-flanking sequence.
Fish Shellfish Immunol.
PUBLISHED: 08-03-2010
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Toll-like receptors (TLRs) are a family of innate immune receptors that recognize molecular patterns associated with microbial pathogens (PAMP), and induce antimicrobial immune responses. Here we report the molecular cloning and characterization of a TLR22 homologue from the large yellow croaker (LycTLR22), an economically important marine fish in China. The complete cDNA of LycTLR22 is 4607 nucleotides (nt) long, encoding a protein of 963 amino acids (aa). The deduced LycTLR22 has a typical TLR domain architecture including 18 leucine-rich repeats (LRRs) (residues 69-638) and one C-terminal LRR domain (residues 698-749) at the extracellular region, and a TIR domain (residues 805-948) in the cytoplasmic region. The three highly conserved regions among the family of bony fish were found in LycTLR22 (box 1, Y(806)DAFISY(812); box 2, L(837)C-RD-PG(846); box 3, W(938) surrounded by basic residue). The deduced LycTLR22 protein has 61.2%, 55.0%, 43.8%, 43.5%, 38.6% and 36.4% identity to Japanese flounder, fugu, Atlantic salmon, rainbow trout, zebrafish and goldfish TLR22, respectively. Genomic analysis showed that LycTLR22 gene consisted of four exons and three introns, which is similar to its homologue in Japanese flounder. The 5-flanking region of LycTLR22 gene, containing several putative transcription factor binding sites and an interferon-sensitive response element (ISRE), exhibits promoter activity. LycTLR22 gene was constitutively expressed in anterior kidney, spleen, liver, heart, intestine and blood, but not in gills and muscle. Upon stimulation with poly (I:C), the LycTLR22 expression was obviously upregulated in anterior kidney and spleen tissues, and also in primary anterior kidney cells of large yellow croaker, suggesting a role for LycTLR22 in the immune response induced by poly (I:C).
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Cloning, characterization, and expression analysis of orange-spotted grouper (Epinephelus coioides) ILF2 gene (EcILF2).
Fish Shellfish Immunol.
PUBLISHED: 06-10-2010
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Interleukin-2 enhancer binding factor 2 (ILF2), also named as nuclear factor 45 (NF45), plays important roles in regulating interleukin-2 expression in mammals. In the present study, a novel ILF2 gene (designated EcILF2) was cloned and well characterized from orange-spotted grouper, Epinephelus coioides. The full-length EcILF2 cDNA is composed of 1544 bp and encodes a polypeptide of 387 amino acids with 98% identity to ILF2 of Atlantic salmon. The genomic DNA of EcILF2 consists of 14 exons and 13 introns, with a length of approximately 6.9 kb. EcILF2 contains two conserved domains including an RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain. Recombinant EcILF2 was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-EcILF2 serum preparation. Subcellular localization analysis revealed that EcILF2 was distributed predominantly in the nucleus. Realtime quantitative PCR (RT-qPCR) analysis revealed a broad expression of EcILF2, with a relative strong expression in skin, liver, brain, head kidney and spleen. The expression of EcILF2 was differentially up-regulated after stimulation with Vibrio vulnificus, Staphylococcus aureus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Furthermore, EcILF2 was able to activate human IL-2 promoter in different cell lines and promote the endogenous IL-2 transcription in human H9 T cells. These results suggest that EcILF2 is potentially involved in grouper immune responses to invasion of bacterial and viral pathogens.
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Identification and characterization of Singapore grouper iridovirus (SGIV) ORF162L, an immediate-early gene involved in cell growth control and viral replication.
Virus Res.
PUBLISHED: 06-29-2009
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Singapore grouper iridovirus (SGIV) is a major pathogen resulting in heavy economic losses to grouper aquaculture. In this study, SGIV ORF162L encoding a putative homolog of ICP46 was identified and characterized. Interestingly, ICP46 could be found in all sequenced iridoviruses and is considered as a core gene of the family Iridoviridae. SGIV ICP46 was classified as an immediate-early (IE) gene during in vitro infection using drug inhibition analysis, reverse transcription polymerase chain reaction and Western blot analysis. Subcellular localization revealed that SGIV ICP46 was distributed predominantly in the cytoplasm. Furthermore, SGIV ICP46 proved to be a structural protein of the nucleocapsid; its overexpression could promote the growth of grouper embryonic cells and contribute to SGIV replication. This is the first report of the characterization of a putative ICP46 homolog and these results should offer important insights into the pathogenesis of iridoviruses.
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Molecular cloning, characterization and expression analysis of a C-type lectin (Ec-CTL) in orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
PUBLISHED: 06-16-2009
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C-type lectins play crucial roles in pathogen recognition, innate immunity, and cell-cell interactions. In this study, a new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-CTL was composed of 840 bp with a 651 bp open reading frame (ORF) that encodes a 216-residue protein. The deduced amino acid sequence of Ec-CTL possessed all conserved features crucial for the fundamental structure, such as the four cysteine residues (Cys(71), Cys(152), Cys(167), Cys(175)) involved in the formation of disulphide bridges and the potential Ca(2+)/carbohydrate-binding sites. Ec-CTL contains a signal peptide and a single carbohydrate recognition domain (CRD). The genomic DNA of the gene consists of three exons and two introns. Ec-CTL showed high similarity of 54% to the C-type lectin of killifish Fundulus heteroclitus. Ec-CTL mRNA is predominately expressed in liver and skin, and lower expressed in kidney, intestine, heart, brain and spleen. The expression of Ec-CTL was differentially up-regulated in orange-spotted grouper challenged with Saccharomyces cerevisiae, Vibrio vulnificus, Staphyloccocus aureus and Singapore grouper iridovirus (SGIV). Recombinant mature Ec-CTL (rEc-CTL) was expressed in E. coli BL21, purified and characterized as a typical Ca(2+)-dependent carbohydrate-binding protein possessing hemagglutinating activity. It bound to all examined bacterial and yeast strains, and aggregated with S. cerevisiae, V. vulnificus and S. aureus in a Ca(2+)-dependent manner.
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Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae.
BMC Genomics
PUBLISHED: 05-14-2009
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Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in cultured soft-shelled turtles (Trionyx sinensis). To our knowledge, the only molecular information available on STIV mainly concerns the highly conserved STIV major capsid protein. The complete sequence of the STIV genome is not yet available. Therefore, determining the genome sequence of STIV and providing a detailed bioinformatic analysis of its genome content and evolution status will facilitate further understanding of the taxonomic elements of STIV and the molecular mechanisms of reptile iridovirus pathogenesis.
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Characterization of Singapore grouper iridovirus (SGIV) ORF086R, a putative homolog of ICP18 involved in cell growth control and virus replication.
Arch. Virol.
PUBLISHED: 03-17-2009
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Singapore grouper iridovirus (SGIV), as a causative agent of serious systemic disease, causes significant economic losses in grouper aquaculture. In this study, a novel ICP18 homolog encoded by SGIV ORF086R was identified and characterized. Strikingly, ICP18 homologs can be found in all ranaviruses, but not in other sequenced large DNA viruses. SGIV ICP18 is an immediate-early gene and begins to be transcribed as early as 2 h post-infection (p.i.). Western blotting indicated that SGIV ICP18 is translated as early as 6 h p.i. and is a viral non-envelope protein. Subcellular localization analysis revealed that the SGIV ICP18 displays a finely punctate cytoplasmic pattern. Furthermore, overexpression of SGIV ICP18 can promote the growth of grouper embryonic cells (GP) and contribute to SGIV replication. These results should offer important insights into the pathogenesis of ranaviruses.
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Molecular cloning, characterization of one key molecule of teleost innate immunity from orange-spotted grouper (Epinephelus coioides): serum amyloid A.
Fish Shellfish Immunol.
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The orange-spotted grouper (Epinephelus coioides), a favorite marine food fish, is widely cultured in China and Southeast Asian countries. However, little is known about its acute phase response (APR) caused by viral diseases. Serum amyloid A (SAA) is a major acute phase protein (APP). In this study, a new SAA homologous (EcSAA) gene was cloned from grouper, E. coioides, by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA sequence of SAA was 508 bp and contained a 363 bp open reading frame (ORF) coding for a protein of 121 aa. Similar to other fish known SAA genes, the EcSAA gene contained four exons and three introns. Quantitative real-time PCR analysis revealed that EcSAA mRNA is predominately expressed in liver and gill of grouper. Furthermore, the expression of EcSAA was differentially up-regulated in liver after infection with Staphyloccocus aureus, Vibrio vulnificus, Vibrio parahaemolyticus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Recombinant EcSAA (rEcSAA) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-EcSAA serum preparation. The rEcSAA fusion protein was demonstrated to bind to all tested bacteria and yeast, and inhibit the replication of SGIV. Overexpression of EcSAA in grouper spleen (GS) cells could also inhibit the replication of SGIV. These results suggest that EcSAA may be an important molecule in the innate immunity of grouper.
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Grouper translationally controlled tumor protein prevents cell death and inhibits the replication of Singapore grouper iridovirus (SGIV).
Fish Shellfish Immunol.
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Translationally controlled tumor protein (TCTP) is an important molecule involved in multiple biological processes, such as cell growth, cell cycle progression, malignant transformation, and enhancement of the anti-apoptotic activity. In this study, the TCTP from orange-spotted grouper Epinephelus coioides (Ec-TCTP) was cloned and characterized. The full-length cDNA of Ec-TCTP was comprised of 1057 bp with a 510 bp open reading frame that encodes a putative protein of 170 amino acids. Recombinant Ec-TCTP (rEc-TCTP) was expressed in Escherichia BL21 (DE3) and purified for mouse anti-Ec-TCTP serum preparation. The rEc-TCTP fusion protein was demonstrated to possess antioxidant activity, which conferred resistance to H(2)O(2) damage. Quantitative real-time PCR analysis revealed that Ec-TCTP mRNA is predominately expressed in the liver, and the expression was up-regulated in the liver of grouper after viral challenge with Singapore grouper iridovirus (SGIV). Intracellular localization revealed that Ec-TCTP expression was distributed predominantly in the cytoplasm. Although human TCTP has a role in apoptosis regulation, it is not known if grouper TCTP has any role in apoptosis regulation. Strikingly, grouper TCTP, when overexpressed in fathead minnow (FHM) cells, protected them from cell death induced by cycloheximide (CHX). In addition, overexpressed Ec-TCTP in grouper spleen (GS) cells inhibited the replication of SGIV. These results suggest that Ec-TCTP may play a critical role in their response to SGIV infection, through regulation of a cell death pathway that is common to fish and humans.
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Immunogenicity and protective effects of inactivated Singapore grouper iridovirus (SGIV) vaccines in orange-spotted grouper, Epinephelus coioides.
Dev. Comp. Immunol.
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Vaccination is one of the best methods against viral diseases. In this study, experimental inactivated Singapore grouper iridovirus (SGIV) vaccines were prepared, and immunogenicity and protection against virus infection of the vaccines were investigated in orange-spotted grouper, Epinephelus coioides. Two kinds of vaccines, including ?-propiolactone (BPL) inactivated virus at 4°C for 12 h and formalin inactivated virus at 4°C for 12 d, was highly protective against the challenge at 30-day post-vaccination and produced relative percent of survival rates of 91.7% and 100%, respectively. These effective vaccinations induced potent innate immune responses mediated by pro-inflammatory cytokines and type I interferon (IFN)-stimulated genes (ISGs). It is noteworthy that ISGs, such as Mx and ISG15, were up-regulated only in the effective vaccine groups, which suggested that type I IFN system may be the functional basis of early anti-viral immunity. Moreover, effective vaccination also significantly up-regulated of the expression of MHC class I gene and produced substantial amount of specific serum antibody at 4 weeks post-vaccination. Taken together, our results clearly demonstrated that effective vaccination in grouper induced an early, nonspecific antiviral immunity, and later, a specific immune response involving both humoral and cell-mediated immunity.
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Isolation and characterization of a thioredoxin domain-containing protein 12 from orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
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Thioredoxin domain-containing protein 12 (Txndc12) belongs to the thioredoxin superfamily, and has roles in redox regulation, defense against oxidative stress, refolding of disulfide-containing proteins, and regulation of transcription factors. In this study, a thioredoxin domain-containing protein 12 was cloned from the marine fish grouper, Epinephelus coioides by RACE PCR, named as Ec-Txndc12. The Ec-Txndc12 encodes 173 amino acid residues with signal peptide in its N-terminal and a thioredoxin (Trx) domain that is homologous with some genes in Mus musculus, Xenopus laveis, etc. Ec-Txndc12 mRNA is predominately expressed in liver, brain and muscle. The expression of Ec-Txndc12 was up-regulated in the liver of grouper challenged with SGIV. In order to elucidate its biological functions, Ec-Txndc12 was recombined and expressed in Escherichia coli BL21 (DE3). The rEc-Txndc12 fusion protein was demonstrated to possess the antioxidant activity. The grouper spleen (GS) cells were treated with a high concentration of rEc-Txndc12 (30 ?g/ml), which significantly enhanced cells viability under oxidative damage caused by viral infection. These results together indicated that Ec-Txndc12 could function as an important antioxidant in a physiological context, and might be involved in the responses to viral challenge.
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Selection and identification of Singapore grouper iridovirus vaccine candidate antigens using bioinformatics and DNA vaccination.
Vet. Immunol. Immunopathol.
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In this study, we described a rapid and efficient method which integrated the bioinformatic prediction and DNA vaccine technology to identify vaccine candidates against Singapore grouper iridovirus (SGIV). The 162 previously defined open reading frames (ORFs) of SGIV were subjected to extensive sequence similarity searches, as well as motif, cellular location, and domain prediction. Based on our analysis, 13 genes were chosen and cloned into the eukaryotic expression vector pcDNA 3.1. In vitro and in vivo expression of these DNA vaccine constructs was examined in Epinephelus akaara spleen cells (EAGS) and immunized fish by Western blot and RT-PCR analysis, respectively. Three weeks after the second booster, immunized fish were challenged with SGIV and the level of protection and survival was assessed. Fish vaccinated with plasmid DNA encoding viral ORF072, ORF039 and ORF036 (designated as pcDNA-72, pcDNA-39 and pcDNA-36, respectively) exhibited 66.7%, 66.7% and 58.3% relative percent survival rates, respectively, in comparison with the control fish. These three DNA vaccines induced innate immune responses, raising significantly high level of Mx expression relative to the fish vaccinated with the empty plasmid at 3 days post-vaccination. Furthermore, recombinant protein from ORF072 was also used to immunize another set of fish and similar protective effect was obtained. Taken together, our results validated the applicability of bioinformatics in genome mining, resulting in the identification of three protective antigens. The promising results obtained in the present study have prompted further testing to improve the immunogenicity of these potential DNA vaccines.
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Cloning, characterization, and expression analysis of a thioredoxin from orange-spotted grouper (Epinephelus coioides).
Dev. Comp. Immunol.
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Thioredoxins (TRXs) are a family of small, highly conserved proteins that are essential for the maintenance of cellular homeostasis. In this study, a thioredoxin gene was cloned from orange-spotted grouper, Epinephelus coioides (designated as Ec-TRX). The full-length cDNA of Ec-TRX was comprised of 767bp with a 327bp open reading frame that encodes a putative protein of 108 amino acids. Quantitative real-time PCR analysis revealed that the Ec-TRX mRNA was distributed abundantly in grouper, E. coioides skin and liver, and the expression in liver was up-regulated after viral challenge with Singapore grouper iridovirus (SGIV). Recombinant Ec-TRX (rEc-TRX) was expressed in Escherichia coli BL21 (DE3) and purified for mouse anti-Ec-TRX serum preparation. The rEc-TRX fusion protein was demonstrated to possess the expected redox activity in enzymatic analysis, and scavenge free radicals and protect supercoiled DNA from oxidative damage induced by a metal-ion catalyzed oxidation reaction. Subcellular localization revealed that Ec-TRX was distributed in both cytoplasm and nucleus. Overexpression of Ec-TRX in grouper spleen (GS) cells could promote the growth of GS cells and inhibit the replication of SGIV. These results suggest that Ec-TRX could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to viral challenge.
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Molecular cloning and characterization of c-type lysozyme gene in orange-spotted grouper, Epinephelus coioides.
Fish Shellfish Immunol.
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Lysozymes are key proteins of the host innate immune system against pathogen infection. In this study, a c-type lysozyme gene (Ec-lysC) was cloned and characterized from orange-spotted grouper, Epinephelus coioides. The full-length Ec-lysC cDNA is composed of 533 bp and encodes a polypeptide of 144-residue protein with 94% identity to lysC of Kelp grouper, Epinephelus bruneus. The genomic DNA of Ec-lysC consists of 4 exons and 3 introns, with a total length of 1897 bp. Amino acid sequence alignment showed that Ec-lysC possessed conserved catalytic residues (Glu50 and Asp67) and "GSTDYGIFQINS" motif. RT-PCR results showed that Ec-lysC transcript was most abundant in head kidney and less in muscle. The expression of Ec-lysC was differentially up-regulated in head kidney after stimulation with lipopolysaccharide (LPS), Vibrio alginolyticus and Singapore grouper iridovirus (SGIV). Subcellular localization analysis revealed that Ec-lysC was distributed predominantly in the cytoplasm. The recombinant Ec-lysC (rEc-lysC) had lytic activities against Gram-positive bacteria Micrococcus lysodeikticus, Staphylococcus aureus, Streptococcus iniae and Gram-negative bacteria V. alginolyticus. The lysozyme acted on M. lysodeikticus cell walls as shown by scanning electron microscopy (SEM). Furthermore, overexpression of Ec-lysC in grouper cells delayed the occurrence of CPE induced by SGIV and inhibited the viral gene transcription significantly. Taken together, Ec-lysC might play an important role in grouper innate immune responses to invasion of bacterial and viral pathogens. C-type lysozyme gene from E. coioides (Ec-lysC) was identified and characterized.
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Antiviral effects of ?-defensin derived from orange-spotted grouper (Epinephelus coioides).
Fish Shellfish Immunol.
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Defensins are a group of small antimicrobial peptides playing an important role in innate host defense. In this study, a ?-defensin cloned from liver of orange-spotted grouper, Epinephelus coioides, EcDefensin, showed a key role in inhibiting the infection and replication of two kinds of newly emerging marine fish viruses, an enveloped DNA virus of Singapore grouper iridovirus (SGIV), and a non-enveloped RNA virus of viral nervous necrosis virus (VNNV). The expression profiles of EcDefensin were significantly (P < 0.001) up-regulated after challenging with Lipopolysaccharide (LPS), SGIV and Polyriboinosinic Polyribocytidylic Acid (polyI:C) in vivo. Immunofluorescence staining observed its intracellular innate immune response to viral infection of SGIV and VNNV. EcDefensin was found to possess dual antiviral activity, inhibiting the infection and replication of SGIV and VNNV and inducting a type I interferon-related response in vitro. Synthetic peptide of EcDefensin (Ec-defensin) incubated with virus or cells before infection reduced the viral infectivity. Ec-defensin drastically decreased SGIV and VNNV titers, viral gene expression and structural protein accumulation. Grouper spleen cells over-expressing EcDefensin (GS/pcDNA-EcDefensin) support the inhibition of viral infection and the upregulation of the expression of host immune-related genes, such as antiviral protein Mx and pro-inflammatory cytokine IL-1?. EcDefensin activated type I IFN and Interferon-sensitive response element (ISRE) in vitro. Reporter genes of IFN-Luc and ISRE-Luc were significantly up-regulated in cells transfected with pcDNA-EcDefenisn after infection with SGIV and VNNV. These results suggest that EcDefensin is importantly involved in host immune responses to invasion of viral pathogens, and open the new avenues for design of antiviral agents in fisheries industry.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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