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ABSTRACT: Megalocytiviruses have three main genotypes, which are represented by ISKNV, RSIV, and TRBIV. To date, the virion-associated proteins of RSIV and TRBIV are still unknown. The spotted knifejaw iridovirus (SKIV) is a newly characterized RSIV-type megalocytivirus. In this study, the virion-associated proteins of SKIV were identified by systemic one-dimensional gel electrophoresis-based proteomic approaches. A total of 49 viral proteins and 33 cellular proteins were associated with the SKIV virions by LC MS/MS, including 18 highly abundant structural proteins that were detected by MALDI TOF/TOF-MS. One highly abundant structural protein of interest was identified as the virus-inducible stress protein (VISP) and further characterized as an envelope protein. However, knockdown of mVISP by siRNA method showed no effect in virion production. The current study is the first to present detailed information on the virion-associated proteins of an RSIV-type megalocytivirus and to identify a novel cellular envelope protein of this virus.
Virology 01/2013; · 3.35 Impact Factor
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ABSTRACT: Infectious spleen and kidney necrosis virus (ISKNV), the type species of genus Megalocytivirus in the family Iridoviridae, is the most important etiological agents in mandarin fish industry in China. Since its first occurrence in China in the early 1990s, there is no effective method to prevent and control this virus. Here we report the successful development of a formalin-killed cell-cultured (FKC) vaccine again ISKNV. Immuno-protection experiments showed that greater than 90% of fish immunized with the FKC vaccine were protected against virulent ISKNV. Sera derived from the immunized fish markedly inhibited the virus infection both in vitro and in vivo. Purified IgM from the immunized fish sera also showed efficient neutralization effects in vivo, strongly suggesting that antibody-mediated immunity may play an important role in the FKC vaccine. Using FKC-immunized fish sera as first antibody, a two-dimensional gel electrophoresis mass spectrometry analysis-based immuno-proteomic method was performed to identify the immunogenic proteins. ORF006L (the major capsid protein), ORF054L, ORF055L, ORF101L, ORF117R, and ORF125R were found to be the major immunogenic proteins of ISKNV. Antibodies generated from these six recombinant viral proteins were able to recognize specifically the corresponding protein fractions from purified virions by Western blot analysis, and five of them (excluding ORF125) showed positive reactions by indirect immunofluorescence assay. In summary, the present study first developed an effective vaccine against ISKNV, and the major immunogenic proteins of ISKNV are also identified. The reported ISKNV immunogenic proteins are important for further development of diagnostic regents and genetic vaccine candidates for all megalocytivirus.
Veterinary Microbiology 11/2012; · 3.33 Impact Factor
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ABSTRACT: Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is a highly infectious causative agent to common carp and koi worldwide. The virus is mainly consisted of European and Asian genotype isolates. To date, no European genotype CyHV-3 has been found emerging in the East and Southeast Asian regions. In late March 2011, an outbreak of CyHV-3 disease occurred in Guangzhou City, Guangdong Province, China, resulting in the deaths of approximately 200 large-sized adult koi within four weeks. One moribund koi was sampled for CyHV-3 isolation. Thus, a CyHV-3 was isolated in KCF-1 cells and designated as KHV-GZ11. Abundant mature or immature virions in infected KCF-1 cells were observed under a transmission electron micrograph. In addition, intra-nuclear inclusion body-like structures with masses of virions were also observed. Based on the TK and ORF136H genes, the sequence analyses revealed that KHV-GZ11 is a distinct European genotype of CyHV-3. Moreover, the infectivity experiment showed that KHV-GZ11 was highly virulent to koi. In summary, we are the first to confirm the emergence of fatal European genotype CyHV-3/KHV in East and Southeast Asia. Our study will provide new insight to explore the virus origin and epidemiology, as well as its pathogenicity.
Veterinary Microbiology 10/2012; · 3.33 Impact Factor
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ABSTRACT: Combined vaccines are immunological products intended for immunization against multifactorial infectious diseases caused by different types or variants of pathogens. In this study, the effectiveness of Vibrio alginolyticus (Va), Vibrio harveyi (Vh), Vibrio vulnificus (Vv) and infectious spleen and kidney necrosis virus (ISKNV), an iridovirus, combined-vaccine (Vibrio and ISKNV combined vaccines, VICV), Va+Vh+Vv inactive vaccine (VIV) and ISKNV whole cell inactive vaccine (IWCIV) in Epinephelus coioides were evaluated using various immunological parameters including antibody titer, serum lysozyme activity (LA), respiratory burst (RB) activity, bactericidal activity (BA) and relative percentage survival (RPS). E. coioides immunized with VICV and challenged with Va+Vh+Vv+ISKNV had an RPS of 80%. The RPS was 73.3% in E. coioides immunized with VIV and challenged with Va+Vh+Vv. E. coioides immunized with IWCIV and challenged with ISKNV had an RPS of 69.6%. Serum LA in the vaccinated group was significantly higher than the control group on days 21 and 28 post-vaccination (P<0.01). The RB activity of head kidney cells in the vaccinated group was significantly higher (P<0.01) compared to that in the control group. However, RB activity of spleen cells in the vaccinated group and the control group were not significantly different (P>0.05). After immunization with VICV, BA values of blood leucocytes and head kidney cells increased significantly more than spleen cells. BA value of blood leucocytes was higher than that in head kidney cells. There were distinct difference between BA values in head kidney cells and in spleen cells (P<0.05) as well as between BA value of blood leucocytes and head kidney cells (P<0.01). E. coioides vaccinated with VICV have significantly higher antibody levels than control groupers (P<0.01). Our study suggests that the VICV candidate can effectively protect groupers against multiple bacterial and viral pathogens.
Veterinary Immunology and Immunopathology 08/2012; 150(1-2):61-8. · 2.08 Impact Factor
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Zengwei Huang,
Xiexiong Deng,
Yinyin Li,
Hongjun Su,
Kunpeng Li,
Zhixun Guo,
Peirui Zheng,
Haidong Xu, Jianguo He,
Qinfen Zhang,
Shaoping Weng
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ABSTRACT: Cryo-electron microscopy was applied to analyze mud crab reovirus (MCRV), which causes 'sleeping disease' in mud crab, Scylla serrata, a marine species cultured in China. We present here the three dimensional structure of MCRV at 13.8Å resolution. The outer capsid shell is composed of 260 trimers with complete T=13 icosahedral symmetry. A major difference between MCRV and previously reported aquareoviruses is that it lacks a pentameric turret structure. These results together with recently published molecular biological evidence (Deng et al., 2012) indicate that, from a structural perspective, MCRV should be classified as a new member of the family Reoviridae.
Virus Research 03/2012; 166(1-2):116-20. · 2.94 Impact Factor
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ABSTRACT: Pacific white shrimp (Litopenaeus vannamei), the major species of farmed shrimps in the world, has been attracting extensive studies, which require more and more genome background knowledge. The now available transcriptome data of L. vannamei are insufficient for research requirements, and have not been adequately assembled and annotated.
This is the first study that used a next-generation high-throughput DNA sequencing technique, the Solexa/Illumina GA II method, to analyze the transcriptome from whole bodies of L. vannamei larvae. More than 2.4 Gb of raw data were generated, and 109,169 unigenes with a mean length of 396 bp were assembled using the SOAP denovo software. 73,505 unigenes (>200 bp) with good quality sequences were selected and subjected to annotation analysis, among which 37.80% can be matched in NCBI Nr database, 37.3% matched in Swissprot, and 44.1% matched in TrEMBL. Using BLAST and BLAST2Go softwares, 11,153 unigenes were classified into 25 Clusters of Orthologous Groups of proteins (COG) categories, 8171 unigenes were assigned into 51 Gene ontology (GO) functional groups, and 18,154 unigenes were divided into 220 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. To primarily verify part of the results of assembly and annotations, 12 assembled unigenes that are homologous to many embryo development-related genes were chosen and subjected to RT-PCR for electrophoresis and Sanger sequencing analyses, and to real-time PCR for expression profile analyses during embryo development.
The L. vannamei transcriptome analyzed using the next-generation sequencing technique enriches the information of L. vannamei genes, which will facilitate our understanding of the genome background of crustaceans, and promote the studies on L. vannamei.
PLoS ONE 01/2012; 7(10):e47442. · 4.09 Impact Factor
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ABSTRACT: The nuclear factor-kappa B (NF-κB) pathways play important roles in innate immune responses. IκB is the main cytoplasmic inhibitor of NF-κB. In this study, we identified the LvCactus gene from Litopenaeus vannamei, which is the first cloned IκB homologue in subphylum Crustacea. LvCactus contains six predicted ankyrin repeats, which show similarities to those of Cactus proteins from insects. LvCactus localizes in cytoplasm and interacts with LvDorsal, an L. vannamei homologue to Drosophila melanogaster Dorsal belonging to class II NF-κB family, to prevent its nuclear translocation. Contrary to that of LvDorsal, over-expression of LvCactus down-regulates the activities of shrimp antimicrobial peptides promoters, suggesting LvCactus is an inhibitor of LvDorsal. The promoter of LvCactus was predicted to contain five putative NF-κB binding motifs, among which four were proved to be bound by LvDorsal by chromatin immunoprecipitation assays. Dual-luciferase reporter assays also showed that transcription of LvCactus was promoted by LvDorsal but inhibited by LvCactus itself, indicating a feedback regulatory pathway between LvCactus and LvDorsal. Expression of LvCactus was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus, and Staphylococcus aureus injections, suggesting an activation response of LvCactus to bacterial and immune stimulant challenges. Differently, the LvCactus expression levels obviously decreased during white spot syndrome virus (WSSV) infection, indicating the feedback regulatory pathway of LvCactus/LvDorsal could be modified by WSSV.
PLoS ONE 01/2012; 7(11):e49711. · 4.09 Impact Factor
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ABSTRACT: Malachite green (MG) had been extensively used worldwide in the past few decades as an effective fungicide, bactericide, ectoparasiticide, and antiprotozoan on fish. It is rapidly and extensively metabolized to leucomalachite green (LMG) in fishes. To study the developmental toxicity of LMG, we evaluated the maternal and embryo/fetal effects of LMG orally administered to Sprague-Dawley rats once daily from day 6-15 of gestation at dose levels of 0, 10, 80, and 160 mg/kg/day. In the current investigation, the unmetabolized compound (LMG) was detected in the plasma of the dams treated with LMG and in the amniotic fluid. At dose levels of 80 and 160 mg/kg/day, LMG induced maternal toxicity, which consisted of severe reduction in maternal weight and food consumption during the treatment period. Doses of 80 and 160 mg/kg/day induced fetal skeletal abnormalities. The major skeletal defects were bipartite ossification of the thoracic centrum. LMG at 160 mg/kg/day also induced marked embryolethality and visceral abnormalities. Based on the results of this study, a dose level of 10mg/kg/day is considered the no-observed-adverse-effect-level (NOAEL) for maternal and fetal developmental toxicity in rats.
Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 12/2011; 49(12):3031-7. · 2.99 Impact Factor
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ABSTRACT: A new continuous cell line (KCF-1) from caudal fin of koi, Cyprinus carpio koi, was developed and sub-cultured more than 100 passages since the present study was initiated in March 2006. KCF-1 predominantly consisted of short fibroblast-like cells and grew well in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Chromosome analysis revealed that 56% of the KCF-1 cells maintained normal diploid chromosome number (2n=100) at Passage 82. Using the KCF-1 cell line, a strain of cyprinid herpesvirus 3 (designated as CyHV-3-QY08) was isolated from the diseased koi. CyHV-3-QY08 continuously propagated in the KCF-1 cells, as confirmed by immunofluorescence assay (IFA) and transmission electron microscopy (TEM). KCF-1 cells infected with CyHV-3-QY08 produced typical cytopathic effects characterized by severe vacuolation, deformation of nuclei, and marginalization of the nuclear chromatin, which are consistent with those of previous reports. CyHV-3-QY08 was purified and subsequently analyzed by SDS-PAGE and TEM. The results showed that the purified virions contained two types of morphologies and were composed of more than 30 obvious viral polypeptides. An infectivity experiment revealed that CyHV-3-QY08 could cause 100% mortality in the infected koi. Based on the genome sequence of CyHV-3-I/U, the CyHV-3(I/U)-ORF136 homologue in CyHV-3-QY08 was cloned and sequenced. Multiple sequence alignments of CyHV-3-I/U-ORF136 homologues showed that CyHV-3-QY08 belonged to the typical Asian genotype. The CyHV-3(I/U)-ORF136 homologue seems to be a novel molecule marker, which can be used to distinguish Asia isolates from Europe-America strains.
Virus Research 08/2011; 161(2):140-9. · 2.94 Impact Factor
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Zhiming Xiang,
Lin Qi,
Weijian Chen,
Chuanfu Dong,
Zhaoyu Liu,
Dong Liu,
Mengli Huang,
Wei Li,
Gan Yang,
Shaoping Weng, Jianguo He
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ABSTRACT: A growing family of cellular proteins encoding for caspase activation and the recruitment domain (CARD) plays a crucial role in immunity by sensing viral infections and signaling antiviral immune defenses. We obtained a MAVS-like protein (named TnMAVS) from Tetradon nigroviridis, which contains a CARD domain, a pro-rich domain, and a TM domain similar to human MAVS. A fluorescence assay showed that TnMAVS was located in the cytoplasm and near by the membrane, and not the mitochondria in FHM cells. As such, it was considered as a new member of MAVS. The TnMAVS was highly expressed in the liver and muscle of T. nigroviridis. In the spleen, TnMAVS was down-regulated when the fish was treated with polyinosinic:polycytidylic acid or challenged with ISKNV, but was not affected by PGN or LPS. The dual luciferase reporter assay revealed that TnMAVS overexpression resulted in the activation of the interferon-sensitive response element and NF-κB signal pathways. In addition, a characteristic TRAF3-associated peptide PVQD was found in the TnMAVS sequence. Co-immunoprecipitation assays indicated that TnMAVS could interact with zfTRAF3 in eukaryotic cells.
Developmental and comparative immunology 04/2011; 35(11):1103-15. · 3.29 Impact Factor
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ABSTRACT: The systemic RNA interference defective protein (SID)-1 plays an important role in dsRNA uptake in cells. We identified the ScSidT2 gene from the mandarin fish (Siniperca chuatsi), which is the first-studied SID-1 homolog in fish. ScSidT2 mRNA is 3380 bp long and contains a 2568-bp open reading frame that encodes an 855-amino-acid protein with an N-terminal signal peptide and ten putative transmembrane domains. Tissue distribution profile in healthy fish and expression profiles of ScSidT2 in infectious spleen and kidney necrosis virus (ISKNV)-infected fish were analyzed. Overexpression of the ScSidT2 protein in fathead minnow (FHM) epithelial cells could remarkably increase the uptake of exogenous dsRNA. In tiger frog virus (TFV)-infected FHM cells, overexpression of ScSidT2 could suppress virus production, and this mechanism could be significantly enhanced by adding virus-specific dsRNA. In mandarin fish fry cells, ISKNV infection and reproduction could be promoted when the ScSidT2 protein was neutralized with specific antisera. These results suggest that ScSidT2 could be associated with the host's antiviral defense mechanism.
Developmental and comparative immunology 02/2011; 35(6):692-701. · 3.29 Impact Factor
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ABSTRACT: Mud crab dicistrovirus (MCDV), a newly identified single-stranded positive RNA virus, is an important pathogen that causes serious economic losses to mud crab aquaculture. In this study, MCDV was purified, and three structural proteins of MCDV were separated by SDS-PAGE. The N-terminal 15 amino acids were sequenced and aligned with the main structural proteins of other dicistrovirus. The three structural proteins were named VP1, VP2 and VP3. Monoclonal antibodies (MAbs) against the two main structural proteins, VP1 and VP2, were prepared, and the two structural proteins were then identified using these MAbs. The results of Western blot analyses demonstrated that five MAbs recognised VP1 and two recognised VP2. The results of immunogold transmission electron microscopy (IEM) revealed that the epitopes of the two structural proteins recognised by the MAbs were located at the outer surface of the virions, which suggested that the two structural proteins are MCDV capsid proteins. The identification of the two structural proteins of MCDV is useful for studying their functions, as well as the mechanism of infection and the pathogenesis of MCDV.
Journal of virological methods 02/2011; 171(2):323-8. · 2.13 Impact Factor
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Zhiming Xiang,
Chuanfu Dong,
Lin Qi,
Weijian Chen,
Lichao Huang,
Zhongsheng Li,
Qiong Xia,
Dong Liu,
Mengli Huang,
Shaoping Weng, Jianguo He
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ABSTRACT: The interferon regulatory factor (IRF) family plays critical roles in a host's virus infection responses. In this study, two IRF family members, zfIRF5 and zfIRF7, are identified in zebrafish. The zfIRF5 protein encodes 297 amino acids without the carboxyl IRF3 domain. We suggest that zfIRF5 is a new kind of splicing variant, following the nine other kinds of IRF5 splicing variants found in mammals. The zfIRF7 protein is identified as a member of the IRF7 family, compared to the human IRF7 protein, the amino acid sequence of zfIRF7 only with 29% identity and devoid a virus activated domain (VAD). There zfIRF5/7 proteins are expressed in all 11 selective zebrafish tissues within 6-120h of embryonic development. Laser confocal microscopy shows that the full length the proteins are separately located in the cytoplasm. Mutation experiments show that the nuclear localization signals (NLS) of zfIRF7 and zfIRF-5 are at the N-terminal and C-terminals, respectively. In the assays, zfIRF7 expression increases during infectious spleen and kidney necrosis virus (ISKNV) infection and by poly(I:C) and LPS injections, both of which activate the transcriptional activity of L8G5-luc plasmids. The over-expression of zfIRF5/7 activates the interferon-stimulated response elements (ISRE) signal pathway. In addition, zfIRF7 can activate IFN-β, zfIRF5/7. Co-immunoprecipitation assays and laser co-confocal microscopy show that the two proteins could interact, and zfIRF7 may stimulate zfIRF5 to move into the nucleus. The co-expression of zfIRF5/IRF7 suppresses the transcriptional activities of IFN-β in HEK293T cells.
Developmental and comparative immunology 12/2010; 34(12):1263-73. · 3.29 Impact Factor
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ABSTRACT: Putative open reading frames (ORFs) encoding laminin-like proteins are found in all members of the genus Megalocytivirus, family Iridoviridae. This is the first study that identified the VP23R protein encoded by ORF23R of the infectious spleen and kidney necrosis virus (ISKNV), a member of these genes of megalocytiviruses. The VP23R mRNA covering the ISKNV genomic coordinates 19547 to 22273 was transcribed ahead of the major capsid protein. Immunofluorescence analysis demonstrated that VP23R was expressed on the plasma membrane of the ISKNV-infected cells and could not be a viral envelope protein. Residues 292 to 576 of VP23R are homologous to the laminin γ1III2-6 fragment, which covers the nidogen-binding site. An immunoprecipitation assay showed that VP23R could interact with nidogen-1, and immunohistochemistry showed that nidogen-1 was localized on the outer membrane of the infected cells. Electron microscopy showed that a virus-mock basement membrane (VMBM) was formed on the surface of the infected cells and a layer of endothelial cells (ECs) was attached to the VMBM. The VMBM contained VP23R and nidogen-1 but not collagen IV. The attached ECs were identified as lymphatic endothelial cells (LECs), which have unique feature of overlapping intercellular junctions and can be stained by immunohistochemistry using an antibody against a specific lymphatic marker, Prox-1. Such infection signs have never been described in viruses. Elucidating the functions of LECs attached to the surface of the infected cells may be useful for studies on the pathogenic mechanisms of megalocytiviruses and may also be important for studies on lymphangiogenesis and basement membrane functions.
Journal of Virology 11/2010; 84(22):11866-75. · 5.40 Impact Factor
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Xiaopeng Xu,
Lichao Huang,
Shaoping Weng,
Jing Wang,
Ting Lin,
Junliang Tang,
Zhongsheng Li,
Qingxia Lu,
Qiong Xia,
Xiaoqiang Yu, Jianguo He
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ABSTRACT: Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus, family Iridoviridae. We have previously established a high mortality ISKNV infection model of zebrafish (Danio rerio). In this study, a nonlethal Tetraodon nigroviridis model of ISKNV infection was established. ISKNV infection did not cause lethal disease in Tetraodon but could infect almost all the organs of this species. Electron microscopy showed ISKNV particles were present in infected tissues. Immunofluorescence and quantitative real-time PCR analysis showed that nearly all the virions and infected cells were cleared at 14 d postinfection. The expression profiles of interferon-γ and tumor necrosis factor-α gene in response to ISKNV infection were significantly different in Tetraodon and zebrafish. The establishment of the nonlethal Tetraodon model of ISKNV infection can offer a valuable tool complementary to the zebrafish infection model for studying megalocytivirus disease, fish immune systems, and viral tropism.
Virology 10/2010; 406(2):167-75. · 3.35 Impact Factor
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ABSTRACT: Mud crab dicistrovirus (MCDV) is a recently identified single-stranded positive RNA virus, which causes serious economic loss. The three structural proteins of MCDV were separated by SDS-PAGE. In this study, a recombinant MCDV-VP3 was successfully expressed by an Escherichia coli expression system. After immunization and cell fusion, three mouse hybridomas (1G6H9, 1E7B8, 4B6E10) producing MAbs against MCDV-VP3 were established. The MAbs obtained were fully characterized using indirect ELISA and Western blot analysis. The ELISA results showed that the titers of MAbs were in the range of 1:3.200×10(3) in culture fluids and 1:2.048×10(5) in ascitic fluids. Using Western blot analyses, we observed the specific characteristic band that defines VP3. It demonstrated that all the MAbs were directed against MCDV-VP3. The results of immunogold transmission electron microscopy (IEM) suggested that MCDV-VP3 is the capsid protein of MCDV. The preparation of MAbs specific to the structure protein of MCDV would be useful for studying the function of the structure proteins and the mechanism of infection and pathogenesis of MCDV.
Hybridoma (2005) 10/2010; 29(5):437-40. · 0.42 Impact Factor
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ABSTRACT: Megalocytiviruses, which belong to the family Iridoviridae, are among the most harmful viruses to cultured fishes. Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus. The genome of ISKNV was sequenced and annotated by He et al. (Virology 291:126-139, 2001) in 2001 and re-annotated by Eaton et al. (Virol. J. 4:11, 2007) in 2007. The ORF092R of ISKNV was identified in the annotations of He et al. but was not reported by Eaton et al. In this study, by using Northern-blot and RACE assays, we identified the ORF092R transcript, indicating that ORF092R was indeed transcribed in the ISKNV-infected cells. Immunofluorescence and Western-blot showed that VP92R protein was expressed in the ISKNV-infected cells, and the molecular mass of VP92R is consistent with the theoretical one. Sequence comparison demonstrated that the VP92R orthologous protein is present in large yellow croaker iridovirus (LYCIV), but not in rock bream iridovirus (RBIV) or orange-spotted grouper iridovirus (OSGIV). Therefore, VP92R may have specific functions during ISKNV pathogenesis and could be a subject for studying the differences between megalocytiviruses.
Virus Genes 10/2010; 41(2):210-7. · 1.85 Impact Factor
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ABSTRACT: VP15R protein, encoded by the 15th open reading frame of infectious spleen and kidney necrosis virus (ISKNV), was identified. VP15R is a 263-residue protein that is first transcribed within 12 h post-infection. The VP15R mRNA is transcribed beginning at ISKNV genomic coordinate 12111, extending 167 bp upstream of the initiation codon. No signal peptides, transmembrane fragments, or nuclear localization signal sequences were predicted in the VP15R sequence. The 102-202 sequence of VP15R is homologous to the 1153-1253 sequence of the filamin C protein of Danio rerio (zebrafish), with an identity of 29%. Immunofluorescence and VP15R-GFP fusion protein subcellular localization assays showed that VP15R is localized in the cytoplasm. Pull-down and MALDI-TOF-TOF/MS assays demonstrated that VP15R can bind to the non-muscle myosin II (NM II) protein. Co-immunoprecipitation assays confirmed that VP15R can bind to the heavy chains of the NM II protein of mandarin fish, mice, and humans.
Archives of Virology 09/2010; 156(1):53-61. · 2.11 Impact Factor
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ABSTRACT: Vitellogenins (Vtgs), the precursors for the yolk proteins, are very important for the embryonic development of teleosts, and have also been studied extensively as biomarkers for environmental estrogenic mimics. The cDNA for a Vtg was isolated from the liver of the female white cloud mountain minnow (Tanichthys albonubes) by 3'- and 5'-RACE methods. It is 4,171 bp in full length, and encodes a putative protein of 1,326 amino acids. This putative Vtg, designated as wcmmVtg, contains complete portions of LVI and PV, but lacks the C-terminal half of LVII and thus belongs to type I vitellogenin. In addition to the liver of the female fish, wcmmVtg was also shown to be expressed in the ovary. During ovarian development, the mRNA expression of wcmmVtg in both the liver and ovary was continuously increased from the previtellogenic to late vitellogenic stages, but then decreased significantly at post-spawning stage. In the male fish, expression of wcmmVtg mRNA was induced in the liver by treatment with E2 (10 and 100 ng/l) for 14 days. These results suggest that the Vtg originated from the ovary of the white cloud mountain minnow may also contribute to the accumulation of yolk proteins during oocyte growth, and that the male white cloud mountain minnow is sensitive to the estrogenic treatment in terms of Vtg mRNA expression, which could also be applied to monitor the environmental estrogenic mimics.
Fish Physiology and Biochemistry 06/2010; 36(2):157-64. · 1.53 Impact Factor
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ABSTRACT: In the present study, we used the infectious spleen and kidney necrosis virus (ISKNV) and zebrafish model system to investigate the inhibitory effect of recombinant zebrafish interferon 1 (zfrIFN1) on acute viral infection and the impact of time of zfrIFN1 administration on its protective efficacy. In vivo experiments showed that administration of recombinant zfrINF1 up-regulated expression of several IFN-stimulated genes within 24 h of injection, and expression levels of these genes dropped to normal levels similar to those in control fish within three days. However, the transcriptions of two viral genes, the major capsid protein and virus protein 48 genes, were significantly inhibited for at least three days, indicating a longer duration of the zfrIFN1-mediated innate immune effect. To evaluate the protective efficacy of zfrIFN1 against ISKNV infection, we compared the relative percentage survival (RPS) of ISKNV-infected zebrafish by intraperitoneally (IP) injecting the fish with zfrIFN1 at different time points before or after infection. IP injection with 1 microg zfrIFN1/g fish body weight at 24, 6 or 0 h before virus infection or 6 h after virus infection significantly improved fish survival. However, IP injection with an equal dose of zfrIFN1 24 h post-infection did not provide significant protection to the fish. Our results suggest that zfrIFN1 is potent in inhibiting ISKNV acute infection and initiating the innate immune response in zebrafish, but its efficiency depends on the time of administration. This study shows the protective effects of interferon against a DNA-virus in fish for the first time and provides information about the efficacy of fish interferon that will prove useful in possible therapeutic applications.
Fish & Shellfish Immunology 05/2010; 29(3):399-406. · 3.32 Impact Factor
