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cloning and expression of a novel serine protease from japanese flounder, paralichthys olivaceus.two different cdna clones of japanese flounder (types i-1 and i-2) with lengths of 1096 and 1572bp, respectively, were found to encode the same serine protease consisting of 244 identical amino acid residues with three putative n-glycosylation sites, an 18-amino acid signal peptide and a 2-amino acid activation peptide. the amino acid sequence of the japanese flounder serine protease shares 39-44% identity to known hematopoietic serine proteases. genomic analysis showed that two different clones ...200717084451
development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative rt-pcr.the aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qrt-pcr) assay using a taqman probe to detect and quantify hirame rhabdovirus (hrv). the results demonstrated that the assay had a detection limit of 100 copies of rna per reaction and a log-linear range up to 10(8) copies of hrv rna. regression analysis demonstrated a significant correlation with an r(2) value of 0.9963 and a slope of -3.18 between the mean c(t) values and hrv crna. this a ...201020723563
modulation of the early immune response against viruses by a teleostean interferon regulatory factor-1 (irf-1).we investigated the role of a teleostean interferon regulatory factor-1 (irf-1) in the regulation of the fish immune system using japanese flounder, paralichthys olivaceus, as a model. fish were intramuscularly vaccinated with a recombinant plasmid expressing the japanese flounder irf-1 (jf irf-1) under the control of the cytomegalovirus immediate/early enhancer (cmv) promoter and were sampled at different days post-immunization. peripheral blood leukocytes (pbls) obtained from the jf irf-1-vacc ...200919101646
complete nucleotide sequence of the hirame rhabdovirus, a pathogen of marine fish.reverse transcription-polymerase chain reaction (rt-pcr) derived clones were constructed for the hirame rhabdovirus (hirrv) ca-9703 strain from korea, and the dna was sequenced. the 3'-end of genomic rna was cloned by poly(a)-tailing of the genomic rna before reverse transcription, and the 5'-end of the genome was cloned by poly(g)- or poly(c)-tailing of the first strand, followed by pcr. the remainder of the genomic dna was cloned by reverse transcription-polymerase chain reaction using primers ...200515567027
difference in japanese flounder, paralichthys olivaceus gene expression profile following hirame rhabdovirus (hirrv) g and n protein dna vaccination.the glycoprotein (g protein) gene, but not other genes, of fish rhabdoviruses, when used as a dna vaccine was previously shown to be highly effective in inducing a protective immune response. in this study we used a dna microarray to examine differential gene expression in japanese flounder (paralichthys olivaceus) in response to a dna vaccine made from the genes of hirame rhabdovirus (rhabdovirus olivaceus) (hirrv) g protein (phrv-g) and nucleocapsid (n) protein (phrv-n). a high level of protec ...200717449275
induction of antiviral state in fish cells by japanese flounder, paralichthys olivaceus, interferon regulatory factor-1.interferon regulatory factor-1 (irf-1) mediates an antiviral state in cells by regulating the expression of the interferon (ifn-alpha/beta) system. to elucidate the role of irf-1 in fish during virus infections, we constructed a recombinant plasmid of the japanese flounder, paralichthys olivaceus irf-1 (jf irf-1) under the control of the cytomegalovirus (cmv) immediate/early enhancer promoter. the antiviral mechanism of jf irf-1 was studied using transfection experiments in a homologous cell lin ...200515722233
establishment and characterization of two new cell lines derived from flounder, paralichthys olivaceus (temminck & schlegel).two new cell cultures from flounder, paralichthys olivaceus (temminck & schlegel), flounder fin (ffn) cells from fin tissue and flounder spleen (fsp) cells from spleen tissue, were established and characterized. the cells multiplied well in eagle's minimum essential medium, supplemented with 10% foetal bovine serum, and have been subcultured more than 100 times, becoming continuous cell lines. modal diploid chromosome number of ffn and fsp cells was 64 and 62, respectively. polymerase chain reac ...200314710758
expression profiling of immune-related genes from japanese flounder paralichthys olivaceus kidney cells using cdna microarrays.a japanese flounder paralichthys olivaceus cdna microarray containing 871 unique cdnas including 91 putative immune-related genes from our est studies was constructed and used to characterize of gene expression of in vitro grown kidney cells stimulated with mitogens such as cona, pma, lps or infected with hirame rhabdovirus (hrv). the numbers of genes whose expressions were increased or decreased by these factors were: 17 by con a, 139 by pma, 76 by lps and 182 by hrv infection. the treatment of ...200515752548
development of a dna vaccine against hirame rhabdovirus and analysis of the expression of immune-related genes after vaccination.intramuscular injection of japanese flounder, paralichthys olivaceus (average weight approximately 2 g) with 1 and 10 microg of a plasmid dna vaccine encoding the hirame rhabdovirus (hirrv) glycoprotein gene (pcmv-hrvg) was found to provide strong protection against hirrv. we also conducted a real-time pcr analysis to quantify immune-related genes, e.g. mhc class ialpha, iialpha, iibeta, tcr-alpha, beta1, beta2 and delta, to characterize the immune response at 1 and 7 days after dna vaccination. ...200415312663
protection of flounder against hirame rhabdovirus (hirrv) with a dna vaccine containing the glycoprotein gene.hirame rhabdovirus (hirrv) is an important virus of cultured flounder (paralichthys olivaceus). we tested the protective immunogenicity of dna-based vaccines against this virus. genes encoding the nucleocapsid protein (n) and the c-terminal half of the glycoprotein (g) were amplified by rt-pcr and separately cloned into the eukaryotic expression vector pcdna 3.1. the g protein expressed by transfected cells was detected by western blot analysis. pcr analyses demonstrated the presence of injected ...200616176849
teleostean il11b exhibits complementing function to il11a and expansive involvement in antibacterial and antiviral responses.interleukin 11 is a class-1 helical cytokine, having the four-helix bundle structure, possessing pleiotropic characteristics involved in physiological processes including blood production, bone formation and placentation. the interleukin 11 paralogues (il11a and il11b) have been identified in fish with only il11a from carp and trout have been characterized and analyzed for its expression thus far. here, we cloned and studied the structure and expression of il11b in japanese flounder (paralichthy ...200818538848
identification of viral induced genes in ig+ leucocytes of japanese flounder paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cdna probes.up-regulated genes of leucocytes expressing immunoglobulin (ig+ leucocytes) of hirame rhabdovirus (hrv)-infected japanese flounder were identified by differential hybridisation, using subtracted and un-subtracted cdna probes. ig+ leucocytes were separated from apparently healthy and hrv-infected japanese flounder by the magnetic beads antibody method using mouse anti-japanese flounder ig monoclonal antibody (mab). a cdna library was constructed from hrv-infected japanese flounder leucocytes, and ...200011081439
isolation and characterization of a rhabdovirus from starry flounder (platichthys stellatus) collected from the northern portion of puget sound, washington, usa.the initial characterization of a rhabdovirus isolated from a single, asymptomatic starry flounder (platichthys stellatus) collected during a viral survey of marine fishes from the northern portion of puget sound, washington, usa, is reported. virions were bullet-shaped and approximately 100 nm long and 50 nm wide, contained a lipid envelope, remained stable for at least 14 days at temperatures ranging from -80 to 5 degrees c and grew optimally at 15 degrees c in cultures of epithelioma papulosu ...200414769907
nucleotide sequence of the 2 matrix protein genes (m1 and m2) of hirame rhabdovirus (hrv), a fish rhabdovirus.we have cloned and sequenced the m1 and m2 protein genes of hirame rhabdovirus (hrv). the m1 protein gene was 684 nucleotides long, encoding 227 amino acids. sequence comparisons between the m1 protein genes of hrv and infectious hematopoietic necrosis virus (ihnv) or viral hemorrhagic septicemia virus (vhsv) revealed similarities of 65.1 and 40.8% at the nucleotide level and 81.1 and 44.8% at the amino acid level. the m2 protein gene was 582 nucleotides long, encoding 193 amino acids. sequence ...19958581014
sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of viral haemorrhagic septicaemia virus, a fish rhabdovirus.to evaluate the genetic diversity of viral haemorrhagic septicaemia virus (vhsv), the sequence of the glycoprotein genes (g) of 11 north american and european isolates were determined. comparison with the g protein of representative members of the family rhabdoviridae suggested that vhsv was a different virus species from infectious haemorrhagic necrosis virus (ihnv) and hirame rhabdovirus (hirrv). at a higher taxonomic level, vhsv, ihnv and hirrv formed a group which was genetically closest to ...19979367370
distribution and variation of nv genes in fish rhabdoviruses.the fish rhabdovirus infectious haematopoietic necrosis virus (ihnv) contains a non-virion (nv) gene between the glycoprotein (g) and polymerase (l) genes on its rna genome. the present study investigated three other fish rhabdovirus genomes and found that the nv gene of hirame rhabdovirus is closely related to the nv of ihnv, whereas the viral haemorrhagic septicemia nv gene showed evidence of significant divergence. most importantly, spring viraemia of carp virus, the only vesiculovirus-like f ...19979010293
the glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: analysis of relationships with other rhabdoviruses.the nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (svcv) and hirame rhabdovirus (hirrv) have been determined from cdna clones generated from viral genomic rna. the svcv glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. the hirrv glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. in sequence comparisons of 15 rhabdoviru ...19968806175
the complete genome structure and phylogenetic relationship of infectious hematopoietic necrosis virus.infectious hematopoietic necrosis virus (ihnv), a member of the family rhabdoviridae, causes a severe disease with high mortality in salmonid fish. the nucleotide sequence (11,131 bases) of the entire genome was determined for the pathogenic wrac strain of ihnv from southern idaho. this allowed detailed analysis of all 6 genes, the deduced amino acid sequences of their encoded proteins, and important control motifs including leader, trailer and gene junction regions. sequence analysis revealed t ...19958578857
identification and characterization of japanese flounder, paralichthys olivaceus interferon-stimulated gene 15 (jf-isg15).previously, using cdna microarray analysis, we demonstrated that an est clone of japanese flounder (paralichthys olivaceus) with homology to mammalian interferon-stimulated gene 15 (isg15) was strongly induced by treatment with dna vaccine encoding the glycoprotein gene of hirame rhabdovirus (hirrv). in this study, we conducted molecular cloning and expression analysis of the japanese flounder isg15 (jf-isg15). jf-isg15 encoded two exons. the first exon was non-coding, while the second exon enco ...201120299096
immunoanalysis of antiviral mx protein expression in japanese flounder (paralichthys olivaceus) cells.mx proteins are interferon-inducible gtpases that possess antiviral properties in vertebrates. japanese flounder (paralichthys olivaceus) mx protein has previously been shown to possess some antiviral activity against rhabdoviruses. a polyclonal antibody was generated against a purified peptide fragment of japanese flounder mx protein that had been produced in an escherichia coli expression system. the pab detected the approximately 71 kda mx protein from japanese flounder (hirame) natural embry ...200415707665
in vitro inhibition of fish rhabdoviruses by japanese flounder, paralichthys olivaceus mx.a homologous fish cell line stably expressing the recombinant japanese flounder mx (jfmx) was infected with hirame rhabdovirus (hirrv) and viral hemorrhagic septicemia virus (vhsv), both of which are negative single-stranded rna viruses belonging to the rhabdoviridae family. analysis of primary transcription of the two rhabdoviruses showed that there was lower expression level and copy number of the viral nucleoprotein transcript in the jfmx-transfected cell line than the infected, control cells ...200314698674
microarray technology is an effective tool for identifying genes related to the aquacultural improvement of japanese flounder, paralichthys olivaceus.molecular techniques are now essential for discovering new functional genes for the improvement of cultured marine organisms. such techniques can be used to obtain expressed sequence tags (est) and, through the use of microarrays, the entire transcriptome. for example, microarrays can be used to reveal biomarkers of health conditions, nutrient changes and immune response in fish and shellfish. est-based microarray chips were constructed for cultured fish and shellfish species including japanese ...201120685186
molecular cloning of a novel interferon regulatory factor in japanese flounder, paralichthys olivaceus.a complementary dna library was constructed in lambda zap ii using messenger rna from the leukocytes of some heterocloned japanese flounder, paralichthys olivaceus, that had been artificially infected with hirame rhabdovirus (hrv). a cloned flounder interferon regulatory factor (designated firf) cdna was found to be 1746 bp in length, with an open reading frame of 297 amino acids. the overall amino acid sequence of firf had approximately 40% identity with the previously reported avian and mammal ...19989628009
engineered virus-encoded pre-microrna (pre-mirna) induces sequence-specific antiviral response in addition to nonspecific immunity in a fish cell line: convergence of rnai-related pathways and ifn-related pathways in antiviral response.transfection with synthesized virus-specific small interfering rnas (sirnas) efficiently inhibits viral replication in viral-infected fish cell lines, implying the involvement of rna interference (rnai)-related pathways in the antiviral response of fish cells. here, we demonstrate that plasmid expressing virus-encoded pre-micrornas (pre-mirnas) can also inhibit viral replication through these pathways. by incorporating sequences encoding mirnas specific to major capsid protein (mcp) gene of red ...200818687362
characterization and antiviral function of a cytosolic sensor gene, mda5, in japanese flounder, paralichthys olivaceus.cytosolic pattern recognition receptors such as retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) play an important role in sensing viral rnas. the receptor encoded by melanoma differentiation-associated gene 5 (mda5), an rlr, recognizes viral rna in the cytoplasm and enhances antiviral response in host cells. the full-length mda5 gene in japanese flounder, paralichthys olivaceus was cloned and found to have 11,251 nucleotides. mda5 transcript abundance was significantly increased in ...201021185857
analysis and characterization of the complete genomic sequence of the chinese strain of hirame rhabdovirus. 201121241324
hirame rhabdovirus (hirrv) as the cause of a natural disease outbreak in cultured black seabream (acanthopagrus schlegeli) in korea.in 2015, a high mortality rate of about 40% was observed in black seabream (acanthopagrus schlegeli) on a farm on the southern coast of korea. most of the diseased fish showed a hemorrhage of the mouth, pale liver, petechial hemorrhaging in the internal fat, and an enlarged spleen. other than alella sp., no parasites or bacteria were isolated from the diseased fish, and all of the tissue filtrates produced cytopathic effects (cpes) in fhm and chse-214 cells. a polymerase chain reaction analysis ...201526315687
first isolation of hirame rhabdovirus from freshwater fish in europe.a rhabdovirus was isolated in cell culture inoculated with tissue material from diseased grayling, thymallus thymallus (l.), originating from a fish farm affected by a mortality episode in poland. diagnostics tests showed that the virus was not related to novirhabdoviruses known in europe, nor to vesiculovirus-like species, except perch rhabdovirus (prhv) with which it shared moderate serological relations. however, rt-pcr with prhv probes gave negative results. to identify the virus, a random-p ...201423962315
inhibition of hirame rhabdovirus growth by rna aptamers.rna aptamers are artificial nucleic acids that specifically bind to a wide variety of targets. they are an effective tool for pharmaceutical research and development of antiviral agents. here, we describe four hirame rhabdovirus (hirrv)-rna aptamers (h1, h2, h3 and h4) that we obtained from an in vitro process called the systematic evolution of ligands by exponential enrichment (selex). the hirrv-rna aptamers specifically bind to hirrv. hirame natural embryo (hinae) cells treated with virus and ...201222943666
antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (vhsv) purified in large amounts from insect larvae.abstract:201121693048
gene expression profile of hirrv g and n protein gene vaccinated japanese flounder, paralichthys olivaceus during hirrv infection.the glycoprotein (g protein) gene, but not the nucleocapsid protein (n protein) gene, of the hirame rhabdovirus (hirrv) was previously shown to be highly effective in inducing a protective immune response in japanese flounder (paralichthys olivaceus) when used as a dna vaccine. our previous cdna microarray analysis demonstrated that interferon-stimulated genes (isgs) were strongly induced by the hirrv g protein gene (phrv-g) but not by the n protein gene (phrv-n). however, the molecular basis fo ...201120371118
isolation and identification of a new strain of hirame rhabdovirus (hirrv) from japanese flounder paralichthys olivaceus in china.hirame rhabdovirus virus (hirrv) is a rhabdovirus that causes acute hemorrhage disease in fish culture, resulting in a great economic loss in parts of asia and europe.201728388934
outbreak of hirame rhabdovirus infection in cultured spotted sea bass lateolabrax maculatus on the western coast of korea.in this study, we determined the cause of a disease outbreak in spotted sea bass, lateolabrax maculatus reared in culture cages on the western coast of korea in 2013. the major signs in the diseased fish exhibited were haemorrhaging on the membranes of the abdomen, gastrointestinal organs and opercular gills, as well as an enlarged spleen. no external morphological signs of infection were visible, except for a darkening in colour. no parasites or pathological bacteria were isolated from the dise ...201627371509
a survey of expressed genes in the leukocytes of japanese flounder, paralichthys olivaceus, infected with hirame rhabdovirus.we constructed a cdna library of japanese flounder, paralichthys olivaceus, leukocytes that were infected with hirame rhabdovirus (hrv) in order to analyze some of the genes that are induced and expressed by virus infection in the immune system. four hundred and fifty-two partial sequences representing 300 cdna clones were obtained from the 5' and/or 3' ends of inserts derived from the japanese flounder leukocyte cdna library. about three-quarters of the 300 cdna clones (217 clones, 72.3%) repre ...200010689095
molecular characterization of the glycoproteins from two warm water rhabdoviruses: snakehead rhabdovirus (shrv) and rhabdovirus of penaeid shrimp (rps)/spring viremia of carp virus (svcv).we have determined the complete coding sequences for the glycoprotein (g) genes from two rhabdoviruses that infect warm water aquatic animals, the snakehead rhabdovirus (shrv) and rhabdovirus of penaeid shrimp (rps). surprisingly, the g nucleotide sequence from rps, a virus which has been isolated from diseased shrimp in hawaii on numerous occasions, was over 99% identical to the g nucleotide sequence from spring viremia of carp virus (svcv), a fish virus from europe and asia. this is the first ...199910518707
sequences of 596 cdna clones (565,977 bp) of japanese flounder (paralichthys olivaceus) leukocytes infected with hirame rhabdovirus.: we have partially sequenced 785 sequences of 596 independent complementary dna clones isolated from a cdna library of japanese flounder leukocytes infected with hirame rhabdovirus. these sequences consist of a total of 565,977 base pairs. the average size of the sequenced lengths was 721 bp. of 596 clones, 386 (64.8%) were identified as previously reported genes by the blastn and blastx programs. about 30% of the identified clones could be recognized by only the blastx program. a total of 251 ...199910525682
cloning and analysis of expression of mx cdna in japanese flounder, paralichthys olivaceus.myxovirus resistance (mx) is an interferon (ifn)-induced protein that specifically prevents growth, in vivo and in vitro, of certain classes of virus. we isolated the mx cdna from a leukocyte cdna library obtained from a japanese flounder infected with hirame rhabdovirus (hrv). the mx cdna was found to contain 2385 bp coding for 620 amino acids. the amino acid sequence had approximately 51 and 78% identities to the sequences of human and rainbow trout mx cdnas, respectively. we propose that the ...200010736524
molecular cloning and antiviral activity of ifn-β promoter stimulator-1 (ips-1) gene in japanese flounder, paralichthys olivaceus.the mitochondrial adaptor, ifn-β promoter stimulator-1 (ips-1), also known as mavs/visa/cardif, plays a key role in the signal transduction of the rig-1/mda5 pathway to induce the production of interferons (ifns) and other cytokines. in the present study, japanese flounder (paralichthys olivaceus) ips-1 cdna was cloned from japanese flounder spleen using pcr-based methods. the full-length cdna has 2235 nucleotides and encodes a polypeptide of 641 amino acids. the putative japanese flounder ips-1 ...201020800685
evolutional conservation of molecular structure and antiviral function of a viral rna receptor, lgp2, in japanese flounder, paralichthys olivaceus.lgp2 is an important intracellular receptor that recognizes viral rnas in innate immunity. to understand the mechanism of viral rna recognition, we cloned an lgp2 cdna and gene in japanese flounder (paralichthys olivaceus). viral hemorrhagic septicemia virus-induced expressions of lgp2 mrna were evaluated in vivo and in vitro by quantitative real-time pcr (q-pcr) using primers based on the clone sequences. the expression of lgp2 mrna in the kidney dramatically increased at 3 d postinfection. the ...201021098234
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