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genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene.a molecular phylogenetic analysis of 25 isolates of fish nodaviruses, the causative agents of viral nervous necrosis of marine fish, was performed based on the nucleotide sequences (427 bases) of the coat protein gene. these fish nodaviruses were classified into four clusters: tiger puffer nervous necrosis virus, striped jack nervous necrosis virus, berfin flounder nervous necrosis virus, and red-spotted grouper nervous necrosis virus.19979097459
temperature effect on nervous necrosis virus infection in grouper cell line and in grouper larvae.this preliminary study elucidates the in vitro and in vivo effects of temperature on grouper nervous necrosis virus (gnnv) infection. a novel continuous cell line derived from the fin tissue of a grouper (epinephelus coioides, hamilton), named as gf-1 cell line, was used. cytopathic effect was observed in gnnv-infected gf-1 cells incubated at 24-32 degrees c after viral adsorption, but not at 20 degrees c or 37 degrees c even though the viral adsorption temperature was 28 degrees c. viral protei ...199910509721
cloning of the fish cell line ssn-1 for piscine nodaviruses.six cell clones were derived from the ssn-1 cell line, which is composed of a mixed cell population and persistently infected with a c-type retrovirus (snrv). these clones were susceptible to 4 piscine nodavirus strains belonging to different genotypes (sjnnv, rgnnv, tpnnv and *bfnnv [striped jack, redspotted grouper, tiger puffer and barfin flounder nervous necrosis viruses]). three clones, designated a-6, e-9, and e-11, were highly permissive to nodavirus infection and production. the virus-in ...200011145456
sequence comparison and phylogenetic analysis of fish nodaviruses based on the coat protein gene.we have amplified by reverse transcription-polymerase chain reaction (rt-pcr) and sequenced a 605-bp fragment covering the variable region of the coat protein gene of fish nodaviruses infecting european sea bass, dicentrarchus labrax (n = 24), and shi drum, umbrina cirrosa (n = 2), in the mediterranean basin. nine new isolates were identified and their sequences were combined with sequences in the literature to produce three different data sets. the first set, based on amino acid sequences, was ...200111504420
characterization of virus-like particles assembled in a recombinant baculovirus system expressing the capsid protein of a fish nodavirus.betanodaviruses are causative agents of neurological disorders in several species of fish. we cloned and sequenced the rna2 segment of two grouper viruses isolated from epinephelus malabaricus (malabaricus grouper nervous necrosis virus, mgnnv) and epinephelus lanceolatus (dragon grouper nervous necrosis virus, dgnnv). the sequences of the two rnas were 99% identical and comparison with previously sequenced rna2 segments of fish nodaviruses striped jack nervous necrosis virus, atlantic halibut v ...200111883005
determination of the complete nucleotide sequences of rna1 and rna2 from greasy grouper (epinephelus tauvina) nervous necrosis virus, singapore strain.the complete nucleotide sequences of rna1 and rna2 from greasy grouper (epinephelus tauvina) nervous necrosis virus (ggnnv), singapore strain, were determined. 5' race and rna ligation were used to obtain the complete nucleotide sequences of the 5' and 3' non-coding regions (ncrs). ggnnv rna1 was determined to be 3103 nt long, containing an orf of 982 aa, while ggnnv rna2 was determined to be 1433 nt long, containing an orf of 338 aa. both ggnnv rnas are longer than those of other published beta ...200111172107
interference of the life cycle of fish nodavirus with fish retrovirus.interference of the life cycle of grouper nervous necrosis virus (gnnv), a member of the nodaviridae, genus betanodavirus, by snakehead retrovirus (snrv) has been studied in vitro. sgf-1, a new fish cell line that is persistently infected with snrv, was induced by inoculating snrv into the grouper fin cell line gf-1. culture supernatants and cell pellets from both gnnv-infected sgf-1 and gf-1 cells were collected and employed for virus productivity analysis. the yields of gnnv rna and capsid pro ...200212237429
in vitro efficiency of intra- and extracellular immunization with mouse anti-ygnnv antibody against yellow grouper nervous necrosis virus.mouse monoclonal antibody (mab-18) against yellow grouper nervous necrosis virus (ygnnv) coat protein was developed and assayed for its neutralization ability. in the present study, we cloned and sequenced the cdnas encoding heavy (gamma) and light (kappa) chains by constructing a cdna library of the mab-18 hybridoma. three expression vectors, pcmv-nnv-18h (gamma), pcmv-nnv-18l (kappa), and pcmv-nnv-18hl (both gamma and kappa chains) were constructed and successfully expressed in grouper brain ( ...200212163274
virus-like particles of a fish nodavirus display a capsid subunit domain organization different from that of insect nodaviruses.the structure of recombinant virus-like particles of malabaricus grouper nervous necrosis virus (mgnnv), a fish nodavirus isolated from the grouper epinephelus malabaricus, was determined by electron cryomicroscopy (cryoem) and three-dimensional reconstruction at 23-a resolution. the cryoem structure, sequence comparison, and protein fold recognition analysis indicate that the coat protein of mgnnv has two domains resembling those of tomato bushy stunt virus and norwalk virus, rather than the ex ...200212021370
establishment of cell lines from a tropical grouper, epinephelus awoara (temminck & schlegel), and their susceptibility to grouper irido- and nodaviruses.four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, epinephelus awoara (temminck & schlegel). optimum media and temperature conditions for maximum growth were standardized. the eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. the viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees c). besides these four cell lines, previously established grouper bra ...200312962209
serological relationships among genotypic variants of betanodavirus.betanodaviruses, the causative agents of viral nervous necrosis or viral encephalopathy and retinopathy, are divided into 4 genotypes based on the coat protein gene (rna2). in the present study, serological relationships among betanodavirus genotypic variants were examined by virus neutralization tests using rabbit antisera raised against purified virions of strains representative of each genotype. all 20 isolates examined shared epitopes for neutralizing, but they fell into 3 major serotypes (a ...200314735917
genetic and antigenic analysis of betanodaviruses isolated from aquatic organisms in taiwan.viral nervous necrosis (vnn) is a worldwide disease among marine fishes. in taiwan, nnn disease was first identified in 2 species of hatchery-reared grouper, epinephelus fuscogutatus and e. akaaya in 1994. since then, increasing mortalities have occurred among groupers epinephelus spp., and also among european eels anguilla anguilla l., yellow-wax pompano trachinotus falcatus, firespot snapper lutaanus erythropterus b., barramundi lates calcarifer, cobias rachycentron canadum, humpback groupers ...200313677508
susceptibility of cultured juveniles of several marine fish to the sevenband grouper nervous necrosis virus.piscine nodaviruses (betanodaviruses) have been tentatively divided into four genotypes (sjnnv, rgnnv, tpnnv and bfnnv) and it is suggested that host specificity is different among these genotypes. in the present study, a betanodavirus [sevenband grouper nervous necrosis virus (sgnnv)] belonging to the redspotted grouper nervous necrosis virus (rgnnv) genotype, to which most betanodaviruses from warm water fish are identified, was evaluated for its pathogenicity to hatchery-reared juveniles of s ...200312962219
infection competition against grouper nervous necrosis virus by virus-like particles produced in escherichia coli.dragon grouper (epinephelus lanceolatus) nervous necrosis virus (dgnnv) comprises 180 copies of capsid protein that encapsulate a bipartite genome of single-stranded (+)-rnas. this study reports that virus-like particles (vlps) are formed in escherichia coli expressing the full-length orf encoding the dgnnv capsid protein. two sizes of vlps are observed. the heavier particles resemble the native piscine nodavirus in size and stain permeability, while the lighter ones are approximately two-thirds ...200312771428
genomic classification of new betanodavirus isolates by phylogenetic analysis of the coat protein gene suggests a low host-fish species specificity.viral encephalopathy and retinopathy is a devastating disease that causes neurological disorders and high mortality in a large number of cultivated marine fish species around the world. it is now established that several viral strains classified in the genus betanodavirus of the family nodaviridae are the aetiological agents of this disease. betanodaviruses can be classified into four genotypes based on the coat protein gene sequence. here, the coat protein genes of the three major strains isola ...200415448371
nodavirus infections in israeli mariculture.viral encephalopathy and retinopathy (ver) infections were diagnosed in five fish species: epinephelus aeneus, dicentrarchus labrax, sciaenops ocellatus, lates calcarifer and mugil cephalus cultured on both the red sea and mediterranean coasts of israel during 1998-2002. spongiform vacuolation of nervous tissue was observed in histological sections of all examined species. with transmission electron microscopy, paracrystalline arrays and pieces of membrane-associated non-enveloped virions measur ...200415291788
membrane association of greasy grouper nervous necrosis virus protein a and characterization of its mitochondrial localization targeting signal.localization of rna replication to intracellular membranes is a universal feature of positive-strand rna viruses. the betanodavirus greasy grouper (epinephelus tauvina) nervous necrosis virus (ggnnv) is a positive-rna virus with one of the smallest genomes among rna viruses replicating in fish cells. to understand the localization of ggnnv replication complexes, we generated polyclonal antisera against protein a, the ggnnv rna-dependent rna polymerase. protein a was detected at 5 h postinfection ...200415163743
inoculation of balb/c mice with fish-pathogenic nodaviruses.to investigate the pathogenicity of fish-pathogenic nodaviruses (betanodaviruses) in mammals, weanling mice were inoculated with striped jack nervous necrosis virus (sjnnv; the type species of the genus betanodavirus) or redspotted grouper nervous necrosis virus (rgnnv), which belong to different genotypes. after inoculation with 10(7.5) tcid50, either intramuscularly or intraperitoneally, mice remained clinically normal for the 14-day observation period. in a subsequent short-term (72 h) experi ...200415003479
pcr-based detection of betanodaviruses from cultured and wild marine fish with no clinical signs.betanodaviruses are the causative agents of viral nervous necrosis (vnn) or viral encephalopathy and retinopathy (ver) in cultured marine fish. a total of 131 apparently healthy fish from 30 species were collected in two geographically remote aquaculture areas, yashima bay (kagawa prefecture) and tamanoura bay (nagasaki prefecture), in japan. the brains of fish were examined by reverse transcriptase-polymerase chain reaction (rt-pcr) and nested pcr to detect the coat protein gene of betanodaviru ...200415482425
early endocytosis pathways in ssn-1 cells infected by dragon grouper nervous necrosis virus.many fish undergo betanodavirus infection. to study the infection process of dragon grouper nervous necrosis virus (dgnnv), native virus and virus-like particles (vlps) were used to analyse the binding and internalization in ssn-1 cells. the binding of dgnnv and vlps to ssn-1 cells was demonstrated using western blotting and immunofluorescence microscopy. as estimated by indirect elisa, the dgnnv particles bound ssn-1 cells in a dose-dependent manner up to 8 x 10(4) particles per cell. the bindi ...200516099914
production of monoclonal antibodies against grouper nervous necrosis virus (gnnv) and development of an antigen capture elisa.five (2 igg, 3 igm) monoclonal antibodies (mabs) against the g9508ks strain of grouper nervous necrosis virus (gnnv) were produced and characterized. all 5 mabs showed positive signals in the retina of gnnv-infected grouper larvae and in the cytoplasm of gnnv-infected gf-1 cells using immunohistochemistry staining. two mabs reacted with the denatured capsid protein derived from gnnv-infected gf-1 cells in western blot analysis, but did not react with the gnnv recombinant capsid protein expressed ...200515759800
inhibition of nervous necrosis virus propagation by fish mx proteins.mx proteins are interferon induced, antiviral proteins, expressed in response to treatment with double stranded rna or virus infection. here we report the cloning, sequencing, and antiviral property of three forms of mx genes, mxi, mxii, and mxiii from grouper (epinephelus coioides). multiple comparison of grouper mx amino acid sequences revealed the conservation of mx putative gtp-binding domain, dynamin family signature, and leucine zipper motif. we have established a new cell line, grouper br ...200617069756
detection of betanodavirus in juvenile barramundi, lates calcarifer (bloch), by antigen capture elisa.betanodavirus infection of fish has been responsible for mass mortalities in aquaculture hatcheries worldwide. betanodaviruses possess a bipartite single-stranded rna genome consisting of the 3.1 kb rna1 encoding an rna-dependent rna polymerase and the b2 protein, while the 1.4 kb rna2 encodes the viral nucleocapsid protein, alpha. a panel of six monoclonal antibodies against the alpha protein of greasy grouper nervous necrosis virus (ggnnv) was developed for use in diagnostics. all antibodies r ...200616866926
betanodavirus infection in the freshwater model fish medaka (oryzias latipes).betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite, positive-sense rna genomes. as their genomes are the smallest and simplest among viruses, betanodaviruses have been studied in detail as model viruses by using a genetic-engineering system, as has occurred with the insect alphanodaviruses, the other members of the family nodaviridae. however, studies of virus-host interactions have been limited, as betanodaviruses basically infect marine fish at early de ...200616847129
temperature-dependency of betanodavirus infection in ssn-1 cell line.this study examined the in vitro effects of temperature on betanodavirus infection in the ssn-1 cell line. a betanodavirus isolated from moribund sea bass fry dicentrarchus labrax farmed in the adriatic sea and characterised as a rgnnv (redspotted grouper nervous necrosis virus) genotype was used. virus-infected ssn-1 cells were incubated at temperatures between 10 and 30 degrees c and observed for cytopathic effects daily for 15 d. cell-free and cell-associated viral growth were evaluated by 50 ...200616610592
betanodavirus induces phosphatidylserine exposure and loss of mitochondrial membrane potential in secondary necrotic cells, both of which are blocked by bongkrekic acid.in this study, we show how the red spotted grouper nervous necrosis virus (rgnnv) causes loss of mitochondrial membrane potential and promotes host secondary apoptotic necrosis. rgnnv viral proteins such as protein alpha (42 kda) and protein a (110 kda) were quickly expressed between 12 h and 24 h postinfection (p.i.) in gl-av cells. annexin v staining revealed that the nnv infection of gl-av cells induced phosphatidylserine (ps) externalization and development of bulb-like vesicles (bleb format ...200616430940
betanodavirus b2 is an rna interference antagonist that facilitates intracellular viral rna accumulation.betanodaviruses are small positive-sense bipartite rna viruses that infect a wide variety of fish species and are notorious for causing lethal outbreaks in juvenile fish hatcheries worldwide. the function of a small nonstructural protein, b2, encoded by the subgenomic rna3 of betanodaviruses, has remained obscure. greasy grouper nervous necrosis virus, a betanodavirus model, was used to develop a facile dna-based reverse genetics system that recapitulated the virus infection cycle, and we used t ...200616352533
immune response against grouper nervous necrosis virus by vaccination of virus-like particles.the grouper is a high-value fish in the seafood market. grouper nervous necrosis virus (gnnv) causes mass mortality, near 100% in larvae and juveniles, which has great economic impact on the aquaculture of the marine fish. since vaccination is one of the best methods against viral diseases, grouper epinephelus lanceolatus was injected with virus-like particles (vlps) of gnnv at different dosages and injection frequencies. the anti-sera of vaccinated fish were analyzed with antigen-capture elisa ...200616797803
enhanced viability of a nervous necrosis virus-infected stable cell line over-expressing a fusion product of the zfbcl-xl and green fluorescent protein genes.nervous necrosis virus (nnv) infection induces host cell apoptosis by an ill-understood process. we utilized a fusion between enhanced green fluorescent protein (egfp) and the zfbcl-x(l) gene in gl-av cells to select for zfbcl-x(l) stable cell lines and to assess the effectiveness of the anti-apoptotic protein bcl-x(l) in circumventing nnv-induced cell death. stable egfp and egfp-bcl-x(l)-expressing clones were obtained at high purity within 2.5-3 months. in the latter, the egfp-bcl-x(l) fusion ...200616768715
high expression of capsid protein of red-spotted grouper nervous necrosis virus in an avian cell line requires viral rna2 non-coding regions. 200717584441
rna-dependent rna polymerase from atlantic halibut nodavirus contains two signals for localization to the mitochondria.nodaviruses encode an rna-dependent rna polymerase called protein a that is responsible for replication of the viral rna segments. the intracellular localization of protein a from a betanodavirus isolated from atlantic halibut (ahnv) was studied in infected fish cells and in transfected mammalian cells expressing myc-tagged wild type protein a and mutants. in infected cells protein a localized to cytoplasmic structures resembling mitochondria and in transfected mammalian cells the ahnv protein a ...200717602779
anti-bcl-2 family members, zfbcl-x(l) and zfmcl-1a, prevent cytochrome c release from cells undergoing betanodavirus-induced secondary necrotic cell death.nervous necrosis virus (nnv)-induced, host cell apoptosis mediates secondary necrosis by an ill-understood process. in this study, redspotted grouper nervous necrosis virus (rgnnv) is shown to induce mitochondria-mediated necrotic cell death in gl-av cells (fish cells) via cytochrome c release, and anti-apoptotic proteins are shown to protect these cells from death. western blots revealed that cytochrome c release coincided with disruption of mitochondrial ultrastructure and preceded necrosis, b ...200717245642
screening of freshwater fish species for their susceptibility to a betanodavirus.betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense rna genomes. because the genomes are the smallest and simplest among viruses, betanodaviruses have been well studied using a reversed genetics system as model viruses. however, studies of virus-host interactions have progressed slowly because permissive hosts for betanodaviruses (basically larvae and juveniles of marine fish) are only available for limited periods of the year and are not ...200717972753
molecular detection of betanodaviruses from apparently healthy wild marine invertebrates.one hundred eighteen samples (21 species) of wild marine invertebrates were collected from western and southern coastal area of korean peninsula. four of 78 (18 species) samples collected at namhae (south) area were positive for nodavirus in nested pcr test. of the 40 samples (5 species) collected at hwanghae (west) areas, all samples were negative for nodavirus in both rt-pcr and nested pcr tests. positive nested pcr results were obtained from the following species: charybdis bimaculata charybd ...200818076900
molecular detection of betanodavirus in wild marine fish populations in korea.viral nervous necrosis (vnn) is a worldwide disease affecting several species of cultured marine fish. in korea, vnn has been identified in several species of cultured marine fish. in this study, the authors present data of the amplified nested polymerase chain reaction product (420 bp) of 21 nodavirus strains from different species of apparently healthy wild marine fish on the southern coast of korea. phylogenetic analysis based on the partial nucleotide sequence (177 bases) of the rna2 coat pr ...200818182506
zebrafish anti-apoptotic protein zfbcl-xl can block betanodavirus protein alpha-induced mitochondria-mediated secondary necrosis cell death.betanodavirus protein alpha induces cell apoptosis or secondary necrosis by a poorly understood process. in the present work, red spotted grouper nervous necrosis virus (rgnnv) rna 2 was cloned and transfected into tissue culture cells (gf-1) which then underwent apoptosis or post-apoptotic necrosis. in the early apoptotic stage, progressive phosphatidylserine externalization was evident at 24h post-transfection (p.t.) by annexin v-fluos staining. tunel assay revealed apoptotic cells at 24-72 h ...200818276161
genetic analysis of betanodaviruses in subclinically infected aquarium fish and invertebrates.betanodaviruses causing viral nervous necrosis (vnn) have been detected and isolated from several species of cultured marine fish worldwide. in korea, vnn was identified in several species of cultured marine fish. this study presents data on the amplified nested pcr product (420 bp) of 11 nodavirus strains from different species of apparently healthy aquarium fish and invertebrates collected in one private commercial aquarium in korea. phylogenetic analyses based on the partial nucleotide sequen ...200818293033
enhanced propagation of fish nodaviruses in bf-2 cells persistently infected with snakehead retrovirus (snrv).fish nodaviruses are causative agents of viral nervous necrosis causing high mortality in cultured marine fishes around the world. the first successful isolation of fish nodavirus was made with ssn-1 cells, which are persistently infected with snakehead retrovirus (snrv). in the present study, a bf-2 cell line persistently infected with snrv (pi-bf-2) was established to evaluate the influence of snrv on the production of fish nodavirus. the pi-bf-2 cells were slightly more slender than bf-2 cell ...200818429438
establishment of reverse genetics system of betanodavirus for the efficient recovery of infectious particles.betanodaviruses, a member of the family nodaviridae, have small positive-stranded bipartite rna genomes and are the causal agent of viral nervous necrosis in marine-farmed fish. to facilitate the study of betanodavirus, infectious cdna clones of its two genomic rnas were generated. the full-length cdna of the new redspotted grouper nervous necrosis virus strain (sg2001nag) rna1 and rna2 were co-transcribed by t7 rna polymerase in baby hamster kidney cells expressing t7 rna polymerase. the transc ...200818508134
efficient propagation of betanodavirus in a murine astrocytoma cell line.betanodavirus, a bipartite rna virus of fishes and a member of nodaviridae family, targets nervous tissues and is the causative agent of viral nervous necrosis in marine farmed fish. betanodavirus is thought to be propagated only in fish cells because betanodavirus has only been isolated in fish and it is not well propagated in mammalian culture cells. however, the host specificity of betanodavirus has not yet been well analyzed. to analyze the host specificity of betanodavirus, various mammalia ...200818556083
variable region of betanodavirus rna2 is sufficient to determine host specificity.betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense rna genomes. the viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called t4 region) of the smaller genomic segment rna2 (1.4 kb). betanodaviruses have marked host specificity, although the primary structures of the viral rnas and encoded proteins are similar among the viruses. we have previously demonstrated, usin ...200818589996
role of the dxxdxd motif in the assembly and stability of betanodavirus particles.piscine betanodavirus possesses a bipartite genome of single-stranded (+)rnas. rna2 cdna of dragon grouper nervous necrosis virus (dgnnv) has been expressed previously to form virus-like particles (vlps), which are highly similar to the native virion. experiments with calcium-chelating or reducing/oxidizing reagents showed that the dgnnv vlps required only calcium for particle assembly. with the recombinant vlps, site-directed mutagenesis can be employed to investigate the roles of calcium-bindi ...200818626568
comparisons among the complete genomes of four betanodavirus genotypes.betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense rna genomes and have been classified (based on analysis of rna2 sequences) into 4 genotypes: tiger puffer nervous necrosis virus (tpnnv), barfin flounder nervous necrosis virus (bfnnv), striped jack nervous necrosis virus (sjnnv), and redspotted grouper nervous necrosis virus (rgnnv). full-length genomes of tpnnv and bfnnv were sequenced for the first time in this study. their sequence d ...200818717064
potential for the replication of the betanodavirus redspotted grouper nervous necrosis virus in human cell lines.the determination of the host ranges of viruses is important because of the possible emergence of infectious agents, which may result from the zoonotic transmission of animal viruses to humans. the family nodaviridae, whose members are non-enveloped, positive-stranded bipartite rna viruses, is comprised of the genera alphanodavirus and betanodavirus, whose members predominantly infect insects and fish, respectively. the alphanodaviruses can also infect suckling mice and suckling hamsters, result ...200817906832
neutralizing antibody levels for protection against betanodavirus infection in sevenband grouper, epinephelus septemfasciatus (thunberg), immunized with an inactivated virus vaccine.abstract an inactivated betanodavirus, red-spotted grouper nervous necrosis virus (rgnnv), is a vaccine candidate for viral nervous necrosis (vnn). the present study was conducted to examine inoculation doses of the vaccine and neutralizing antibody titre levels to protect fish against vnn. young sevenband grouper, epinephelus septemfasciatus, averaging 25.4 g, were immunized at 25 degrees c water temperature by a single intraperitoneal injection of formalin-inactivated rgnnv. fish immunized at ...200919490392
viral encephalopathy and retinopathy of dicentrarchus labrax and sparus aurata farmed in tunisia.viruses belonging to the nodaviridae family cause disease worldwide among a large number of species of marine fish, and have been described in all continents. in the present study, a total of 69 farmed tunisian sea bass (dicentrarchus labrax) and 24 sea bream (sparus aurata) samples were tested monthly for the detection of betanodavirus. the virus was identified in both species using indirect immunofluorescence assays (ifat) and rt-pcr. in addition sequence analysis of part of the coat protein g ...200918982421
phylogenetic analysis of betanodavirus isolates from australian finfish.in australia, disease caused by betanodavirus has been reported in an increasing number of cultured finfish since the first report of mortalities in 1990. partial coat protein gene sequences from the t2 or t4 regions of 8 betanodaviruses from barramundi lates calcarifer, sleepy cod oxyeleotris lineolata, striped trumpeter latris lineata, barramundi cod cromileptes altivelis, australian bass macquaria novemaculata and gold-spotted rockcod epinephelus coioides from several australian states were d ...200920099409
genetic analysis and pathogenicity of betanodavirus isolated from wild redspotted grouper epinephelus akaara with clinical signs.diseased wild redspotted grouper epinephelus akaara were collected from seto inland sea, ehime prefecture, in august 2002. fish showed erratic swimming behavior and inflation of the swim bladder. the fish brains were positive for nodavirus in both rt-pcr and nested pcr. the sequence of the nested pcr product (177 nt) was closely related to that of a known betanodavirus, redspotted grouper nervous necrosis virus. when juvenile sevenband grouper e. septemfasciatus were challenged intravitreously w ...200919130166
betanodavirus non-structural protein b1: a novel anti-necrotic death factor that modulates cell death in early replication cycle in fish cells.the functions of the betanodavirus non-structural protein b1 is still unknown. we examined b1 expression patterns and investigated novel cell death regulatory functions for this viral protein following rgnnv infection in fish cells. the b1 gene (336 nt) was cloned from the redspotted grouper nervous necrosis virus (rgnnv) genome. b1 mrna was rapidly expressed in the fish cells from viral rna3 at 12 h post-infection (p.i.). at the protein level, expression was low at 12 h p.i., and then increased ...200919136133
protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, epinephelus septemfasciatus (thunberg).an aquabirnavirus (abv) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (rgnnv)] were investigated for their potential to prevent rgnnv-induced viral nervous necrosis (vnn) in the sevenband grouper, epinephelus septemfasciatus (thunberg). three groups of fish were injected intramuscularly with abv, intraperitoneally with inactivated rgnnv (irgnnv) or with both abv and irgnnv. at 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscu ...200919261047
immune responses of asian sea bass, lates calcarifer bloch, against an inactivated betanodavirus vaccine.asian sea bass, lates calcarifer (bloch), exhibited strong immune responses against a single injection of the formalin-inactivated red-spotted grouper nervous necrosis virus (rgnnv), a betanodavirus originally isolated in japan. fish produced neutralizing antibodies at high titre levels from days 10 (mean titre 1:480) to 116 (1:1280), with the highest titre at day 60 post-vaccination (1:4480). when fish were challenged with the homologous rgnnv at day 54 post-vaccination, there were no mortaliti ...200919364387
outbreak of betanodavirus infection in tilapia, oreochromis niloticus (l.), in fresh water.a betanodavirus associated with a massive mortality was isolated from larvae of tilapia, oreochromis niloticus, maintained in fresh water at 30 degrees c. histopathology revealed vacuolation of the nervous system, suggesting an infection by a betanodavirus. the virus was identified by indirect fluorescent antibody test in the ssn1 cell line and further characterized by sequencing of a pcr product. sequencing of the t4 region of the coat protein gene indicated a phylogenetic clustering of this is ...200919500206
comparative analysis of both genomic segments of betanodaviruses isolated from epizootic outbreaks in farmed fish species provides evidence for genetic reassortment.sequencing of the full coding region of both genomic segments of seven betanodavirus strains isolated from different farmed species in spain and portugal revealed that six were reassortants, exhibiting a red-spotted grouper nervous necrosis virus (rgnnv)-type rna1 and a striped jack nervous necrosis virus (sjnnv)-type rna2. analysis of sequences of reassortant strains at both the genomic and protein levels revealed the existence of differences compared with type strains of both genotypes. these ...200919710256
fish immunization using a synthetic double-stranded rna poly(i:c), an interferon inducer, offers protection against rgnnv, a fish nodavirus.viral nervous necrosis (vnn), caused by a fish nodavirus, is one of the most serious fish diseases worldwide. here we report a unique vaccination method in sevenband grouper epinephelus septemfasciatus using a synthetic double-stranded rna polyinosinic polycytidylic acid (poly(i:c)), an interferon inducer, followed by challenge with a live fish nodavirus. fish injected with poly(i:c) at 200 microg fish(-1) were highly protected from artificial challenge with red-spotted grouper nervous necrosis ...200919326792
identification of rna regions that determine temperature sensitivities in betanodaviruses.betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense rna genomes. the larger genomic segment, rna1 (~3.1 kb), encodes an rna-dependent rna polymerase (protein a), and the smaller genomic segment rna2 (~1.4 kb) codes for the coat protein. these viruses can be classified into four genotypes, designated striped jack nervous necrosis virus (sjnnv), redspotted grouper nervous necrosis virus (rgnnv), tiger puffer nervous necrosis virus (tpnnv), ...201020582605
rgnnv induces mitochondria-mediated cell death via newly synthesized protein dependent pathway in fish cells.the rna nervous necrosis virus induces necrotic cell death in fish; however, the molecular mechanism remains unknown. in this study, we demonstrated that beta-nodavirus-induced mitochondria-mediated dependent cell death is through newly synthesized protein dependent pathway in replication cycle. we determined that newly synthesized protein dependent pathway is required for red-spotted grouper nervous necrosis virus (rgnnv)-induced cell death. uv irradiation of the virus effectively blocked viral ...201020641154
detection of red-spotted grouper nervous necrosis virus by loop-mediated isothermal amplification.red-spotted grouper nervous necrosis virus (rgnnv) causes high mortality in marine fish larvae cultured in china. to control better an outbreak of this virus, a rapid, specific and sensitive detection method based on loop-mediated isothermal amplification (lamp) was developed. a set of four primers, two outer and two inner, was designed from rgnnv genome rna. the lamp reaction mix was optimized. the method was specific as no cross-reaction was observed between white spot syndrome virus, koi herp ...201019755132
optimisation and validation of a real-time reverse transcriptase-polymerase chain reaction assay for detection of betanodavirus.a rt-qpcr assay that was developed and optimised for detection of betanodaviruses was validated for use as a diagnostic test for viral nervous necrosis disease of fish. four betanodavirus genotypes were detected but the sensitivity was greatest for redspotted grouper nervous necrosis virus (rgnnv). the analytical sensitivity was 10-1000-fold greater than that of a nested rt-pcr assay and the limit of detection was <0.4 tcid(50) units per reaction. the assay was highly repeatable (standard deviat ...201019891987
complete sequencing of tunisian redspotted grouper nervous necrosis virus betanodavirus capsid gene and rna-dependent rna polymerase gene.finfish nodaviruses (betanodaviruses) can cause highly destructive infections in numerous species of farmed marine fish larvae and juveniles worldwide. the betanodavirus genome consists of two single-stranded positive-sense rna molecules (rna1 and rna2). the virus can be classified into four genotypes based on the partial sequences of the coat protein (cp) gene (t2 and t4 regions). currently, genomic sequence information for rna1 regions of rna2 outside of t2 and t4 is less well documented. this ...201019912455
protective immunity against viral nervous necrosis (vnn) in brown-marbled grouper (epinephelus fuscogutattus) following vaccination with inactivated betanodavirus.viral nervous necrosis (vnn) caused by betanodaviruses has been recently implicated in serious mortalities of groupers in the grow-out culture system. a safe and effective vaccine against this disease is urgently needed. this study demonstrates that a single intramuscular vaccination with formalin-inactivated philippine strain of piscine betanodavirus (genotype: redspotted grouper nervous necrosis virus; rgnnv) induces potent immune responses and substantial protective immunity against an intram ...201020018247
susceptibilities of medaka (oryzias latipes) cell lines to a betanodavirus.betanodaviruses, members of the family nodaviridae, have bipartite, positive-sense rna genomes and are the causal agents of viral nervous necrosis in many marine fish species. recently, the viruses were shown to infect a few freshwater fish species including a model fish medaka (oryzias latipes). although virological study using cultured medaka cells would provide a lot of insight into virus-fish interactions in molecular aspects, no such cells have yet been tested for virus susceptibility.201020624282
betanodavirus b2 causes atp depletion-induced cell death via mitochondrial targeting and complex ii inhibition in vitro and in vivo.the betanodavirus non-structural protein b2 is a newly discovered necrotic death factor with a still unknown role in regulation of mitochondrial function. in the present study, we examined protein b2-mediated inhibition of mitochondrial complex ii activity, which results in atp depletion and thereby in a bioenergetic crisis in vitro and in vivo. expression of protein b2 was detected early at 24 h postinfection with red-spotted grouper nervous necrosis virus in the cytoplasm. later b2 was found i ...201020870718
a combined rt-pcr and dot-blot hybridization method reveals the coexistence of sjnnv and rgnnv betanodavirus genotypes in wild meagre (argyrosomus regius).to detect the possible coexistence of striped jack nervous necrosis virus (sjnnv) and red-spotted grouper nervous necrosis virus (rgnnv) genotypes in a single fish, a methodology based on the combination of pcr amplification and blot hybridization has been developed and applied in this study.201020497277
roles of cysteines cys115 and cys201 in the assembly and thermostability of grouper betanodavirus particles.the virus-like particle (vlp) assembled from capsid subunits of the dragon grouper nervous necrosis virus (dgnnv) is very similar to its native t = 3 virion. in order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of dgnnv virions, we recombinantly cloned mutant vlps by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all s-s bonds. the mutant vlps of c187a and c331a ...201020446029
development and partial characterization of new marine cell line from brain of asian sea bass lates calcarifer for virus isolation.a new cell line, asian sea bass brain (asbb), was derived from the brain tissue of asian sea bass lates calcarifer. this cell line was maintained in leibovitz l-15 media supplemented with 10% fetal bovine serum (fbs). the asbb cell line was subcultured more than 60 times over a period of 15 mo. the asbb cell line consists predominantly of fibroblastic-like cells and was able to grow at temperatures between 20°c and 30°c with an optimum temperature of 25°c. the growth rate of these cells increase ...201021082288
betanodavirus up-regulates chaperone grp78 via er stress: roles of grp78 in viral replication and host mitochondria-mediated cell death.whether viral pathogens that induce er stress responses benefit the host or the virus remains controversial. in this study we show that betanodavirus induced er stress responses up-regulate grp78, which regulates the viral replication and host cellular mitochondrial-mediated cell death. betanodavirus (redspotted grouper nervous necrosis virus, rgnnv) infection resulted in the following increased er stress responses in fish gf-1 grouper fin cells: (1) ire-1 and atf-6 sensors at 48 h post-infectio ...201121170590
optimization of betanodavirus culture and enumeration in striped snakehead fish cells.an optimized culture method for detection of infection of fish with the red spotted grouper nervous necrosis virus (rgnnv) genotype of betanodavirus in striped snakehead (ssn-1, channa striatus) cells is described. inoculation of fish tissue homogenates at the same time or within 4 hr of seeding the ssn-1 cells was as sensitive as the method recommended by the world organization for animal health, where homogenates were adsorbed onto an established cell monolayer. such modification halved the ti ...201121908274
genome analysis of betanodavirus from cultured marine fish species in malaysia.betanodavirus is the causative agent of the viral nervous necrosis (vnn) or viral encephalopathy and retinopathy disease in marine fish. this disease is responsible for most of the mass mortalities that occurred in marine fish hatcheries in malaysia. the genome of this virus consists of two positive-sense rna molecules which are the rna1 and rna2. the rna1 molecule contains the rdrp gene which encodes for the rna-dependent rna polymerase and the rna2 molecule contains the cp gene which encodes f ...201122051446
Betanodavirus induces oxidative stress-mediated cell death that prevented by anti-oxidants and zfcatalase in fish cells.The role of oxidative stress in the pathogenesis of RNA nervous necrosis virus infection is still unknown. Red-spotted grouper nervous necrosis virus (RGNNV) induced free radical species (ROS) production at 12-24 h post-infection (pi; early replication stage) in fish GF-1 cells, and then at middle replication stage (24-48 h pi), this ROS signal may upregulate some expressions of the anti-oxidant enzymes Cu/Zn SOD and catalase, and eventually expression of the transcription factor Nrf2. Furthermo ...201121991373
tissue distribution of red spotted grouper nervous necrosis virus (rgnnv) genome in experimentally infected juvenile european seabass (dicentrarchus labrax).the distribution of viral genome in the tissues of juvenile european seabass (dicentrarchus labrax) during the course of a red spotted grouper nervous necrosis virus (rgnnv) infection has not yet been described. the present study addresses this and indicates which target organs may be involved in viral replication. this information should enable more accurate detection of virus in asymptomatic carriers, and in turn help to control the spread of the disease. the aim of this study was to examine t ...201121783331
distribution of red-spotted grouper nervous necrosis virus (rgnnv) antigens in nervous and non-nervous organs of european seabass(dicentrarchus labrax) during the course of an experimental challenge.the distribution of red-spotted grouper nervous necrosis virus (rgnnv) antigens was examined by immunohistochemistry in the nervous and non-nervous organs of juvenile european seabass (dicentrarchus labrax) during the course of an intramuscular infection. histological changes resulting from the infection were evaluated from 3 days to 2 months post-infection. the specific antibody response was also studied 2 months post-challenge. viral proteins were present throughout the experimental period in ...201223271176
betanodavirus of marine and freshwater fish: distribution, genomic organization, diagnosis and control measures.the family nodaviridae include the genera alphanodavirus and the betanodavirus which are non-enveloped, single stranded rna viruses. alphanodavirus include the insect viruses while betanodavirus include species that are responsible for causing disease outbreaks in hatchery-reared larvae and juveniles of a wide variety of marine and freshwater fish throughout the world and has impacted fish culture over the last decade. according to international committee on taxonomy of viruses, the genus betano ...201223997435
development of a reverse transcription loop-mediated isothermal amplification assay for detecting nervous necrosis virus in olive flounder paralichthys olivaceus.in this study, a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (nnv) in olive flounder, paralichthys olivaceus, in korea. a set of six specific primers was designed to target the rna 2 gene encoding the coat protein of korean nnv strains. the rt-lamp reaction successfully detected nnv after 30 min at 65 degrees c. when the sensitivities among rt-lamp, rt-pcr, and nested rtpc ...201222580323
complete sequence of a viral nervous necrosis virus (nnv) isolated from red-spotted grouper (epinephelus akaara) in china.a nodavirus isolated from red-spotted grouper (epinephelus akaara) larvae in china has been subjected to genome analysis. the full-length genome sequences of rna1 and rna2 were determined, and the 5'-non-coding region (ncr) and 3'ncr sequences were determined by 5' rapid amplification of cdna ends (race) and 3'race. rna1 is 3,103 nt in length and contains a 982-amino-acid open reading frame (orf) encoding protein a with a calculated molecular mass of 110.74 kda. rna2 is 1,433 nt long and contain ...201222270757
Molecular epidemiology and evolutionary dynamics of betanodavirus in southern Europe.Viral encephalopathy and retinopathy (VER) is one of the most devastating diseases for marine aquaculture, and similarly represents a threat to wild fish populations because of its high infectivity and broad host range. Betanodavirus, the causative agent of VER, is a small non-enveloped virus with a bipartite RNA genome comprising the RNA1 and RNA2 segments. We partially sequenced both RNA1 and RNA2 from 120 viral strains isolated from 2000 to 2009 in six different countries in Southern Europe. ...201222036789
viral susceptibility, transfection and growth of spb--a fish neural progenitor cell line from the brain of snubnose pompano, trachinotus blochii (lacépède).this study investigates the susceptibilities of the spb cell line to fish viruses including giant seaperch iridovirus (gsiv-k1), red sea bream iridovirus (rsiv-ku), grouper nervous necrosis virus (gnnv-k1), chum salmon reovirus (csv) and eel herpesvirus (hva). gsiv-k1, rsiv-ku and csv replicated well in spb cells, with a significant cytopathic effect and virus production. however, the cells were hva and gnnv refractory. to examine the ability of spb cells to stably express foreign protein, expre ...201323305502
first report of viral nervous necrosis-induced mass mortality in hatchery-reared larvae of clownfish, amphiprion sebae bleeker.frequent mortality was observed in the larval rearing facility of marine clownfish, amphiprion sebae bleeker. mortality resulted in 80% loss of larval stock. moribund larvae showed clinical signs typical of viral nervous necrosis, such as uncoordinated, corkscrew-like swimming behaviour, hypersensitivity to stimuli, darkening of body and assembly into large groups, similar to bunches of grapes. the aetiology of the disease was confirmed by gross observation of clinical signs, histopathology and ...201324117972
molecular cloning, expression and functional analysis of isg15 in orange-spotted grouper, epinephelus coioides.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 im ...201323403156
molecular epidemiology of betanodaviruses isolated from sea bass and sea bream cultured along the tunisian coasts.viral nervous necrosis (vnn) is a serious viral disease affecting farmed sea bass (dicentrarchus labrax). only scarce molecular data are available on the disease-causing betanodavirus populations in tunisia. therefore, we carried out the first molecular survey of betanodaviruses in farmed sea bass and sea bream (sparus aurata) along the tunisian coasts. among 81 samples from five farms, 20 tested positive with rt-pcr, not only in clinical cases but also in asymptomatic fish before and during out ...201323288625
genetic characterization of betanodavirus isolates from asian seabass lates calcarifer (bloch) in india.betanodavirus has been detected in asian seabass in india. molecular characterization of the isolates on the basis of the full-length viral rna2 sequence was performed. subsequent phylogenetic analysis with sequences from members of the four species in the genus betanodavirus revealed that the present isolates are closely related to members of the species redspotted grouper nervous necrosis virus. the analysis also revealed that the rna2 sequence was not responsible for acute symptoms in seabass ...201323224758
medaka haploid embryonic stem cells are susceptible to singapore grouper iridovirus as well as to other viruses of aquaculture fish species.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 la ...201323828270
chromatographically-purified capsid proteins of red-spotted grouper nervous necrosis virus expressed in saccharomyces cerevisiae form virus-like particles.nervous necrosis viruses (nnvs) cause mass mortality of marine fish, leading to large economic losses for aquaculturists. a promising vaccine candidate for preventing nnv infection is the nnv virus-like particle (vlp), which is a structure resulting from assembly of recombinant nnv capsid protein. nnv capsid proteins have been expressed in insect cells and the escherichia coli expression system, and purified by non-scalable protocols such as ultracentrifugation on sucrose and cesium chloride den ...201323537792
comparative pathogenicity study of ten different betanodavirus strains in experimentally infected european sea bass, dicentrarchus labrax (l.).viral encephalopathy and retinopathy (ver), otherwise known as viral nervous necrosis (vnn), is a severe pathological condition caused by rna viruses belonging to the nodaviridae family, genus betanodavirus. the disease, described in more than 50 fish species worldwide, is considered as the most serious viral threat affecting marine farmed species in the mediterranean region, thus representing one of the bottlenecks for further development of the aquaculture industry. to date, four different gen ...201423662921
isolation and characterization of a nodavirus associated with mass mortality in asian seabass (lates calcarifer) from the west coast of india.a fish nodavirus was detected in the juveniles of asian seabass (lates calcarifer) during a massive outbreak in the seabass cage culture farm located in the south west coast of india. the clinical signs of the disease included anorexia, inflated abdomen, exophthalmia, darkening of the whole body, erratic swimming and cork-screw type movement followed by death. the dead and the moribund fish were analyzed for nodavirus by reverse transcriptase-polymerase chain reaction (rt-pcr) using specific pri ...201425674617
oral immunization with whole yeast producing viral capsid antigen provokes a stronger humoral immune response than purified viral capsid antigen.weak antibody responses to protein antigens after oral immunization remain a serious problem. yeasts have a rigid cell wall and are inherently resistant to harsh conditions, suggesting that recombinant antigens made in yeast could have a greater chance of making contact with the immune cells of the gastrointestinal (gi) tract in intact form. we compared antibody responses to oral immunization with purified recombinant antigen, used in the conventional manner, and responses to whole recombinant y ...201424251903
complete genome sequence of a betanodavirus isolated from half-smooth tongue sole (cynoglossus semilaevis).betanodavirus, commonly called nervous necrosis virus (nnv) of fish, has emerged as a major constraint on marine aquaculture worldwide. here, we report the complete genome sequence of a betanodavirus (strain cscn128) isolated from diseased half-smooth tongue sole (cynoglossus semilaevis) in china. the genome sequence of strain cscn128 shares ≥98.7% similarity with seven-band grouper nervous necrosis virus from japan. phylogenetic analysis indicates that strain cscn128 belongs to the red-spotted ...201425523771
influence of temperature on mx gene expression profiles and the protection of sevenband grouper, epinephelus septemfasciatus, against red-spotted grouper nervous necrosis virus (rgnnv) infection after poly (i:c) injection.influence of temperature on the susceptibility of fish against virus infection has been studied for a decade. recent reports have been shown the effects of rearing temperatures on the fish immune system against virus infection. however, the roles of temperature in regulation of type i interferon (ifn) system has not yet been investigated. thus, the effects of temperature on type i ifn response were investigated in this study using poly (i:c) injection in sevenband grouper and mx gene was used as ...201425107694
development of simple, rapid and sensitive detection assay for grouper nervous necrosis virus using real-time loop-mediated isothermal amplification.a quantitative rapid detection method based on loop-mediated isothermal amplification has been developed for red-spotted grouper nervous necrosis virus (rgnnv). the nested polymerase chain reaction (pcr) assay is the mainstream inspection of the brooder in the hatchery. in this study, a real-time loop-mediated isothermal amplification (lamp) method has been applied for rgnnv detection, known as a high-speed gene amplification procedure. of the three temperatures (60 °c, 63 °c and 65 °c) attempte ...201425073724
isolation and characterization of tumor necrosis factor receptor-associated factor 6 (traf6) from grouper, epinephelus tauvina.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-tr ...201424811008
crystallization and x-ray diffraction of virus-like particles from a piscine betanodavirus.dragon grouper nervous necrosis virus (dgnnv), a member of the genus betanodavirus, causes high mortality of larvae and juveniles of the grouper fish epinephelus lanceolatus. currently, there is no reported crystal structure of a fish nodavirus. the dgnnv virion capsid is derived from a single open reading frame that encodes a 338-amino-acid protein of approximately 37 kda. the capsid protein of dgnnv was expressed to form virus-like particles (vlps) in escherichia coli. the vlp shape is t = 3 q ...201425084387
ghsc70 is involved in the cellular entry of nervous necrosis virus.nervous necrosis virus (nnv) is a devastating pathogen of cultured marine fish and has affected more than 40 fish species. nnv belongs to the betanodaviruses of nodaviridae and is a nonenveloped icosahedral particle with 2 single-stranded positive-sense rnas. to date, knowledge regarding nnv entry into the host cell remains limited, and no nnv-specific receptor protein has been published. using grouper fin cell line gf-1 and purified nnv capsid protein in a virus overlay protein binding assay (v ...201425320288
identification of orange-spotted grouper (epinephelus coioides) interferon regulatory factor 3 involved in antiviral immune response against fish rna virus.interferon regulatory factor 3 (irf3) is an important transcription factor which regulates the expression of interferon (ifn) and ifn-stimulated genes (isgs) following virus recognition. in this study, a novel irf3 gene was cloned from grouper epinephelus coioides (ecirf3) and its effects against singapore grouper iridovirus (sgiv) and red spotted grouper nervous necrosis virus (rgnnv) was investigated. the full-length of ecirf3 cdna was composed of 2513 bp and encoded a polypeptide of 458 amino ...201525463297
first generic one step real-time taqman rt-pcr targeting the rna1 of betanodaviruses.the detection of betanodavirus genomic components is a major issue for diagnostics and control of viral nervous necrosis (vnn), a devastating disease affecting fish worldwide. despite a number of published molecular-based tests, most of them targeting the rna2 molecule of the virus, diagnostics is still a challenge due to the high genetic diversity within this genus. in the present study, a new one-step real-time rt-pcr (rrt-pcr), targeting rna1 of most genotypes of betanodaviruses, was proposed ...201525311184
infections of nervous necrosis virus in wild and cage-reared marine fish from south china sea with unexpected wide host ranges.the concerns about the impact of the nervous necrosis virus (nnv) infections in wild fish have been raised. this paper presents the results of quarterly surveys of nnv in wild and cage-reared marine fish from south china sea. samples of 892 wild fish belonging to 69 species and 381 cage-reared fish belonging to 11 species were collected and were detected by seminested pcr and nested pcr. in the case of seminested pcr, the positive signal was detected in 3.0% and 3.1% samples of wild and cage-rea ...201524943478
establishment of a new cell line from the heart of giant grouper, epinephelus lanceolatus (bloch), and its application in toxicology and virus susceptibility.a new marine fish cell line, derived from the heart of giant grouper, epinephelus lanceolatus (bloch), was established and characterized. the cell line was designated as elgh and subcultured with more than 60 passages. the elgh cells were mainly composed of fibroblast-like cells and multiplied well in leibovitz's l-15 medium supplemented with 10% foetal bovine serum (fbs) at 28 °c. chromosome analysis indicated that the modal chromosome number was 48. the fluorescent signals were detected in elg ...201524372271
genetic characterization of a betanodavirus isolated from a clinical disease outbreak in farm-raised tilapia oreochromis niloticus (l.) in thailand.betanodavirus infection was diagnosed in larvae of farm-raised tilapia oreochromis niloticus (l.), in central thailand. extensive vacuolar degeneration and neuronal necrosis were observed in histological sections with positive immunohistochemical staining for betanodavirus. molecular phylogenetic analysis was performed based on the nucleotide sequences (1333 bases) of the capsid protein gene. the virus strain was highly homologous (93.07-93.88%) and closely related to red-spotted grouper nervous ...201524164433
in vitro and in vivo characterization of molecular determinants of virulence in reassortant betanodavirus.we previously reported that betanodavirus reassortant strains [redspotted grouper nervous necrosis virus/striped jack nervous necrosis virus (sjnnv)] isolated from senegalese sole (solea senegalensis) exhibited a modified sjnnv capsid amino acid sequence, with changes at aa 247 and 270. in the current study, we investigated the possible role of both residues as putative virulence determinants. three recombinant viruses harbouring site-specific mutations in the capsid protein sequence, rss160.032 ...201525626678
involvement of fish signal transducer and activator of transcription 3 (stat3) in nodavirus infection induced cell death.the janus kinase (jak)-signal transducer and activator of transcription (stat) signaling pathway is an important signaling pathway activated by interferons in response to virus infection. fish stat3 has been demonstrated to be involved in singapore grouper iridovirus (sgiv) infection and virus induced paraptosis, but its effects on the replication of other fish viruses still remained uncertain. here, the roles of grouper stat3 (ec-stat3) in red spotted grouper nervous necrosis virus (rgnnv) infe ...201525555814
crystal structures of a piscine betanodavirus: mechanisms of capsid assembly and viral infection.betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. the structure of a t = 3 grouper nervous necrosis virus-like particle (gnnv-lp) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 å resolution. each capsid protein (cp) shows three major domains: (i) the n-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (s-domain), a jelly-roll structure; and (iii) the protrusion domain (p-domain ...201526491970
establishment and application of cross-priming isothermal amplification coupled with lateral flow dipstick (cpa-lfd) for rapid and specific detection of red-spotted grouper nervous necrosis virus.red-spotted grouper nervous necrosis virus (rgnnv) is an important pathogen that causes diseases in many species of fish in marine aquaculture. the larvae and juveniles are more easily infected by rgnnv and the cumulative mortality is as high as 100 % after being infected with rgnnv. this virus imposes a serious threat to aquaculture of grouper fry. this study aimed to establish a simple, accurate and highly sensitive method for rapid detection of rgnnv on the spot.201526409445
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