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
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
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
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
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
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
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
betanodavirus induces phosphatidylserine exposure and loss of mitochondrial membrane potential in secondary necrotic cells, both of which are blocked by bongkrekic 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
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
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
molecular detection of betanodaviruses from apparently healthy wild marine 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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
developing immunological methods for detecting macrobrachium rosenbergii nodavirus and extra small virus using a recombinant protein preparation.macrobrachium rosenbergii nodavirus (mrnv) and extra small virus (xsv) have been identified as the causative agents for white tail disease (wtd) of m. rosenbergii. in this study, the gene sequences encoding mrnv and xsv capsid proteins were separately ligated into the pgex-4t-3 expression vector and transformed into escherichia coli. after induction, glutathione-s-transferase (gst)-tagged mrnv and xsv fusion proteins were obtained with molecular masses of 68 and 43 kda, respectively. specific po ...201626263892
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
inoculation of balb/c mice with fish-pathogenic 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
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
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
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
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
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
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
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
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
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
detection of red-spotted grouper nervous necrosis virus by loop-mediated isothermal 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
antiviral function of tachyplesin i against iridovirus and nodavirus.antimicrobial peptides (amps) are ubiquitously found in living organisms and are an important component in innate immune response. tachyplesin i is a potent antimicrobial peptide isolated from the hemocytes of the horseshoe crab, tachypleus tridentatus. previous studies have shown that the 17-residue peptide exhibits a wide spectrum of antimicrobial activity against gram-negative and gram-positive bacteria, fungi, protozoa, and viruses. however, the efficiencies and defense mechanisms of the tac ...201627633674
a formalin-inactivated immunogen against viral encephalopathy and retinopathy (ver) disease in european sea bass (dicentrarchus labrax): immunological and protection effects.the european sea bass (dicentrarchus labrax) is an important farmed fish species in the mediterranean area, very sensitive to the infection by encephalopathy and retinopathy virus (verv), or betanodavirus, which causes massive mortalities. effective vaccines to fight the pathology are not yet available and in this work we describe a promising intraperitoneal immunization route against verv of sea bass juveniles. we performed intraperitoneal and immersion immunization trials with a verv (isolate ...201627590537
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
phylogenetic analysis of betanodavirus isolates from australian 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
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
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
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
optimization of betanodavirus culture and enumeration in striped snakehead fish 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
grouper trim13 exerts negative regulation of antiviral immune response against nodavirus.the tripartite motif (trim)-containing proteins have attracted particular attention to their multiple functions in different biological processes. trim13, a member of the trim family, is a ring domain-containing e3 ubiquitin ligase which plays critical roles in diverse cellular processes including cell death, cancer and antiviral immunity. in this study, a trim13 homolog from orange spotted grouper, epinephelus coioides (ectrim13) was cloned and characterized. the full-length of ectrim13 cdna en ...201627235367
water temperature affects pathogenicity of different betanodavirus genotypes in experimentally challenged dicentrarchus labrax.betanodaviruses are the causative agents of a highly infectious disease of fish known as viral nervous necrosis (vnn). to date, 4 different nervous necrosis virus (nnv) genotypes have been described, but natural reassortant viruses have also been detected, which further increase viral variability. water temperature plays an important role in determining the appearance and the severity of vnn disease. we assessed the effect of temperature (20°, 25° and 30°c) on mortality and virus load in the bra ...201627225206
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
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
age dependency of nervous necrosis virus infection in barramundi lates calcarifer (bloch).age-dependent susceptibility to nervous necrosis virus (nnv) was demonstrated for barramundi (lates calcarifer). the experiment used juvenile barramundi produced from a single spawning that were challenged consecutively by immersion with a redspotted grouper nervous necrosis virus (rgnnv) isolate. the dose and environmental conditions (35 ppt salinity and 30 °c) were constant. fish and water were sampled longitudinally for histopathology and rt-qpcr analysis to examine the evolution of the disea ...201728117491
antiviral function of grouper mda5 against iridovirus and nodavirus.melanoma differentiation-associated gene 5 (mda5) is a critical member of retinoic acid-inducible gene i (rig-i)-like receptor (rlr) family which can recognize viral rna and enhances antiviral response in host cells. in this study, a mda5 homolog from orange spotted grouper (epinephelus coioides) (ecmda5) was cloned, and its roles on grouper virus infection were characterized. the full-length ecmda5 cdna encoded a polypeptide of 982 amino acids with 74% identity with mda5 homolog from rock bream ...201627050314
establishment and characterization of a cell line from the head kidney of golden pompano trachinotus ovatus and its application in toxicology and virus susceptibility.a cell line derived from the head kidney of golden pompano trachinotus ovatus (tohk) was established and characterized in this study. the tohk cells grew most rapidly at 28° c and the optimum foetal bovine serum concentration in l-15 medium was 10%. the tohk cells have a diploid chromosome number of 2n = 54. the transfection efficiency of tohk cells was 7·5% at the 15th passage and 72% at the 40th passage. the transfection efficiency in tohk cells was high, so these cells are suitable for foreig ...201728271507
characterization of micrornas in orange-spotted grouper (epinephelus coioides) fin cells upon red-spotted grouper nervous necrosis virus infection.nervous necrosis virus (nnv), one of the most prevalent fish pathogens, has caused fatal disease of viral nervous necrosis (vnn) in many marine and freshwater fishes, and resulted in heavy economic losses in aquaculture industry worldwide. however, the molecular mechanisms underlying the pathogenicity of nnv remain elusive. in this study, the expression profiles of microrna (mirna) were investigated in grouper fin (gf-1) cells infected with red-spotted grouper nervous necrosis virus (rgnnv) via ...201728232192
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
generation and characterization of novel dna aptamers against coat protein of grouper nervous necrosis virus (gnnv) with antiviral activities and delivery potential in grouper cells.nervous necrosis virus (nnv) infected larvae and juveniles of more than 50 fish species, resulting in mortality rates of greater than 95%. however, there is no efficient method to control nnv infections. aptamers generated by selective evolution of ligands by exponential enrichment (selex) are short, single-stranded nucleic acid oligomers. they display a high degree of affinity and specificity for many targets, such as viruses and viral proteins. in this study, three novel dna aptamers (a5, a10, ...201626892075
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
fish trim39 regulates cell cycle progression and exerts its antiviral function against iridovirus and nodavirus.the tripartite motif (trim)-containing proteins exert important immune regulatory roles through regulating different signaling pathways in response to different stimuli. trim39, a member of the trim family, is a ring domain-containing e3 ubiquitin ligase which could regulate cell cycle progression and apoptosis. however, the antiviral activity of trim39 is not explored. here, a trim39 homolog from grouper, epinephelus coioides (ectrim39) was cloned, and its effects on cell cycle progression and ...201626784918
molecular cloning and expression analysis of small ubiquitin-like modifier (sumo) genes from grouper (epinephelus coioides).small ubiquitin-like modifier (sumo) is a group of proteins binding to lysine residues of target proteins and thereby modifying their stability, activity and subcellular localization. in the present study, two sumo homolog genes (ecsumo1 and ecsumo2) from grouper (epinephelus coioides) were cloned and characterized. the full-length sequence of ecsumo1 was 749 bp in length and contained a predicted open reading frame of 306 bp encoding 101 amino acids with a molecular mass of 11.34 kda. the full- ...201626616235
quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass dicentrarchus labrax.viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in europe. this work describes a reliable and sensitive indirect elisa assay to detect betanodavirus in biological samples using a polyclonal antiserum (pab 283) against the 283/i09 virus strain, the most common red-spotted grouper nervous necrosis virus (rgnnv) genotype in the mediterranean are ...201626610431
inhibition of nervous necrosis virus propagation by fish 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
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
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
expression profile analysis of mir-221 and mir-222 in different tissues and head kidney cells of cynoglossus semilaevis, following pathogen infection.half-smooth tongue sole (cynoglossus semilaevis) is an important marine commercial fish species in china, which suffers from widespread disease outbreaks. recently, in this regard, our group identified immune-related micrornas (mirnas) of c. semilaevis following vibrio anguillarum infection. furthermore, mirna microarray was utilized to characterize the immune roles of important mirna candidates in response to bacterial infection. therefore, in the present study, we characterized mir-221 and mir ...201626420296
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
epidemiological characterization of vnnv in hatchery-reared and wild marine fish on hainan island, china, and experimental infection of golden pompano (trachinotus ovatus) juveniles.the current epidemiological situation of viral nervous necrosis virus (vnnv) on hainan island was investigated. a total of 490 hatchery-reared fish and 652 wild fish were sampled for vnnv detection from march 2013 to may 2014. positive detection rates of 84.53% (153/181) and 0.97 % (3/309) were obtained in diseased and healthy hatchery-reared samples, respectively, by conventional rt-pcr. however, using more-sensitive nested rt-pcr, the positive detection rates in healthy hatchery-reared fish re ...201526350771
complete genome sequence of a fish nervous necrosis virus isolated from sea perch (lateolabrax japonicus) in china.we sequenced and analyzed the complete genome of a fish nervous necrosis virus isolated from diseased sea perch (lateolabrax japonicus) in guangdong province, china. the virus genome contains rna1 (3,103 bp) and rna2 (1,433 bp). phylogenetic analysis shows that the virus belongs to the redspotted grouper nervous necrosis virus genotype of betanodavirus.201526044411
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
expression profiling analysis of the microrna response of cynoglossus semilaevis to vibrio anguillarum and other investigate the roles of micrornas (mirna) of cynoglossus semilaevis in response to vibrio anguillarum that were previously identified using high-throughput sequencing, microarray analyses was performed on three small rna libraries (cg, nosg, and hosg) prepared from c. semilaevis immune tissues. in total, of 1279 designed probes, 739 (57.78 %) were detectable. the expression levels of these mirnas were analyzed using pairwise comparisons among the three libraries, and a total of 99 mirnas wer ...201525715708
identification and characterization of the melanoma differentiation - associated gene 5 in sea perch, lateolabrax japonicus.the rig-i-like receptors family is a group of cytosolic rna helicase proteins that can recognize viral rna via binding to pathogen associated molecular pattern motifs within rna ligands. a novel vertebrate rlr counterpart named ljmda5 was firstly identified from the marine fish sea perch lateolabrax japonicus in this study. the full-length cdna of ljmda5 is 3750 bp and encodes a polypeptide of 988 amino acids, containing two n-terminal tandem caspase activation and recruitment domains, a dexh (a ...201627039216
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
establishment and characterization of a mid-kidney cell line derived from golden pompano trachinotus ovatus, a new cell model for virus pathogenesis and toxicology studies.golden pompano trachinotus ovatus, a popularly cultured and commercially important marine fish worldwide, has been recognized as a promising candidate for mariculture. however, outbreaks of infectious bacterial or viral diseases and environmental deterioration have led to great economic losses in t. ovatus aquaculture recently. in our research, we established a new mid-kidney cell line, designated as tok, from golden pompano, t. ovatus. the optimized growth temperature and working concentration ...201627981423
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
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
transcriptomic profiles of striped snakehead fish cells (ssn-1) infected with red-spotted grouper nervous necrosis virus (rgnnv) with an emphasis on apoptosis pathway.nervous necrosis virus (nnv), the causative agent of viral nervous necrosis (vnn) disease, has caused mass mortality of cultured marine and freshwater fish worldwide, resulting in enormous economic losses in the aquaculture industry. however, the molecular mechanisms underlying the pathogenicity of nnv are still poorly understood. in this study, the transcriptomic profiles of striped snakehead fish (channa striatus) cells (ssn-1) infected with red-spotted grouper nnv (rgnnv) were investigated us ...201727914997
identification of sea perch (lateolabrax japonicus) ribonucleoprotein ptb-binding 1 involved in antiviral immune response against rgnnv.rig-i-like receptors (rlrs) can recognize viral rna and initiate innate antiviral response. in earlier studies, we demonstrated that rlrs were implicated in the antiviral immunity against rgnnv in the seawater fish sea perch (lateolabrax japonicus). however, potential regulators of rlrs-mediated signaling pathways involved in rgnnv infection remain unclear. in this study, a novel ribonucleoprotein ptb-binding 1 (raver1) of sea perch (ljraver1) was identified for the first time. the cdna of ljrav ...201727876623
understanding the interaction between betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy.over the last three decades, the causative agent of viral encephalopathy and retinopathy (ver) disease has become a serious problem of marine finfish aquaculture, and more recently the disease has also been associated with farmed freshwater fish. the virus has been classified as a betanodavirus within the family nodaviridae, and the fact that betanodaviruses are known to affect more than 120 different farmed and wild fish and invertebrate species, highlights the risk that betanodaviruses pose to ...201626997200
cell culture isolation of piscine nodavirus (betanodavirus) in fish-rearing seawater.piscine nodavirus (betanodavirus) is the causative agent of viral nervous necrosis (vnn) in a variety of cultured fish species, particularly marine fish. in the present study, we developed a sensitive method for cell culture isolation of the virus from seawater and applied the method to a spontaneous fish-rearing environment. the virus in seawater was concentrated by an iron-based flocculation method and subjected to isolation with e-11 cells. a real-time reverse transcriptase pcr (rt-pcr) assay ...201626896128
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
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
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
establishment and characterization of a novel cell line from the brain of golden pompano (trachinotus ovatus).golden pompano is a commercially important marine fish that is widely cultured in china, japan, and southeast asian countries but has been seriously threatened by pathogen. a novel cell line (togb) derived from the brain of golden pompano trachinotus ovatus was established and characterized in this study. togb cell line showed high virus susceptibility, especially grouper nervous necrosis virus (gnnv) and singapore grouper iridovirus (sgiv). as one of the most devastating viruses in marine fish ...201626822430
talens-mediated gene disruption of myostatin produces a larger phenotype of medaka with an apparently compromised immune system.although myostatin, a suppressor of skeletal muscle development and growth, has been well studied in mammals, its function in fish remains unclear. in this study, we used a popular genome editing tool with high efficiency and target specificity (talens; transcription activator-like effector nucleases) to mutate the genome sequence of myostatin (mstn) in medaka (oryzias latipes). after the talen pair targeting olmyostatin was injected into fertilized medaka eggs, mutant g0 fish carrying different ...201626578247
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
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
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
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