Viral nervous necrosis (VNN) is a worldwide disease among teleost fish. In the mainland of China, VNN was first identified in 2 species of hatchery-reared groupers, Epinephelus akaara and E. coioides. In the present s...Viral nervous necrosis (VNN) is a worldwide disease among teleost fish. In the mainland of China, VNN was first identified in 2 species of hatchery-reared groupers, Epinephelus akaara and E. coioides. In the present study, samples were collected from larvae of E. akaara with signs of VNN in Dayawan bay which is located in southern China. The complete viral coat protein gene was amplified using extracted total RNA as template. The reverse transcription polymerase chain reaction (RT-PCR) amplification was performed using primers containing a heterologous restriction site for NotI. PCR products were cloned into pcDNA3 vector and sequenced. The results indicated that the coat protein gene of Dayawan isolate (RG-CN) was 1056 bases in length and contained a single open reading frame (ORF) of 1017 bases encoding a protein of 338 amino acids. The sequence similarities between the coat protein gene of RG-CN and other 8 isolates of NNV from Dayawan, Taiwan, Japan, Singapore and France were 79.1%-99.5% at the nucleotide level and 83.7%-100% at the amino acid level, respectively.The homology between RG-CN and the other 5 isolates from groupers was high and relatively lower between RG-CN and guppy nervous necrosis virus (GNNV), sea bass nervous necrosis virus (DIEV), but more divergences existed between RG-CN and striped jack nevous necrosis virus (SJNNV). Compared with SJNNV, RG-CN and the other 7 isolates all lacked 6 nucleotides and 2 amino acids in the same positions. Based on the result of molecular phylogenetic analysis, Dayawan isolate belongs to red-spotted grouper nervous necrosis virus (RGNNV) genotype.展开更多
In recent years,piscine nodaviruses have emerged as major pathogens of a wide range of larval and juvenile marine finfish resulting in high mortality in aquaculture worldwide.Affected fish exhibit a range of neurologi...In recent years,piscine nodaviruses have emerged as major pathogens of a wide range of larval and juvenile marine finfish resulting in high mortality in aquaculture worldwide.Affected fish exhibit a range of neurological signs,such as erratic swimming behaviour with the associated microscopic lesions of necrosis and vacuolation of the central nervous tissues and retina.Numerous round-shaped,unenveloped and 25-30 nm in diameter virus particles were found in the cytoplasm of affected retinal and nerve cells.Nodaviruses have a bipartite genome of positive-sense RNA,with RNA1 encoding the RNA-dependent RNA polymerase and RNA2 encoding the capsid protein.Both RNA are capped,but not polyadenylated.The family Nodaviridae comprises two genera: Alphanodavirus and Betanodavirus,members of which primarily infect insects and fish,respectively.Therefore,betanodavirus is also named piscine nodavirus.At present,piscine nodaviruses are divided into four genotypes based on partial sequences of the coat protein gene.ELISA and RT-PCR amplification have been developed as specific diagnostic methods for the detection of the virus.Antibodies to striped jack(Pseudocaranx dentex) nervous necrosis(SJNNV) were found in 65% of plasma samples collected from wild and domestic brood stocks of striped jack,suggesting that the virus is very prevalent.Viral antigens were detected in eggs,larvae,and ovaries of hatchery-reared and wild spawner fish,suggesting both horizontal and vertical modes of transmission of the virus.Selection of nodavirus-free spawners using ELISA for detection of antigens and RT-PCR techniques have successfully reduced incidences of the virus infections in juvenile sea bass(Dicentrarchus labrax),striped jack and barfin flounder(Verasper moseri).The SSN-1 and GF cell lines have been successfully used in isolating piscine nodaviruses.Although there are many papers describing the molecular characteristics of betanodavirus,our knowledge of the genomic attributes of these viruses is still limited.Vaccination studies are being undertaken by a number of researchers and need to be fostered.In particular,the use of passive immunization of broodfish with homologous and heterologous,high-titre antisera are worthy of investigation.展开更多
[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The r...[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The recombinant expression vector pRSET A-MCP was constructed and transformed into BL21(DE3)plysS to express proteins with induction in different media, at different pH, or at different temperatures. [Result] The expression level of recombinant bacteria reached a peak with induction under the following condition: SOB or LB medium, pH 7.0, 37 ℃ while the fusion protein was about 44.5 kD in molecular weight. [Conclusion] This study provided a basis for the development of RGNNV-MCP vaccine.展开更多
文摘Viral nervous necrosis (VNN) is a worldwide disease among teleost fish. In the mainland of China, VNN was first identified in 2 species of hatchery-reared groupers, Epinephelus akaara and E. coioides. In the present study, samples were collected from larvae of E. akaara with signs of VNN in Dayawan bay which is located in southern China. The complete viral coat protein gene was amplified using extracted total RNA as template. The reverse transcription polymerase chain reaction (RT-PCR) amplification was performed using primers containing a heterologous restriction site for NotI. PCR products were cloned into pcDNA3 vector and sequenced. The results indicated that the coat protein gene of Dayawan isolate (RG-CN) was 1056 bases in length and contained a single open reading frame (ORF) of 1017 bases encoding a protein of 338 amino acids. The sequence similarities between the coat protein gene of RG-CN and other 8 isolates of NNV from Dayawan, Taiwan, Japan, Singapore and France were 79.1%-99.5% at the nucleotide level and 83.7%-100% at the amino acid level, respectively.The homology between RG-CN and the other 5 isolates from groupers was high and relatively lower between RG-CN and guppy nervous necrosis virus (GNNV), sea bass nervous necrosis virus (DIEV), but more divergences existed between RG-CN and striped jack nevous necrosis virus (SJNNV). Compared with SJNNV, RG-CN and the other 7 isolates all lacked 6 nucleotides and 2 amino acids in the same positions. Based on the result of molecular phylogenetic analysis, Dayawan isolate belongs to red-spotted grouper nervous necrosis virus (RGNNV) genotype.
文摘In recent years,piscine nodaviruses have emerged as major pathogens of a wide range of larval and juvenile marine finfish resulting in high mortality in aquaculture worldwide.Affected fish exhibit a range of neurological signs,such as erratic swimming behaviour with the associated microscopic lesions of necrosis and vacuolation of the central nervous tissues and retina.Numerous round-shaped,unenveloped and 25-30 nm in diameter virus particles were found in the cytoplasm of affected retinal and nerve cells.Nodaviruses have a bipartite genome of positive-sense RNA,with RNA1 encoding the RNA-dependent RNA polymerase and RNA2 encoding the capsid protein.Both RNA are capped,but not polyadenylated.The family Nodaviridae comprises two genera: Alphanodavirus and Betanodavirus,members of which primarily infect insects and fish,respectively.Therefore,betanodavirus is also named piscine nodavirus.At present,piscine nodaviruses are divided into four genotypes based on partial sequences of the coat protein gene.ELISA and RT-PCR amplification have been developed as specific diagnostic methods for the detection of the virus.Antibodies to striped jack(Pseudocaranx dentex) nervous necrosis(SJNNV) were found in 65% of plasma samples collected from wild and domestic brood stocks of striped jack,suggesting that the virus is very prevalent.Viral antigens were detected in eggs,larvae,and ovaries of hatchery-reared and wild spawner fish,suggesting both horizontal and vertical modes of transmission of the virus.Selection of nodavirus-free spawners using ELISA for detection of antigens and RT-PCR techniques have successfully reduced incidences of the virus infections in juvenile sea bass(Dicentrarchus labrax),striped jack and barfin flounder(Verasper moseri).The SSN-1 and GF cell lines have been successfully used in isolating piscine nodaviruses.Although there are many papers describing the molecular characteristics of betanodavirus,our knowledge of the genomic attributes of these viruses is still limited.Vaccination studies are being undertaken by a number of researchers and need to be fostered.In particular,the use of passive immunization of broodfish with homologous and heterologous,high-titre antisera are worthy of investigation.
基金Supported by Science and Technology Planning Project of Guangdong Province,China(2003B21502,2005B20301016)~~
文摘[Objective] To optimize the prokaryotic expression of MCP gene of red-spotted grouper nervous necrosis virus. [Method] The MCP gene was amplified from red-spotted grouper nervous necrosis viral genome by RT-PCR. The recombinant expression vector pRSET A-MCP was constructed and transformed into BL21(DE3)plysS to express proteins with induction in different media, at different pH, or at different temperatures. [Result] The expression level of recombinant bacteria reached a peak with induction under the following condition: SOB or LB medium, pH 7.0, 37 ℃ while the fusion protein was about 44.5 kD in molecular weight. [Conclusion] This study provided a basis for the development of RGNNV-MCP vaccine.
