Viral nervous necrosis (VNN) causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is i...Viral nervous necrosis (VNN) causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction (RT-PCR) was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necro- sis virus (NNV), in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), spring viraemia of carp virus (SVCV), epizootic haematopoietic necrosis virus (EHNV), and large yellow croaker iridovirus (LYC1V). With this method, the orange-spotted grouper (Epinephelus coioides) fry from hatcheries with or without incidence of the VN- N epidemic in Fujian Province were detected. The results showed that all or 93% of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40% or 25% of fry from the t- wo hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.展开更多
On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus( OGNNV), China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OG...On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus( OGNNV), China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV. The major capsid protein ( MCP)gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction (RT-PCR) and ligated into the pET32a expression plasmid. The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa. Recombinant protein with a molecular mass of 57.4 kDa was expressed in E. coli BL21 (DE3). Vaccine was prepared from the recombinant protein expressed in recombinant cells. The juvenile orange-spotted groupers (8 cm in average length) were immunized by intraperitoneal injection. Group A was challenged with infected tissue filtrates 25 d post-vaccination. The mortality in the vaccined group ( A1,30% ) was a little higher than the unvaccined group ( B2, 27.8% ). Group B was challenged after three vaccine injections. The mortality in the vaccined group (B1, 16.7% ) was lower than the unvaccined group (132, 27.8% ), And the relative percentage survival (RPS) value of vaccined group, compared with the unvaccined group, was 40%. The anti-recombinant protein sera with a 1 : 100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4 ℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper. Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish ( Group C1, mortality of 18.18% ), compared with the fish ( Group C2, mortality of 40% ) which received infected tissue filtrates treated with control serum. Results implied the potential use of the capsid protein in immunization against OGNNV.展开更多
Pseudomonas plecoglossicida is the pathogen responsible for visceral white spot disease in large yellow croaker(Larimichthys crocea)and orangespotted grouper(Epinephelus coioides).Previously,RNA sequencing showed that...Pseudomonas plecoglossicida is the pathogen responsible for visceral white spot disease in large yellow croaker(Larimichthys crocea)and orangespotted grouper(Epinephelus coioides).Previously,RNA sequencing showed that P.plecoglossicida flgK gene expression was significantly up-regulated in orange-spotted grouper spleens during infection.To explore the role of flgK in P.plecoglossicida pathogenicity,RNA interference(RNAi)was performed to silence the P.plecoglossicida flgK gene,and the mutant(flgK-RNAi strain)with the best silencing efficiency(89.40%)was chosen for further study.Results showed that flgK gene silencing significantly attenuated P.plecoglossicida motility,adhesion,and biofilm formation.Compared to those fish infected with the wild-type strain of P.plecoglossicida,orange-spotted grouper infected with the flgK-RNAi strain showed a 55%increase in the survival rate and a one-day delay in time of first death,with fewer pathogens in the spleen and fewer white spots on the spleen surface.RNAi of flgK significantly affected the transcriptome and metabolome of the spleen in infected orange-spotted grouper.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that the C-type lectin receptor signaling pathway was the most significantly changed immune-related pathway and the mitogen-activated protein kinase(MAPK)signaling pathway was related to multiple immunerelated pathways.Furthermore,arginine biosynthesis and glycerophospholipid metabolism were the most significantly changed metabolism-related pathways.These findings suggest that flgK is a virulence gene of P.plecoglossicida.Furthermore,flgK appears to be involved in the regulation of motility,adhesion,and biofilm formation in P.plecoglossicida,as well as in the regulation of inflammatory and immune responses of orange-spotted grouper to P.plecoglossicida infection.展开更多
基金The National Basic Research Program under contract No.2012CB114402the National High Technology Research and Development Program of China (863 Program) under contract No.2012AA092202Scientific Research Project of Marine Public Welfare Industry of China under contract No.200905020-07
文摘Viral nervous necrosis (VNN) causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction (RT-PCR) was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necro- sis virus (NNV), in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), spring viraemia of carp virus (SVCV), epizootic haematopoietic necrosis virus (EHNV), and large yellow croaker iridovirus (LYC1V). With this method, the orange-spotted grouper (Epinephelus coioides) fry from hatcheries with or without incidence of the VN- N epidemic in Fujian Province were detected. The results showed that all or 93% of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40% or 25% of fry from the t- wo hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.
基金This work was supported by the National"863"Projects of China under contract No.2001AA601010the Natural Science Foundation of Jinan University,China under contract No.51204062.
文摘On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus( OGNNV), China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV. The major capsid protein ( MCP)gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction (RT-PCR) and ligated into the pET32a expression plasmid. The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa. Recombinant protein with a molecular mass of 57.4 kDa was expressed in E. coli BL21 (DE3). Vaccine was prepared from the recombinant protein expressed in recombinant cells. The juvenile orange-spotted groupers (8 cm in average length) were immunized by intraperitoneal injection. Group A was challenged with infected tissue filtrates 25 d post-vaccination. The mortality in the vaccined group ( A1,30% ) was a little higher than the unvaccined group ( B2, 27.8% ). Group B was challenged after three vaccine injections. The mortality in the vaccined group (B1, 16.7% ) was lower than the unvaccined group (132, 27.8% ), And the relative percentage survival (RPS) value of vaccined group, compared with the unvaccined group, was 40%. The anti-recombinant protein sera with a 1 : 100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4 ℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper. Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish ( Group C1, mortality of 18.18% ), compared with the fish ( Group C2, mortality of 40% ) which received infected tissue filtrates treated with control serum. Results implied the potential use of the capsid protein in immunization against OGNNV.
基金supported by the National Natural Science Foundation of China(31972836)Natural Science Foundation of Fujian Province(2021J01828)Open Fund of Fujian Province Key Laboratory of Special Aquatic Formula Feed(TMKJZ2101)。
文摘Pseudomonas plecoglossicida is the pathogen responsible for visceral white spot disease in large yellow croaker(Larimichthys crocea)and orangespotted grouper(Epinephelus coioides).Previously,RNA sequencing showed that P.plecoglossicida flgK gene expression was significantly up-regulated in orange-spotted grouper spleens during infection.To explore the role of flgK in P.plecoglossicida pathogenicity,RNA interference(RNAi)was performed to silence the P.plecoglossicida flgK gene,and the mutant(flgK-RNAi strain)with the best silencing efficiency(89.40%)was chosen for further study.Results showed that flgK gene silencing significantly attenuated P.plecoglossicida motility,adhesion,and biofilm formation.Compared to those fish infected with the wild-type strain of P.plecoglossicida,orange-spotted grouper infected with the flgK-RNAi strain showed a 55%increase in the survival rate and a one-day delay in time of first death,with fewer pathogens in the spleen and fewer white spots on the spleen surface.RNAi of flgK significantly affected the transcriptome and metabolome of the spleen in infected orange-spotted grouper.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that the C-type lectin receptor signaling pathway was the most significantly changed immune-related pathway and the mitogen-activated protein kinase(MAPK)signaling pathway was related to multiple immunerelated pathways.Furthermore,arginine biosynthesis and glycerophospholipid metabolism were the most significantly changed metabolism-related pathways.These findings suggest that flgK is a virulence gene of P.plecoglossicida.Furthermore,flgK appears to be involved in the regulation of motility,adhesion,and biofilm formation in P.plecoglossicida,as well as in the regulation of inflammatory and immune responses of orange-spotted grouper to P.plecoglossicida infection.