斜带石斑鱼(Epinephelus coioides)病毒性神经坏死病毒的纯化分析及检测方法建立
|
摘要
根据GenBank中已有的鱼类病毒性神经坏死病毒(NNV)RNA2基因序列,设计引物,从福建厦门具有典型NNV发病症状的斜带石斑鱼中克隆了RNA2基因的全长序列,并将序列提交到GenBank获得登录号为MF510920,命名为XMNNV。系统进化树分析结果表明XMNNV与RGNNV聚类在一起,与SJNNV、BFNNV和TPNNV等其他鱼类神经坏死病毒亲缘关系较远,说明本研究分离得到的XMNNV属于RGNNV基因型。通过超速离心方法对病毒进行提纯,得到了纯化的NNV病毒。电镜观察结果表明,病毒粒子直径20～25 nm,结构为正二十面体,与已经报道的NNV结构一致。通过对XMNNV与其它RGNNV RNA2基因进行序列比对,在保守区设计引物,运用RT-PCR方法建立了RGNNV的PCR检测方法,该方法灵敏度高达67 copies/μL。纯化的病毒对E11(条纹月鳢细胞系)和大黄鱼肌肉细胞进行感染,结果表明该病毒可以感染这两种鱼类细胞。E11细胞被感染病毒后,细胞出现空泡化,并最终导致细胞分解死亡;大黄鱼肌肉细胞感染后,细胞变圆,慢慢从培养皿壁脱落,最终解体死亡。另外,对感染后细胞进行PCR检测,结果显示为阳性,进一步确定了分离的NNV具有感染这两种细胞的能力。本研究通过电镜观察和PCR检测两种方法确定了患病石斑鱼携带NNV,通过对两种鱼类细胞的感染实验,确定了该病毒具有一定的感染能力。综上,本研究为石斑鱼NNV疾病的诊断提供了有效的方法,对石斑鱼NNV疾病的预防具有一定的指导意义。
Primers were designed based on various grouper nervous necrosis virus RNA2 genome sequence obtained from GeneBank.The coat protein gene of Fujian Xiamen Epinephelus coioides nervous necrosis virus was cloned and sequenced,while the sequence was submitted to the GenBank database under accession number MF510920.In the phylogenetic tree,the coat protein gene from XMNNV formed distinct group with that from RGNNV,but separated to the SJNNV,BFNNV and TPNNV nervous necrosis virus.Based on the result of molecular phylogenetic analysis,XMNNV was found belonged to red-spotted grouper nervous necrosis virus(RGNNV)genotype.The virus particles were purified by ultracentrifugation.The results showed that it was an icosahedral virus with a mean diameter of 20～25 nm by electron microscopy,consistent with the reported structure of NNV.According to the sequence alignment results of XMNNV and the other RGNNV RNA2,the specific primers were designed according to the highly conservative sequence of capsid protein gene of viral nervous necrosis virus.A reverse-transcription polymerase chain reaction(RT-PCR)assay for fish nervous necrosis virus was developed,the sensitivity of the method was 67 copies/μL.The isolated virus was used to infect E11 cell line and Larimichthys crocea muscle cells,and both cells showed sensitive for the isolate.While incubated with E11 cell,CPE was developed and vacuolation after incubation.L.crocea muscle cell was round and shedding off the wall,when broken up and died eventually.Besides,the infected samples were cloned and sequenced.The result of PCR indicated that the isolated virus was NNV.According to the transmission electron microscopy and PCR assay,we suspected the E.coioides was infected by red-spotted grouper nervous necrosis virus.This research provided effective method for the diagnosis and will be used in the field of prevention of grouper disease.
Primers were designed based on various grouper nervous necrosis virus RNA2 genome sequence obtained from GeneBank.The coat protein gene of Fujian Xiamen Epinephelus coioides nervous necrosis virus was cloned and sequenced,while the sequence was submitted to the GenBank database under accession number MF510920.In the phylogenetic tree,the coat protein gene from XMNNV formed distinct group with that from RGNNV,but separated to the SJNNV,BFNNV and TPNNV nervous necrosis virus.Based on the result of molecular phylogenetic analysis,XMNNV was found belonged to red-spotted grouper nervous necrosis virus(RGNNV)genotype.The virus particles were purified by ultracentrifugation.The results showed that it was an icosahedral virus with a mean diameter of 20～25 nm by electron microscopy,consistent with the reported structure of NNV.According to the sequence alignment results of XMNNV and the other RGNNV RNA2,the specific primers were designed according to the highly conservative sequence of capsid protein gene of viral nervous necrosis virus.A reverse-transcription polymerase chain reaction(RT-PCR)assay for fish nervous necrosis virus was developed,the sensitivity of the method was 67 copies/μL.The isolated virus was used to infect E11 cell line and Larimichthys crocea muscle cells,and both cells showed sensitive for the isolate.While incubated with E11 cell,CPE was developed and vacuolation after incubation.L.crocea muscle cell was round and shedding off the wall,when broken up and died eventually.Besides,the infected samples were cloned and sequenced.The result of PCR indicated that the isolated virus was NNV.According to the transmission electron microscopy and PCR assay,we suspected the E.coioides was infected by red-spotted grouper nervous necrosis virus.This research provided effective method for the diagnosis and will be used in the field of prevention of grouper disease.
引文
[1]王大鹏,曹占旺,谢达祥,等.石斑鱼的研究进展[J].南方农业学报,2012,43(7):1058-1065.
[2]陈大玮,邓利,刘志刚,等.斜带石斑鱼抗菌肽hepcidin基因克隆及其成熟肽的原核融合表达[J].南方水产科学,2011,7(1):1-7.
[3]Qin Q W,Chang S F,Ngoh-Lim G H,et al.Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina[J].Diseases of A-quatic Organisms,2003,53(1):1-9.
[4]Qin Q W,Wu T H,Jia T L,et al.Development and characterization of a new tropical marine fish cell line from grouper,Epinephelus coioides susceptible to iridovirus and nodavirus[J].Journal of Virological Methods,2006,131(1):58-64.
[5]Georgiadis M P,Gardner I A,Hedrick R P.The role of epidemiology in the prevention,diagnosis,and control of infectious diseases of fish[J].Preventive Veterinary Medicine,2001,48(4):287-302.
[6]罗鸣,陈傅晓,刘龙龙,等.我国石斑鱼养殖疾病的研究进展[J].水产科学,2013,32(9):549-554.
[7]Qin Q W,Lam T J,Sin Y M,et al.Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper,Epinephelus tauvina[J].Journal of Virological Methods,2001,98(1):17-24.
[8]LüL,Zhou S Y,Chen C,et al.Complete genome sequence analysis of an iridovirus isolated from the orange-spotted grouper,Epinephelus coioides[J].Virology,2005,339(1):81-100.
[9]Hill B J,Way K.Serological classification of infectious pancreatic necrosis(IPN)virus and other aquatic birnaviruses[J].Annual Review of Fish Diseases,1995,(5):55-77.
[10]Mori K,Nakai T,Muroga K,et al.Properties of a new virus belonging to nodaviridae found in larval striped jack(Pseudocaranx dentex)with nervous necrosis[J].Virology,1992,187(1):368-71.
[11]Aspehaug V,Devold M,Nylund A.The phylogenetic relationship of nervous necrosis virus from halibut(Hippoglossus hippoglossus)[J].BulletinEuropean Association of Fish Pathologists,1999,19(5):196-202.
[12]Bovo G,Nishizawa T,Maltese C,et al.Viral encephalopathy and retinopathy of farmed marine fish species in Italy[J].Virus Research,1999,63(1-2):143-146.
[13]Breuil G,Mouchel O,Fauvel C,et al.Sea bass Dicentrarchus labrax nervous necrosis virus isolates with distinct pathogenicity to sea bass larvae[J].Diseases of Aquatic Organisms,2001,45(1):25-31.
[14]Munday B L,Kwang J,Moody N.Betanodavirus infections of teleost fish:A review[J].Journal of Fish Diseases,2002,25(3):127-142.
[15]Nishizawa T,Furuhashi M,Nagai T,et al.Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene[J].Applied and Environmental Microbiology,1997,63(4):1633-1636.
[16]陈信忠,龚艳清,苏永全,等.闽南地区紫石斑(Epinephelus lanceolatus)幼鱼爆发性传染病的病原学研究[J].厦门大学学报(自然科学版),2004,43(4):557-562.
[17]Tamura K,Stecher G,Peterson D,et al.MEGA6:Molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,2013,30(12):2725-2729.
[18]Saitou N,Nei M.The Neighbor-joining method:Anew method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4):406-425.
[19]Grove S,Johansen R,Dannevig B H,et al.Experimental infection of Atlantic halibut Hippoglossus hippoglossus with nodavirus:tissue distribution and immune response[J].Diseases of Aquatic Organisms,2003,53(3):211-221.
[20]Iwamoto T,Mise K,Mori K,et al.Establishment of an infectious RNA transcription system for Striped jack nervous necrosis virus,the type species of the betanodaviruses[J].Journal of General Virology,2001,82(11):2653-2662.
[21]林克冰,方琼珊,吴建绍,等.石斑鱼神经坏死病毒传播途径阻断的初步研究[J].福建水产,2011,33(5):15-19.
[22]Kuo H C,Wang T Y,Hsu H H,et al.Nervous necrosis virus replicates following the embryo development and dual infection with iridovirus at juvenile stage in grouper[J].Plos One,2012,7(4):e36183.
[23]Chi S C,Lo B J,Lin S C.Characterization of grouper nervous necrosis virus(GNNV)[J].Journal of Fish Diseases,2001,24(1):3-13.
[24]Nishizawa T,Mori K,Nakai T,et al.Polymerase chain reaction(PCR)amplification of RNA of striped jack nervous necrosis virus(SJNNV)[J].Diseases of aquatic organisms,1994,18(2):103-107.
[25]Valle L D,Toffolo V,Lamprecht M,et al.Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR[J].Veterinary Microbiology,2005,110(3-4):167-179.
[26]Mekata T,Satoh J,Inada M,et al.Development of simple,rapid and sensitive detection assay for grouper nervous necrosis virus using real-time loop-mediated isothermal amplification[J].Journal of Fish Diseases,2015,38(10):873-879..
[27]Toffolo V,Negrisolo E,Maltese C,et al.Phylogeny of betanodaviruses and molecular evolution of their RNA polymerase and coat proteins[J].Molecular Phylogenetics Evolution,2007,43(1):298-308.
[28]黄剑南,林蠡,翁少萍,等.赤点石斑鱼诺达病毒的纯化及其衣壳蛋白的Western-blot分析[J].南方水产科学,2005,1(5):33-36.
[29]Totland G K,Grotmol S,Morita Y,et al.Pathogenicity of nodavirus strains from striped jack Pseudocaranx dentex and Atlantic halibut Hippoglossus hippoglossus,studied by waterborne challenge of yolk-sac larvae of both teleost species[J].Diseases of A-quatic Organisms,1999,38(3):169-175.
[2]陈大玮,邓利,刘志刚,等.斜带石斑鱼抗菌肽hepcidin基因克隆及其成熟肽的原核融合表达[J].南方水产科学,2011,7(1):1-7.
[3]Qin Q W,Chang S F,Ngoh-Lim G H,et al.Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina[J].Diseases of A-quatic Organisms,2003,53(1):1-9.
[4]Qin Q W,Wu T H,Jia T L,et al.Development and characterization of a new tropical marine fish cell line from grouper,Epinephelus coioides susceptible to iridovirus and nodavirus[J].Journal of Virological Methods,2006,131(1):58-64.
[5]Georgiadis M P,Gardner I A,Hedrick R P.The role of epidemiology in the prevention,diagnosis,and control of infectious diseases of fish[J].Preventive Veterinary Medicine,2001,48(4):287-302.
[6]罗鸣,陈傅晓,刘龙龙,等.我国石斑鱼养殖疾病的研究进展[J].水产科学,2013,32(9):549-554.
[7]Qin Q W,Lam T J,Sin Y M,et al.Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper,Epinephelus tauvina[J].Journal of Virological Methods,2001,98(1):17-24.
[8]LüL,Zhou S Y,Chen C,et al.Complete genome sequence analysis of an iridovirus isolated from the orange-spotted grouper,Epinephelus coioides[J].Virology,2005,339(1):81-100.
[9]Hill B J,Way K.Serological classification of infectious pancreatic necrosis(IPN)virus and other aquatic birnaviruses[J].Annual Review of Fish Diseases,1995,(5):55-77.
[10]Mori K,Nakai T,Muroga K,et al.Properties of a new virus belonging to nodaviridae found in larval striped jack(Pseudocaranx dentex)with nervous necrosis[J].Virology,1992,187(1):368-71.
[11]Aspehaug V,Devold M,Nylund A.The phylogenetic relationship of nervous necrosis virus from halibut(Hippoglossus hippoglossus)[J].BulletinEuropean Association of Fish Pathologists,1999,19(5):196-202.
[12]Bovo G,Nishizawa T,Maltese C,et al.Viral encephalopathy and retinopathy of farmed marine fish species in Italy[J].Virus Research,1999,63(1-2):143-146.
[13]Breuil G,Mouchel O,Fauvel C,et al.Sea bass Dicentrarchus labrax nervous necrosis virus isolates with distinct pathogenicity to sea bass larvae[J].Diseases of Aquatic Organisms,2001,45(1):25-31.
[14]Munday B L,Kwang J,Moody N.Betanodavirus infections of teleost fish:A review[J].Journal of Fish Diseases,2002,25(3):127-142.
[15]Nishizawa T,Furuhashi M,Nagai T,et al.Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene[J].Applied and Environmental Microbiology,1997,63(4):1633-1636.
[16]陈信忠,龚艳清,苏永全,等.闽南地区紫石斑(Epinephelus lanceolatus)幼鱼爆发性传染病的病原学研究[J].厦门大学学报(自然科学版),2004,43(4):557-562.
[17]Tamura K,Stecher G,Peterson D,et al.MEGA6:Molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution,2013,30(12):2725-2729.
[18]Saitou N,Nei M.The Neighbor-joining method:Anew method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4):406-425.
[19]Grove S,Johansen R,Dannevig B H,et al.Experimental infection of Atlantic halibut Hippoglossus hippoglossus with nodavirus:tissue distribution and immune response[J].Diseases of Aquatic Organisms,2003,53(3):211-221.
[20]Iwamoto T,Mise K,Mori K,et al.Establishment of an infectious RNA transcription system for Striped jack nervous necrosis virus,the type species of the betanodaviruses[J].Journal of General Virology,2001,82(11):2653-2662.
[21]林克冰,方琼珊,吴建绍,等.石斑鱼神经坏死病毒传播途径阻断的初步研究[J].福建水产,2011,33(5):15-19.
[22]Kuo H C,Wang T Y,Hsu H H,et al.Nervous necrosis virus replicates following the embryo development and dual infection with iridovirus at juvenile stage in grouper[J].Plos One,2012,7(4):e36183.
[23]Chi S C,Lo B J,Lin S C.Characterization of grouper nervous necrosis virus(GNNV)[J].Journal of Fish Diseases,2001,24(1):3-13.
[24]Nishizawa T,Mori K,Nakai T,et al.Polymerase chain reaction(PCR)amplification of RNA of striped jack nervous necrosis virus(SJNNV)[J].Diseases of aquatic organisms,1994,18(2):103-107.
[25]Valle L D,Toffolo V,Lamprecht M,et al.Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR[J].Veterinary Microbiology,2005,110(3-4):167-179.
[26]Mekata T,Satoh J,Inada M,et al.Development of simple,rapid and sensitive detection assay for grouper nervous necrosis virus using real-time loop-mediated isothermal amplification[J].Journal of Fish Diseases,2015,38(10):873-879..
[27]Toffolo V,Negrisolo E,Maltese C,et al.Phylogeny of betanodaviruses and molecular evolution of their RNA polymerase and coat proteins[J].Molecular Phylogenetics Evolution,2007,43(1):298-308.
[28]黄剑南,林蠡,翁少萍,等.赤点石斑鱼诺达病毒的纯化及其衣壳蛋白的Western-blot分析[J].南方水产科学,2005,1(5):33-36.
[29]Totland G K,Grotmol S,Morita Y,et al.Pathogenicity of nodavirus strains from striped jack Pseudocaranx dentex and Atlantic halibut Hippoglossus hippoglossus,studied by waterborne challenge of yolk-sac larvae of both teleost species[J].Diseases of A-quatic Organisms,1999,38(3):169-175.