中国狂犬病毒P和M基因分子生物学特征研究
摘要
本研究从国内外研究较少的狂犬病毒P、M基因出发,针对中国广西、贵州、湖南、江苏、安徽和云南六省区狂犬病人、犬阳性脑组织标本通过RT-PCR方法扩增目的基因来测定P、M基因编码区全序,利用来源于GenBank中不同国家地区代表不同基因型的狂犬病毒株P、M基因序列信息在分子水平进行P、M基因序列同源性分析、主要功能位点比较和种系发生分析,探讨中国狂犬病毒P、M基因分子生物学特征,为进一步研究狂犬病毒的分子结构提供基础数据。
中国狂犬病毒P基因核苷酸与氨基酸序列同源性分别为81.8～100%和82.9～99.7%;M基因核苷酸与氨基酸序列同源性分别为88.7～100%和95.0～100%。PP与胞浆动力蛋白轻链(LC8)作用位点、PP与LP主要作用区域前19位氨基酸都高度保守,特定的蛋白激酶磷酸化丝氨酸位点、PP与NP相互作用主要区域第209～216位氨基酸仅个别位点发生变化,中部高变区第50～75位氨基酸位于核酸外导信号结构域内;MP调节病毒RNA转录和复制功能的第58位氨基酸、在病毒晚期芽生过程中起调节作用的PPxY结构域均高度保守,高变区第17～22位氨基酸位于N末端潜在的抗原决定簇内。P、M基因核苷酸水平的变异区存在于氨基酸水平同样的位置,核苷酸序列的变异多为同义突变。P、M基因系统进化分析显示所有中国标本P、M基因序列与其它基因1型序列形成独立的进化分支,具有中国地域特点:来源于同一省区的标本构成同一亚分支,趋向于同一进化群。
对P、M基因的研究结果表明中国狂犬病毒均属于基因1型,序列同源性都比较高,具有共同的进化途径和基因结构特点,序列间差异规律明显,病毒分布具有独特的中国地域性特征,但序列的差异并不完全与病毒分离的行政地理区域相关,未严格按照行政地理分群。六省区狂犬病毒不但在同一省区内循环传播,而且在各个省区相邻地域之间相互传播,中国与东南亚国家的狂犬病毒株P、M基因具有较近的亲缘关系,中国云南省个别毒株可能和泰国毒株来源于共同的狂犬病毒。六省区所有狂犬病毒街毒株都来源于人和犬,人、犬分离株P、M基因全序列无明显差异,进化呈现由犬到人的方向,从病毒的来源角度证实了犬对人狂犬病的重要影响。
Although the bulk of data and research on RV now exist in the public archives, the relatively few research about the characteristics of both phosphoprotein(PP)and matrix protein(MP)gene of rabies virus are available. In this study, the DFA-positive human and dog samples were collected from six provinces including Guangxi, Guizhou, Hunan, Jiangsu, Anhui and Yunnan. The Nucleotide sequences of the PP and MP genes which were amplified by RT-PCR techniques were determined and compared with other known RV isolates in GenBank. The similarity, functional sites and phylogenetic analyses were performed, which are helpful to the further research on molecular biology and structure of rabies viruses in China.
The results show that the similarity of nucleotide and amino acid sequences of PP gene are 81.8～100% and 82.9～99.7% respectively while those of MP gene are 88.7～100% and 95.0～100% respectively. The highly conserved region of P gene is the interactive region between PP and endochylema dynein light chain 8(LC8), and the first 19 amino acids in the interactive region between PP and LP(large protein). There is a few substitution sites in the P gene such as the specific Ser phosphorylation sites and some sites among the interactive region ( aa 209-216 ) between PP and NP(nucleoprotein).The variable region(aa 50-75)located in the signal domain. The analyses of M gene show the 58th amino acid residue and the PPxY domain are highly conserved, while the 17th-22nd amino acid residues are located in the potential antigen determinant in the end of NP.
According to the analyses of P and M gene, all the tested RV isolates belong to rabies virus genotype1. Both P and M gene are highly conserved and they take on a co-evolutional property. Although the distribution of Chinese isolates is regionally, it is not strictly correlated to administrational areas. The rabies virus strains not only circulated in their original province but also spread to the adjoining provinces. Around the national boundary between China and Southeast Asians, the rabies virus isolates are tightly related to each other genetically especially between Yunnan province and Thailand. All the RV strains isolated from dog and human samples, and phylogenetic analyses indicates the tendency of RV evolution is from dogs to humans, which confirms the important role of dog RV in human rabies.
中国狂犬病毒P基因核苷酸与氨基酸序列同源性分别为81.8～100%和82.9～99.7%;M基因核苷酸与氨基酸序列同源性分别为88.7～100%和95.0～100%。PP与胞浆动力蛋白轻链(LC8)作用位点、PP与LP主要作用区域前19位氨基酸都高度保守,特定的蛋白激酶磷酸化丝氨酸位点、PP与NP相互作用主要区域第209～216位氨基酸仅个别位点发生变化,中部高变区第50～75位氨基酸位于核酸外导信号结构域内;MP调节病毒RNA转录和复制功能的第58位氨基酸、在病毒晚期芽生过程中起调节作用的PPxY结构域均高度保守,高变区第17～22位氨基酸位于N末端潜在的抗原决定簇内。P、M基因核苷酸水平的变异区存在于氨基酸水平同样的位置,核苷酸序列的变异多为同义突变。P、M基因系统进化分析显示所有中国标本P、M基因序列与其它基因1型序列形成独立的进化分支,具有中国地域特点:来源于同一省区的标本构成同一亚分支,趋向于同一进化群。
对P、M基因的研究结果表明中国狂犬病毒均属于基因1型,序列同源性都比较高,具有共同的进化途径和基因结构特点,序列间差异规律明显,病毒分布具有独特的中国地域性特征,但序列的差异并不完全与病毒分离的行政地理区域相关,未严格按照行政地理分群。六省区狂犬病毒不但在同一省区内循环传播,而且在各个省区相邻地域之间相互传播,中国与东南亚国家的狂犬病毒株P、M基因具有较近的亲缘关系,中国云南省个别毒株可能和泰国毒株来源于共同的狂犬病毒。六省区所有狂犬病毒街毒株都来源于人和犬,人、犬分离株P、M基因全序列无明显差异,进化呈现由犬到人的方向,从病毒的来源角度证实了犬对人狂犬病的重要影响。
Although the bulk of data and research on RV now exist in the public archives, the relatively few research about the characteristics of both phosphoprotein(PP)and matrix protein(MP)gene of rabies virus are available. In this study, the DFA-positive human and dog samples were collected from six provinces including Guangxi, Guizhou, Hunan, Jiangsu, Anhui and Yunnan. The Nucleotide sequences of the PP and MP genes which were amplified by RT-PCR techniques were determined and compared with other known RV isolates in GenBank. The similarity, functional sites and phylogenetic analyses were performed, which are helpful to the further research on molecular biology and structure of rabies viruses in China.
The results show that the similarity of nucleotide and amino acid sequences of PP gene are 81.8～100% and 82.9～99.7% respectively while those of MP gene are 88.7～100% and 95.0～100% respectively. The highly conserved region of P gene is the interactive region between PP and endochylema dynein light chain 8(LC8), and the first 19 amino acids in the interactive region between PP and LP(large protein). There is a few substitution sites in the P gene such as the specific Ser phosphorylation sites and some sites among the interactive region ( aa 209-216 ) between PP and NP(nucleoprotein).The variable region(aa 50-75)located in the signal domain. The analyses of M gene show the 58th amino acid residue and the PPxY domain are highly conserved, while the 17th-22nd amino acid residues are located in the potential antigen determinant in the end of NP.
According to the analyses of P and M gene, all the tested RV isolates belong to rabies virus genotype1. Both P and M gene are highly conserved and they take on a co-evolutional property. Although the distribution of Chinese isolates is regionally, it is not strictly correlated to administrational areas. The rabies virus strains not only circulated in their original province but also spread to the adjoining provinces. Around the national boundary between China and Southeast Asians, the rabies virus isolates are tightly related to each other genetically especially between Yunnan province and Thailand. All the RV strains isolated from dog and human samples, and phylogenetic analyses indicates the tendency of RV evolution is from dogs to humans, which confirms the important role of dog RV in human rabies.
引文
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5 Tordo,N.Characteristics and molecular biology of the rabies virus[R]. World Health Organization,Geneva,Switzerland,1996,28-51.
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10 Hopper PT, Lunt RA, Gould AR, et al, P. T. Hopper, et al. A new lyssavirus--the first endemic rabies related virus recognized in Australia[J]. Bull Inst Pasteur,1997, 95:209-218.
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12 Badrane H, Bahloul C, Perrin P, et al. Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity[J].J Virol,2005,75: 3268-3276.
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14 Ertl H C, Fischer S, Flamand A. Phosphorylation of the N and M1 proteins of rabies virus[J].Virol,1985,66:2285-2289.
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20 Yves Jacob, Eleonore Real, and Noel Tordo. Fuctional Interaction Mao oflyssavirus phosphoprotein:Identification of the Minimal Transcription Domains[J]. Virol,2001, 75:9613-9622.
21 Gigant B,Iseni F,Gaudin Y,et al. Neither phosphorylation nor the amino-ternimal part of rabies virus phosphoprotein os required for its oligomerization[J]. GenVirol,2000, 81:1757-1761.
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2 Willoughby R E Jr, Tieves K S, Hoffman G M, et al.Survial after treatment of rabies with induction of coma[J]. N Engl J Med,2005,352:2508-2514.
3 唐青,杨为松.狂犬病毒分子生物学及遗传变异性研究进展[J].中国人兽共患杂志,1999. 15(2):77-81.
4 Reagan R L,Chang S,Brueckner A L.Studies by electron microscopy of a fixed (Pasteur) rabies virus,a street rabies virus and the egg adapted Flury strain of rabies[J]. Tex Rep Biol Med.1955,13(2):356-61.
5 Tordo,N.Characteristics and molecular biology of the rabies virus[R]. World Health Organization,Geneva,Switzerland,1996,28-51.
6 Wiktor T J,Gyorgy E,Schlumberger D,et al.Antigenic properties of rabies virus components[J]. Journal of Immunology,1973,110:269-276.
7 Willam H Wunner, Jovi K Larson, Bernhard Dietzschold, et al. The molecular biology of rabies viruses[J].Reviews of infectious diseases.1988,10(4):S771-S784.
8 Bourhy H, Kissi B, Tordo N. Molecular diversity of the Lyssavirus genus[J]. Virology, 1993,194:70-81.
9 Bourthy H, Kissi B, Lafon M, et al. Antigenic and molecular characterization of bat rabies virus in Europe[J], J Clin Microbiol,1992,30,2419-2426
10 Hopper PT, Lunt RA, Gould AR, et al, P. T. Hopper, et al. A new lyssavirus--the first endemic rabies related virus recognized in Australia[J]. Bull Inst Pasteur,1997, 95:209-218.
11 金奇.医学分子病毒学[M].北京:科学技术出版社.2001.614-632.
12 Badrane H, Bahloul C, Perrin P, et al. Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity[J].J Virol,2005,75: 3268-3276.
13 袁慧君,王三虎,秦鄂德.狂犬病病毒分子生物学研究进展[J].生物技术通讯,2004,15(6): 596-599.
14 Ertl H C, Fischer S, Flamand A. Phosphorylation of the N and M1 proteins of rabies virus[J].Virol,1985,66:2285-2289.
15 Emerson, S U, and M Schubert. Location of the binding domain for the RNA polymerase L and the ribonucleocapsid template within different haves of the NS phosphoprotein of vesicular stomatitis virus[J].Proc Natl Acad Sci USA,1987,84:5655-5659.
16 Gao Y,J Lenard.Multimerization and transcriptional activation of the phosphoprotein(P) of vesicular stomatis virus by casein kinase-Ⅱ[J].EMBO,1995, 14:1240-1247.
17 Gupta A K,Blondel D,Choudhary S. The Phosphoprotein of Rabies Virus Is Phosphorylated by a Unique Celluar Protein Kinase and Specific Isomers of Protein Kinase C[J]. Virol, 2000,74(1):91-98.
18 Ashim K. Gupta, Danielle Blondel,Suresh Choudhary,et al.The Phosphoprotein of Rabies Virus Is Phosphorylated by a Unique Cellular Protein Kinase and Specific Isomers of Protein Kinase C[J]. Virol,2000,74:91-98.
19 Fu Z F, Zheng Y, Wunner W H,et al. Both the N and C- terminal domains of the nominal phosphoprotein of rabies virus are involved in binding to the nucleoprotein[J]. Virol, 94:200:590.
20 Yves Jacob, Eleonore Real, and Noel Tordo. Fuctional Interaction Mao oflyssavirus phosphoprotein:Identification of the Minimal Transcription Domains[J]. Virol,2001, 75:9613-9622.
21 Gigant B,Iseni F,Gaudin Y,et al. Neither phosphorylation nor the amino-ternimal part of rabies virus phosphoprotein os required for its oligomerization[J]. GenVirol,2000, 81:1757-1761.
22 Schoehn G,F Iseni,M Mavrakis,et al.Structure of recombinant rabies virus nucleoprotein-RNA complex and identification of the phosphoprotein binding site[J]. Virol,2001,75:490-498.
23 Larson J K, Wunner W H, Otvos L J, et al.Identification of an immunodominant epitope within the phosphoprtein of rabies virus that is recognized by both class Ⅰ-and class Ⅱ-restricted T cells[J]. Virol,1991,65(11):5673.
24 Gao Y, Greenfield N J, Cleverley D Z. The transcriptional form of the phosphoprotein of vesicular stomatitis virus is a trimer:structure and stability[J]. Biochemistry, 1996,35:14569-14573.
25 M Chenik, M Schnell, K K Conzelmann, et al. Mapping the Interacting Domains between the Rabies Virus Polymerase and Phosphoprotein[J].Virol,1998,72:1925-1930.
26 He!le′ne Raux, Fre!de! ric Iseni, Florence Lafay et al. Mapping of monoclonal antibody epitopes of the rebies virus P protein[J].Journal of general virology, 1997, 78:119-124.
27 He′le`ne Raux, Anne Flamand, and Danielle Blongel. Interaction of the Rabies Virus P Protein with the LC8 Dynein Light Chain[J]. Virol, 2000,10212-10216.
28 Jacob Y, Badrane H, Ceccaldi P E, et al. Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein[J].Virol,2000,74(21):10217-10222.
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