广西6株狂犬病分离毒株P和M基因的克隆和序列分析
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摘要
为了探讨广西狂犬病病毒在分子水平上的变异规律,本试验应用RT-PCR技术,通过NSM1/NSM2引物,对6个广西毒株GXNN2、GXPL、GXPXD、GXQZD、GXYZD和GXNND的P和M基因进行扩增、克隆和测序。将克隆的P和M基因ORF的核苷酸及其推导的氨基酸序列,与本实验室已测定的23株及国内外公开发表的16株狂犬病病毒相应序列进行同源性和遗传进化树分析。结果显示,不管基于P基因还是M基因,进化树分析表明,广西狂犬病分离毒株都可分为3个群:Ⅰ群、Ⅱ群、Ⅲ群。Ⅰ群包括GXHX82、GXHXB、GXLC9、GXQZD、GX08、GXNND、GXHX、GX014、GX195、GXLB、GXSL、GXWXp、GX260、GX01、GX09、GX091和GXLA共17株,Ⅱ群包括GXLCC、GXPL、GXYZD、GXBM、GX219、GX304、GXLA11、GXNN2、GX074、GXPX和GXPXD共11株,Ⅲ群仅有GXN119株。本次克隆的6个毒株中,GXQZD和GXNND株属于Ⅰ群,GXPL、GXYZD、GXNN2和GXPXD属于Ⅱ群。
同源性分析表明,本次克隆的6个毒株P基因编码区核苷酸及其推导的氨基酸序列同源性分别在85.8%—99.6%和91.2%—99.7%之间;M基因的分别在89.7%—100%和96%—100%之间。此6株与之前测序的23个广西毒株P基因编码区核苷酸及其推导的氨基酸序列同源性分别在85%—100%和90.2%—100%之间,M基因的分别在88.5%—100%和95.5%—100%之间。
比较的P基因和M基因的氨基酸特异位点,结果发现,广西毒株P基因氨基酸第69、70、130和131位属于Ⅰ群毒株特异性位点;第134、135、140和151位为Ⅱ群毒株特异性位点;第73、162、167、174、240、253和281位为Ⅲ群特异性位点;第90位为广西Ⅰ、Ⅱ群共有的特异性位点;第57,63和241位属于Ⅰ、Ⅱ、Ⅲ群毒株共有的特异性位点;广西毒株M基因氨基酸第17和20位属于Ⅱ群毒株特异性位点。
To understand the molecular characteristics of rabies virus isolates from Guangxi, we amplified the P gene and M gene of six isolates GXNN2, GXPL, GXPXD, GXQZD, GXYZD and GXNND, by RT-PCR with primers NSM1/NSM2. The amplified cDNA fragments were then cloned and sequenced.
The P and M gene ORF nucleotide sequences of twenty-nine isolates, including twenty-three isolates isolated previously in our laboratory and other Sixteen isolates from China and abroad were compared. Whatever used the P gene or the M gene to construct the phylogenetic tree, both showed that rabies virus isolates from Guangxi could be divided into three groups. Seventeen isolates GXHX82, GXHXB, GXLC9, GXQZD, GX08, GXNND, GXHX, GX014, GX195, GXLB, GXSL, GXWXp, GX260, GX01, GX09, GX091 and GXLA belong to groupⅠ. Eleven isolates GXLCC, GXPL, GXYZD, GXBM, GX219, GX304, GXLA11, GXNN2, GX074, GXPX and GXPXD belong to groupⅡ. GXN119 isolate belongs to groupⅢSix isolates in this experiment, two isolates GXQZD and GXNND belong to groupⅠ, four isolates GXPL, GXYZD, GXNN2 and GXPXD belong to groupⅡ.
The homology analysis of the sequences of nucleotide and their deduced amino acid indicates that of P gene among the six isolates in this experiment range from 85.8% to 99.6% and 91.2% to 99.7, respectively. That of M gene range from 89.7% to 100% and 96% to 100%, respectively. That of P gene between the six isolates and twenty-three isolates in our laboratory range from 85% to 100% and 90.2% to 100%, respectively. That of M gene range from 88.5% to 100% and 95.5% to 100%, respectively.
Additionally, we found that P gene amino acid specific mutation sites in group I are 69, 70, 130 and 131, in group II are 134, 135, 140 and 151, in groupⅢare 73,162,167,174, 240, 253 and 281, the 90 amino acid sites is specific for the group I andⅡ, while the 57, 63 and 241 are specific for the groupⅠ,ⅡandⅢ. M gene amino acid specific mutation sites in group II is 17 and 20.
同源性分析表明,本次克隆的6个毒株P基因编码区核苷酸及其推导的氨基酸序列同源性分别在85.8%—99.6%和91.2%—99.7%之间;M基因的分别在89.7%—100%和96%—100%之间。此6株与之前测序的23个广西毒株P基因编码区核苷酸及其推导的氨基酸序列同源性分别在85%—100%和90.2%—100%之间,M基因的分别在88.5%—100%和95.5%—100%之间。
比较的P基因和M基因的氨基酸特异位点,结果发现,广西毒株P基因氨基酸第69、70、130和131位属于Ⅰ群毒株特异性位点;第134、135、140和151位为Ⅱ群毒株特异性位点;第73、162、167、174、240、253和281位为Ⅲ群特异性位点;第90位为广西Ⅰ、Ⅱ群共有的特异性位点;第57,63和241位属于Ⅰ、Ⅱ、Ⅲ群毒株共有的特异性位点;广西毒株M基因氨基酸第17和20位属于Ⅱ群毒株特异性位点。
To understand the molecular characteristics of rabies virus isolates from Guangxi, we amplified the P gene and M gene of six isolates GXNN2, GXPL, GXPXD, GXQZD, GXYZD and GXNND, by RT-PCR with primers NSM1/NSM2. The amplified cDNA fragments were then cloned and sequenced.
The P and M gene ORF nucleotide sequences of twenty-nine isolates, including twenty-three isolates isolated previously in our laboratory and other Sixteen isolates from China and abroad were compared. Whatever used the P gene or the M gene to construct the phylogenetic tree, both showed that rabies virus isolates from Guangxi could be divided into three groups. Seventeen isolates GXHX82, GXHXB, GXLC9, GXQZD, GX08, GXNND, GXHX, GX014, GX195, GXLB, GXSL, GXWXp, GX260, GX01, GX09, GX091 and GXLA belong to groupⅠ. Eleven isolates GXLCC, GXPL, GXYZD, GXBM, GX219, GX304, GXLA11, GXNN2, GX074, GXPX and GXPXD belong to groupⅡ. GXN119 isolate belongs to groupⅢSix isolates in this experiment, two isolates GXQZD and GXNND belong to groupⅠ, four isolates GXPL, GXYZD, GXNN2 and GXPXD belong to groupⅡ.
The homology analysis of the sequences of nucleotide and their deduced amino acid indicates that of P gene among the six isolates in this experiment range from 85.8% to 99.6% and 91.2% to 99.7, respectively. That of M gene range from 89.7% to 100% and 96% to 100%, respectively. That of P gene between the six isolates and twenty-three isolates in our laboratory range from 85% to 100% and 90.2% to 100%, respectively. That of M gene range from 88.5% to 100% and 95.5% to 100%, respectively.
Additionally, we found that P gene amino acid specific mutation sites in group I are 69, 70, 130 and 131, in group II are 134, 135, 140 and 151, in groupⅢare 73,162,167,174, 240, 253 and 281, the 90 amino acid sites is specific for the group I andⅡ, while the 57, 63 and 241 are specific for the groupⅠ,ⅡandⅢ. M gene amino acid specific mutation sites in group II is 17 and 20.
引文
[1] Hummeler K, Koprowski H, Wiktor TJ. Structure and development of rabies virus in tissue culture. J Virol 1967,1:152-170.
[2] Gould AR, Hyatt AD, Lunt R, et al.Characterization of a novel lyssavirus isolated Pteropid bats in Austrlia. Virus Res 1998,54:165-87.
[3] Badrane H, Bahloul C, Perrin P, et al. Evidence of two lyssavirus phylogroup with distinctpathogenicity and immunogenicity. J Virol 2001, 75:3268-3276.
[4] Kissi B, Tordo N, Bota'hy H. Genetic polymorphism in the rabies nudeoproetin gene. Virol 1995, 209: 526-537.
[5]Zaides V M, Krotova L I, Selimova L M,et al. Reevaluation of the proteins in rabies virus particles. J Virol, 1979, 29: 1226-1228.
[6] Ertl HC, Fischer S, Flamand A. Phosphorylation of the N and Ml proteins of rabies virus. J Virol, 1985, 66: 2285-2289.
[7] Emerson S U, and Schubert M. Location of the binding domains for the RNA polymerase L and the ribonucleocapsid template within different halves of the NS phosphoprotein of vesicular stomatitis virus. Proc. Natl. Acad. Sci. USA. 1987, 84:5655-5659.
[8] Jacob Y, Real E, Tordo N. Functional interaction map of lyssavirus phosphoprotein: identification of the minimal transcription domains. J Virol 2001,75:9613-9622.
[9] Fu ZF, Y Zheng, Wunner WH,et al. Both the N- and the C-terminal domains of thenominal phosphoprotein of rabies virus are involved in binding to the nucleoprotein. Virol 1994, 200: 590-597.
[10]Chenik M, Schnell M, Conzelmann KK, et al. Mapping the Interacting Domains between the Rabies Virus Polymerase and Phosphoprotein. J Virol 1998, 72: 1925-1930.
[11]Ashim K. Gupta, Danielle Blondel. 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.
[12] Raux H, Flamand A, Blondel D. Interaction of the rabies virus P protein with the LC8 dynein light chain. J Virol 2000, 74:10212-10216.
[13] Mebatsion T. Extensive attenuation of rabies virus by simultaneously modifying the dynein light chain binding site in the P protein and replacing Arg333 in the G protein. J Virol 2001, 75:11496-11502.
[14]Jacob Y, Badrane H, Ceccaldi PE, et al. Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein. J Virol 2000, 74:10217-10222.
[15] Chenik M, Chebli K, Blondel D. Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism. J Virol 1995, 69:707-712.
[16]Brzo'zka K, Finke S, and Conzelmann KK, Identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein P interferes with phosphorylation of interferon regulatory factor 3. J Virol 2005, 79: 7673-7681,
[17] Vidy A, Chelbi-Alix M, Blondel D. Rabies virus P protein interacts with STAT1 and inhibits interferon signal transduction pathways. J Virol 2005,79:14411-14420.
[18] Vidy A, El Bougrini J, Chelbi-Alix MK, et al. The nucleocytoplasmic rabies virus P protein counteracts interferon signaling by inhibiting both nuclear accumulation and DNA binding of STAT1. J Virol 2007,81:4255-4263.
[19]Brzozka K, Finke S, Conzelmann KK. Inhibition of interferon signaling by rabies virus phosphoprotein P: activation-dependent binding of STAT1 and STAT2. J Virol 2006,80:2675-2683.
[20] Blondel D, Regad T, Poisson N, et al. Rabies virus P and small P products interact directly with PML and reorganize PML nuclear bodies. Oncogene 2002,21:7957-7970.
[21] Rayssiguier C, Cioe L, Withers E, et al. Cloning of rabies virus matrix protein mRNA and determination of its amino acid sequence. Virus Res 1986,5:177-190.
[22] Mebatsion T, Weiland F, Conzelmann KK. Matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein G. J Virol 1999,73:242-250.
[23] Ronald N. Harty, Jason Paragas, et al. A Proline-Rich Motif within the Matrix Protein of Vesicular Stomatitis Virus and Rabies Virus Interacts with WW Domains of Cellular Proteins: Implications for Viral Budding. J Virol 1999, 73: 2921-2929.
[24] Harty RN, Paragas J,Sudol M, et al. A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding. J Virol 1999, 73:2921-2929.
[25] Harty RN., Brown ME, McGettigan JP, et al. Rhabdoviruses and the cellular ubiquitin- proteasome system: a budding interaction. J Virol 2001, 75: 10623-10629.
[26] Finke S, Mueller-Waldeck R, Conzelmann KK. Rabies virus matrix protein regulates the balance of virus transcription and replication. J Gen Virol 2003, 84:1613-1621.
[27] Stefan Finke and Karl-Klaus Conzelmann. Dissociation of Rabies Virus Matrix Protein Functions in Regulation of Viral RNA Synthesis and Virus Assembly. J Virol 2003,77: 12074-12082.
[28] Raid Kassis,Florence Larrous,Jerome Estaquier,et al. Lyssavirus matrix protein induces apoptosis by a trail-dependent mechanism involving caspase-8. J Virol 2004, 78: 6543— 6555.
[29] Jackson AC. Apoptosis in experinental rabies in bax-deficient mice. Acta Neuropathol 1999, 98(3): 288-294.
[30] Connor JH, and Lyles DS. Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1. J Virol 2002, 76,10177-10187.
[31] Connor JH. and Lyles DS. Inhibition of host and viral translation during vesicular stomatitis virus infection. eIF2 is responsible for the inhibition of viral but not host translation. J Biol Chem 2005, 280,13512 - 13519.
[32] Komarova AV, Real E, Borman AM, et al. Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis. Nucleic Acids Res 2007,35:1522-1532.
[33] Sizova DV, Kolupaeva VG, Pestova TV,et al. Specific interaction of eukaryotic translation initiation factor 3 with the 50 nontranslated regions of hepatitis C virus and classical swine fever virus RNAs. J Virol 1998, 72, 4775 - 4782.
[34] Pisarev AV, Shirokikh NE, and Hellen CU. Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites. C. R. Biol 2005., 328, 589 - 605.
[35] Gupta AK, Banerjee AK. Expression and purification of vesicular stomatitis virus N-P complex from Escherichia coli: role in genome RNA transcription and replication in vitro . J Virol 1997, 71: 4264-4271.
[36] Sacramento D, Badrane H, Bourhy H, et al. Molecular epidemiology of rabies virus in France: comparison with vaccine strains. J Gen Virol, 1992, 73: 1149-58.
[37] Finke S, Cox JH, Conzelmann KK. Differential transcription attenuation of rabies virus genes by intergenic regions: generation of recombinant viruses overexpressing the polymerase gene. J Virol 2000, 74: 7261-7269.
[38]Martinez L.Global infectious disease surveillance.Int.J.Infect.Dis.2000,4:222-228.
[39]张德礼,高步先,朱关福.狂犬病基因工程疫苗研究进展.农业生物技术学报,1994,2(1):14-20.
[40]雷芝樱,吴泰才,吕元聪.1996～2005年广西狂犬病流行病学特点分析.应用预防医学,2007,13(2):94-95.
[2] Gould AR, Hyatt AD, Lunt R, et al.Characterization of a novel lyssavirus isolated Pteropid bats in Austrlia. Virus Res 1998,54:165-87.
[3] Badrane H, Bahloul C, Perrin P, et al. Evidence of two lyssavirus phylogroup with distinctpathogenicity and immunogenicity. J Virol 2001, 75:3268-3276.
[4] Kissi B, Tordo N, Bota'hy H. Genetic polymorphism in the rabies nudeoproetin gene. Virol 1995, 209: 526-537.
[5]Zaides V M, Krotova L I, Selimova L M,et al. Reevaluation of the proteins in rabies virus particles. J Virol, 1979, 29: 1226-1228.
[6] Ertl HC, Fischer S, Flamand A. Phosphorylation of the N and Ml proteins of rabies virus. J Virol, 1985, 66: 2285-2289.
[7] Emerson S U, and Schubert M. Location of the binding domains for the RNA polymerase L and the ribonucleocapsid template within different halves of the NS phosphoprotein of vesicular stomatitis virus. Proc. Natl. Acad. Sci. USA. 1987, 84:5655-5659.
[8] Jacob Y, Real E, Tordo N. Functional interaction map of lyssavirus phosphoprotein: identification of the minimal transcription domains. J Virol 2001,75:9613-9622.
[9] Fu ZF, Y Zheng, Wunner WH,et al. Both the N- and the C-terminal domains of thenominal phosphoprotein of rabies virus are involved in binding to the nucleoprotein. Virol 1994, 200: 590-597.
[10]Chenik M, Schnell M, Conzelmann KK, et al. Mapping the Interacting Domains between the Rabies Virus Polymerase and Phosphoprotein. J Virol 1998, 72: 1925-1930.
[11]Ashim K. Gupta, Danielle Blondel. 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.
[12] Raux H, Flamand A, Blondel D. Interaction of the rabies virus P protein with the LC8 dynein light chain. J Virol 2000, 74:10212-10216.
[13] Mebatsion T. Extensive attenuation of rabies virus by simultaneously modifying the dynein light chain binding site in the P protein and replacing Arg333 in the G protein. J Virol 2001, 75:11496-11502.
[14]Jacob Y, Badrane H, Ceccaldi PE, et al. Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein. J Virol 2000, 74:10217-10222.
[15] Chenik M, Chebli K, Blondel D. Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism. J Virol 1995, 69:707-712.
[16]Brzo'zka K, Finke S, and Conzelmann KK, Identification of the rabies virus alpha/beta interferon antagonist: phosphoprotein P interferes with phosphorylation of interferon regulatory factor 3. J Virol 2005, 79: 7673-7681,
[17] Vidy A, Chelbi-Alix M, Blondel D. Rabies virus P protein interacts with STAT1 and inhibits interferon signal transduction pathways. J Virol 2005,79:14411-14420.
[18] Vidy A, El Bougrini J, Chelbi-Alix MK, et al. The nucleocytoplasmic rabies virus P protein counteracts interferon signaling by inhibiting both nuclear accumulation and DNA binding of STAT1. J Virol 2007,81:4255-4263.
[19]Brzozka K, Finke S, Conzelmann KK. Inhibition of interferon signaling by rabies virus phosphoprotein P: activation-dependent binding of STAT1 and STAT2. J Virol 2006,80:2675-2683.
[20] Blondel D, Regad T, Poisson N, et al. Rabies virus P and small P products interact directly with PML and reorganize PML nuclear bodies. Oncogene 2002,21:7957-7970.
[21] Rayssiguier C, Cioe L, Withers E, et al. Cloning of rabies virus matrix protein mRNA and determination of its amino acid sequence. Virus Res 1986,5:177-190.
[22] Mebatsion T, Weiland F, Conzelmann KK. Matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein G. J Virol 1999,73:242-250.
[23] Ronald N. Harty, Jason Paragas, et al. A Proline-Rich Motif within the Matrix Protein of Vesicular Stomatitis Virus and Rabies Virus Interacts with WW Domains of Cellular Proteins: Implications for Viral Budding. J Virol 1999, 73: 2921-2929.
[24] Harty RN, Paragas J,Sudol M, et al. A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding. J Virol 1999, 73:2921-2929.
[25] Harty RN., Brown ME, McGettigan JP, et al. Rhabdoviruses and the cellular ubiquitin- proteasome system: a budding interaction. J Virol 2001, 75: 10623-10629.
[26] Finke S, Mueller-Waldeck R, Conzelmann KK. Rabies virus matrix protein regulates the balance of virus transcription and replication. J Gen Virol 2003, 84:1613-1621.
[27] Stefan Finke and Karl-Klaus Conzelmann. Dissociation of Rabies Virus Matrix Protein Functions in Regulation of Viral RNA Synthesis and Virus Assembly. J Virol 2003,77: 12074-12082.
[28] Raid Kassis,Florence Larrous,Jerome Estaquier,et al. Lyssavirus matrix protein induces apoptosis by a trail-dependent mechanism involving caspase-8. J Virol 2004, 78: 6543— 6555.
[29] Jackson AC. Apoptosis in experinental rabies in bax-deficient mice. Acta Neuropathol 1999, 98(3): 288-294.
[30] Connor JH, and Lyles DS. Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1. J Virol 2002, 76,10177-10187.
[31] Connor JH. and Lyles DS. Inhibition of host and viral translation during vesicular stomatitis virus infection. eIF2 is responsible for the inhibition of viral but not host translation. J Biol Chem 2005, 280,13512 - 13519.
[32] Komarova AV, Real E, Borman AM, et al. Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis. Nucleic Acids Res 2007,35:1522-1532.
[33] Sizova DV, Kolupaeva VG, Pestova TV,et al. Specific interaction of eukaryotic translation initiation factor 3 with the 50 nontranslated regions of hepatitis C virus and classical swine fever virus RNAs. J Virol 1998, 72, 4775 - 4782.
[34] Pisarev AV, Shirokikh NE, and Hellen CU. Translation initiation by factor-independent binding of eukaryotic ribosomes to internal ribosomal entry sites. C. R. Biol 2005., 328, 589 - 605.
[35] Gupta AK, Banerjee AK. Expression and purification of vesicular stomatitis virus N-P complex from Escherichia coli: role in genome RNA transcription and replication in vitro . J Virol 1997, 71: 4264-4271.
[36] Sacramento D, Badrane H, Bourhy H, et al. Molecular epidemiology of rabies virus in France: comparison with vaccine strains. J Gen Virol, 1992, 73: 1149-58.
[37] Finke S, Cox JH, Conzelmann KK. Differential transcription attenuation of rabies virus genes by intergenic regions: generation of recombinant viruses overexpressing the polymerase gene. J Virol 2000, 74: 7261-7269.
[38]Martinez L.Global infectious disease surveillance.Int.J.Infect.Dis.2000,4:222-228.
[39]张德礼,高步先,朱关福.狂犬病基因工程疫苗研究进展.农业生物技术学报,1994,2(1):14-20.
[40]雷芝樱,吴泰才,吕元聪.1996～2005年广西狂犬病流行病学特点分析.应用预防医学,2007,13(2):94-95.