2008～2014年中国6省市流行的人呼吸道合胞病毒B亚型G蛋白编码基因特征分析
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摘要
人呼吸道合胞病毒(Human respiratory syncytial virus,HRSV)是引起婴幼儿下呼吸道感染的主要病原体之一。HRSV粘附蛋白(Attachment glycoprotein,G)是病毒膜表面糖蛋白,介导保护性免疫,是抗HRSV抗体和疫苗等的研究热点之一。为研究2008～2014年中国HRSV B亚型G蛋白编码基因的基因特征,于2008～2014年收集的363份HRSV阳性临床标本或病毒株中挑选89份代表株进行G蛋白编码基因全长核苷酸序列的扩增和测序,使用Sequencher 5.0进行序列的编辑,使用MEGA 5.0进行序列比对和亲缘关系进化树构建以及氨基酸变异分析。89份代表株分别挑选自中国北京、吉林、广东、河北、湖南、陕西6省市发热呼吸道感染患者鼻咽拭子或病毒分离株,基因型包括BA9、BA10、BAc、BA、CB1及SAB4,覆盖我国近年流行的优势基因型。亲缘关系分析显示,89株代表株可以分为6个分支,同一基因型G蛋白编码基因核苷酸序列荟聚为一个分支;89份代表株之间的核苷酸(氨基酸)遗传距离为0.03(0.04),与原型株CH-18537株的核苷酸(氨基酸)遗传距离为0.08～0.10(0.14～0.18),其中B5分支与原型株的核苷酸(氨基酸)遗传距离最远为0.10(0.18);氨基酸变异主要在第二高变区,其次为第一高变区。在中心保守区(aa164～176)以及CX3C模序区,89条HRSV B亚型G蛋白编码基因氨基酸序列与原型株CH-18537株一致。本研究表明2008～2014年中国HRSV B亚型G蛋白编码基因变异较多,但在中心保守区和CX3C模序区保守,本研究为我国HRSV的病毒学研究提供了基础科学数据。
Human respiratory syncytial virus(HRSV)is one of the main pathogens,leading to the lower respiratory tract infection in children. Attachment glycoprotein(G)is one of the main membrane surface glycoproteins mediating protective immunity as one of research emphases of the anti-HRSV antibodies and vaccines. In order to study the genetic characteristics of the G protein of HRSV B,eighty-nine representative strains were selected from 363 clinical specimens/isolates identified as HRSV-positive collected from 6 Provinces/cities(Beijing,Jilin,Guangdong,Hebei,Hunan and Shaanxi)in China during 2008 and 2014. The G gene sequences were edited by Sequencher software version 5.0. Phylogenetic trees were built using MEGA software version 5.0. The genotypes of 89 sequences were BA9,BA10,BAc,BA,CB1,and SAB4. Phylogenetic analyses showed that the sequences identified as the same genotype clustered into one lineage. The nucleotide(amino acid)p-distance of 89 sequences was 0.03(0.04),the p-distance between the prototype strain CH-18537 and 89 sequences was0.08～0.10(0.14～0.18)and the nucleotide(amino acid)p-distance between B5 lineage and the prototype strain CH-18537 was the farthest[0.10(0.18)]. Mutations occurred mostly in the second hypervariable region of G protein. Eighty-nine Chinese HRSVB G gene amino acid sequences were highly conserved in the CX3 C motif region and central conserved domain(aa164～176)compared with CH-18537 strain. In the present study,the amino acid of HRSVB G protein had more variation,but they were conserved at the region of central conserved domain and CX3 C motif,which provided the basic data for further study on HRSV prevention and control.
Human respiratory syncytial virus(HRSV)is one of the main pathogens,leading to the lower respiratory tract infection in children. Attachment glycoprotein(G)is one of the main membrane surface glycoproteins mediating protective immunity as one of research emphases of the anti-HRSV antibodies and vaccines. In order to study the genetic characteristics of the G protein of HRSV B,eighty-nine representative strains were selected from 363 clinical specimens/isolates identified as HRSV-positive collected from 6 Provinces/cities(Beijing,Jilin,Guangdong,Hebei,Hunan and Shaanxi)in China during 2008 and 2014. The G gene sequences were edited by Sequencher software version 5.0. Phylogenetic trees were built using MEGA software version 5.0. The genotypes of 89 sequences were BA9,BA10,BAc,BA,CB1,and SAB4. Phylogenetic analyses showed that the sequences identified as the same genotype clustered into one lineage. The nucleotide(amino acid)p-distance of 89 sequences was 0.03(0.04),the p-distance between the prototype strain CH-18537 and 89 sequences was0.08～0.10(0.14～0.18)and the nucleotide(amino acid)p-distance between B5 lineage and the prototype strain CH-18537 was the farthest[0.10(0.18)]. Mutations occurred mostly in the second hypervariable region of G protein. Eighty-nine Chinese HRSVB G gene amino acid sequences were highly conserved in the CX3 C motif region and central conserved domain(aa164～176)compared with CH-18537 strain. In the present study,the amino acid of HRSVB G protein had more variation,but they were conserved at the region of central conserved domain and CX3 C motif,which provided the basic data for further study on HRSV prevention and control.
引文
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[7] Boyoglu-Barnum S,Todd S O,Chirkova T,Barnum T R,Gaston K A,Haynes L M,Tripp R A,Moore M L,Anderson L J. An anti-G protein monoclonal anti-body treats RSV disease more effectively than an anti-F monoclonal antibody in BALB/c mice[J]. Virology,2015,483:117-125.
[8] Boyoglu-Barnum S,Todd S O,Meng J,Barnum T R,Chirkova T,Haynes L M,Jadhao S J,Tripp R A,Oo-mens A G,Moore M L,Anderson L J,Perlman S. Mu-tating the CX3C motif in the G Protein should make a live respiratory syncytial virus vaccine safer and more ef-fective[J/OL]. J Virol,2017,91(10):e02059-16.
[9] Van Elden L J,van Loon Am Fau-van der Beek A,van der Beek A Fau-Hendriksen K A W,Hendriksen Ka Fau-Hoepelman A I M,Hoepelman Ai Fau-van Kraaij M G J,van Kraaij Mg Fau-Schipper P,Schip-per P Fau-Nijhuis M,Nijhuis M. Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults[J]. J Clin Microbiol,2003,41(9):4378-4381.
[10]Song J,Wang H,Shi J,Cui A,Huang Y,Sun L,Xiang X,Ma C,Yu P,Yang Z,Li i,Ng T Ig,Zhang Y,Zhang R,Xu W. Emergence of BA9 genotype of hu-man respiratory syncytial virus subgroup B in China from2006 to 2014[J]. Sci Rep,2017,7(1):16765.
[11]Peret T C,Hall Cb Fau-Schnabel K C,Schnabel Kc Fau-Golub J A,Golub Ja Fau-Anderson L J,Ander-son L J. Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community[J]. J Gen Virol,1998,79(Pt 9):2221-2229.
[12]Caidi H,Miao C,Thornburg N J,Tripp R A,Ander-son L J,Haynes L M. Anti-respiratory syncytial virus(RSV)G monoclonal antibodies reduce lung inflamma-tion and viral lung titers when delivered therapeutically in a BALB/c mouse model[J]. Antivir Res,2018,154:149-157.
[13]Collarini E J,Lee F E H,Foord O,Park M,Sperinde G,Wu H,Harriman W D,Carroll S F,Ellsworth S L,Anderson L J,Tripp R A,Walsh E E,Keyt B A,Kauvar L M. Potent high-affinity antibodies for treat-ment and prophylaxis of respiratory syncytial virus de-rived from B cells of infected patients[J]. J Immunol,2009,183(10):6338-6345.
[14]宋金华,王慧玲,石晶,崔爱利,杨子峰,孙利伟,于德山,张蕾,张红,张燕,许文波. 2008-2014年中国8省市流行的人呼吸道合胞病毒A亚型G蛋白编码基因全长序列分析[J].病毒学报,2017,33(6):821-828.DOI:10.13242/j.cnki.bingduxuebao.003253
[15]Kushibuchi I,Kobayashi M,Kusaka T,Tsukagoshi H,Ryo A,Yoshida A,Ishii H,Saraya T,Kurai D,Yama-moto N,Kanou K,Saitoh M,Noda M,Kuroda M,Morita Y,Kozawa K,Oishi K,Tashiro M,Kimura H.Molecular evolution of attachment glycoprotein(G)gene in human respiratory syncytial virus detected in Ja-pan 2008–2011[J]. Infect Genet Evol,2013;18:168-173.
[16]Harcourt J,Alvarez R,Jones L P,Henderson C,An-derson L J,Tripp R A. Respiratory syncytial virus G protein and G protein CX3C motif adversely affect CX3CR1+T cell responses[J]. J Immunol,2006,176(3):1600-1608.
[17]Chirkova T,Boyoglu-Barnum S,Gaston K A,Malik F M,Trau S P,Oomens A G P,Anderson L J. Respira-tory syncytial virus G protein CX3C motif impairs hu-man airway epithelial and immune cell responses[J]. J Virol,2013,87(24):13466-13479.
[18]Chirkova T,Stobart C C,Hartert T V,Lin S,Gaston K A,Anderson L J,Oomens A G P,Boyoglu-Barnum S,Moore M L,Ziady A G,Meng J,Cotton C U.CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells[J]. J Gen Virol,2015,96(9):2543-2556.
[2] Mufson M A,Orvell C,Rafnar B,Norrby E. Two dis-tinct subtypes of human respiratory syncytial virus[J]. J Gen Virol,1985,66(Pt 10):2111-2124.
[3] Song J,Zhang Y,Wang H,Shi J,Sun L,Zhang X,Yang Z,Guan W,Zhang H,Yu P,Xie Z,Cui A,Ng T I,Xu W. Emergence of ON1 genotype of human re-spiratory syncytial virus subgroup A in China between2011 and 2015[J]. Sci Rep,2017,7(1):5501.
[4] Turner Stephen J,Cui G,Zhu R,Qian Y,Deng J,Zhao L,Sun Y,Wang F. Genetic variation in attach-ment glycoprotein genes of human respiratory syncytial virus subgroups A and B in children in recent five consec-utive years[J/OL]. PLoS One,2013,8(9):e75020.
[5] Trento A,ábrego L, Rodriguez-Fernandez R,González-Sánchez M I,González-Martínez F,Delfraro A,Pascale J M,Arbiza J,Melero J A,Lyles D S. Con-servation of G-protein epitopes in respiratory syncytial vi-rus(Group A)despite broad genetic diversity:is anti-body selection involved in virus evolution?[J]. J Virol,2015,89(15):7776-7785.
[6] McLellan J S,Yang Y,Graham B S,Kwong P D.Structure of respiratory syncytial virus fusion glycopro-tein in the postfusion conformation reveals preservation of neutralizing epitopes[J]. J Virol,2011,85(15):7788-7796.
[7] Boyoglu-Barnum S,Todd S O,Chirkova T,Barnum T R,Gaston K A,Haynes L M,Tripp R A,Moore M L,Anderson L J. An anti-G protein monoclonal anti-body treats RSV disease more effectively than an anti-F monoclonal antibody in BALB/c mice[J]. Virology,2015,483:117-125.
[8] Boyoglu-Barnum S,Todd S O,Meng J,Barnum T R,Chirkova T,Haynes L M,Jadhao S J,Tripp R A,Oo-mens A G,Moore M L,Anderson L J,Perlman S. Mu-tating the CX3C motif in the G Protein should make a live respiratory syncytial virus vaccine safer and more ef-fective[J/OL]. J Virol,2017,91(10):e02059-16.
[9] Van Elden L J,van Loon Am Fau-van der Beek A,van der Beek A Fau-Hendriksen K A W,Hendriksen Ka Fau-Hoepelman A I M,Hoepelman Ai Fau-van Kraaij M G J,van Kraaij Mg Fau-Schipper P,Schip-per P Fau-Nijhuis M,Nijhuis M. Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults[J]. J Clin Microbiol,2003,41(9):4378-4381.
[10]Song J,Wang H,Shi J,Cui A,Huang Y,Sun L,Xiang X,Ma C,Yu P,Yang Z,Li i,Ng T Ig,Zhang Y,Zhang R,Xu W. Emergence of BA9 genotype of hu-man respiratory syncytial virus subgroup B in China from2006 to 2014[J]. Sci Rep,2017,7(1):16765.
[11]Peret T C,Hall Cb Fau-Schnabel K C,Schnabel Kc Fau-Golub J A,Golub Ja Fau-Anderson L J,Ander-son L J. Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community[J]. J Gen Virol,1998,79(Pt 9):2221-2229.
[12]Caidi H,Miao C,Thornburg N J,Tripp R A,Ander-son L J,Haynes L M. Anti-respiratory syncytial virus(RSV)G monoclonal antibodies reduce lung inflamma-tion and viral lung titers when delivered therapeutically in a BALB/c mouse model[J]. Antivir Res,2018,154:149-157.
[13]Collarini E J,Lee F E H,Foord O,Park M,Sperinde G,Wu H,Harriman W D,Carroll S F,Ellsworth S L,Anderson L J,Tripp R A,Walsh E E,Keyt B A,Kauvar L M. Potent high-affinity antibodies for treat-ment and prophylaxis of respiratory syncytial virus de-rived from B cells of infected patients[J]. J Immunol,2009,183(10):6338-6345.
[14]宋金华,王慧玲,石晶,崔爱利,杨子峰,孙利伟,于德山,张蕾,张红,张燕,许文波. 2008-2014年中国8省市流行的人呼吸道合胞病毒A亚型G蛋白编码基因全长序列分析[J].病毒学报,2017,33(6):821-828.DOI:10.13242/j.cnki.bingduxuebao.003253
[15]Kushibuchi I,Kobayashi M,Kusaka T,Tsukagoshi H,Ryo A,Yoshida A,Ishii H,Saraya T,Kurai D,Yama-moto N,Kanou K,Saitoh M,Noda M,Kuroda M,Morita Y,Kozawa K,Oishi K,Tashiro M,Kimura H.Molecular evolution of attachment glycoprotein(G)gene in human respiratory syncytial virus detected in Ja-pan 2008–2011[J]. Infect Genet Evol,2013;18:168-173.
[16]Harcourt J,Alvarez R,Jones L P,Henderson C,An-derson L J,Tripp R A. Respiratory syncytial virus G protein and G protein CX3C motif adversely affect CX3CR1+T cell responses[J]. J Immunol,2006,176(3):1600-1608.
[17]Chirkova T,Boyoglu-Barnum S,Gaston K A,Malik F M,Trau S P,Oomens A G P,Anderson L J. Respira-tory syncytial virus G protein CX3C motif impairs hu-man airway epithelial and immune cell responses[J]. J Virol,2013,87(24):13466-13479.
[18]Chirkova T,Stobart C C,Hartert T V,Lin S,Gaston K A,Anderson L J,Oomens A G P,Boyoglu-Barnum S,Moore M L,Ziady A G,Meng J,Cotton C U.CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells[J]. J Gen Virol,2015,96(9):2543-2556.
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