一株B亚型人呼吸道合胞病毒兰州分离株的全基因序列测定及其分子特征分析
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摘要
人呼吸道合胞病毒(Human respiratory syncytial virus,HRSV)是每年引起婴幼儿和老年人下呼吸道感染的主要病毒性病原,在全球造成了高疾病负担,明确RSV流行毒株的遗传背景是其流行病学研究和预防控制的基础。为了解HRSV兰州分离株RSV B/LZ11/12的全基因组序列、分子结构特征及遗传变异情况,本研究对其全基因组进行扩增克隆和测序后,与GenBank中相关的参考毒株序列构建系统发生树并进行遗传进化及相似性分析。针对其主要蛋白F、G和SH的氨基酸进行了比对和差异分析,并对F蛋白的N-糖基化位点进行分析。结果表明B/LZ11/12与B亚型RSV的核苷酸同源性高达99%以上,其基因组全长15 275bp,具有RSV的所有结构特征。遗传进化分析显示,B/LZ11/12与B/GZ/13-730、B/England138/2016和SC2225同属于一个分支,提示亲缘关系最近。G基因遗传进化树表明该毒株属于RSV B亚型BA9基因型。主要氨基酸位点分析显示该毒株的G基因和F基因均具有一个特有的变化,即Glu87Gly和Val243Ala的氨基酸替换。F蛋白N-糖基化位点分析显示该毒株具有RSV F蛋白的5个共有修饰位点,即27～30(NITE)、70～73(NGTD)、116～119(NYTI)、120～123(NTTK)和126～129(NVSI)。相似性分析表明整个基因组最易变异的区域在SH和G基因,其次是NS1、NS2和L基因的3′端。本研究首次获得了RSV B/LZ11/12兰州分离株全基因组序列,并阐明了其基因组分子结构特征,该结果对RSV病毒的基因及氨基酸数据库进行了数据补充,也为我国RSV流行病学数据进行了补充。
Human respiratory syncytial virus(HRSV)is a major viral pathogen causing lower respiratory tract infections in infants,young children and older adults every year. It has caused a high burden of disease worldwide. It is the basis of epidemiological research and prevention and control to identify the genetic background of respiratory syncytial virus(RSV) epidemic strains. In order to understand the whole genome sequence,molecular structure and genetic variation of HRSV Lanzhou isolate RSV B/LZ11/12,the whole genome was amplified,cloned and sequenced. The phylogenetic tree was constructed with reference sequences of other RSV strains in GenBank. Genetic evolution and similarity analysis were carried out. The amino acids of major proteins F,G and SH were compared and analyzed. In addition,the N-glycosylation sites of F protein were analyzed. The results showed that the nucleotide homology between B/LZ11/12 and other B subtype RSV strains was over 99%. The genome length of B/LZ11/12 was 15,275 bp,which showed all the structural characteristics of RSV. Genetic evolution analysis showed that B/LZ11/12 belonged to the same branch as B/GZ/13-730,B/England 138/2016 and SC225. The genetic evolution tree of G gene indicated that the strain belonged to BA9 genotype of RSV B subtype. The analysis of major amino acid loci showed that the G gene and F gene of the strain had unique change,that is amino acid substitution of Glu87 Gly and Val243 Ala. Nglycosylation site analysis of F protein showed that the strain contains five common modification sites in RSV F protein,namely 27～30(NITE),70～73(NGTD),116～119(NYTI),120～123(NTTK)and 126～129(NVSI). Similarity analysis showed that highly variable regions of the whole genome were SH and G genes,followed by NS1,NS2 and L genes at the 3′ end. The whole genome sequence of RSV B/LZ11/12 Lanzhou isolate was obtained for the first time and its molecular structure was elucidated.
Human respiratory syncytial virus(HRSV)is a major viral pathogen causing lower respiratory tract infections in infants,young children and older adults every year. It has caused a high burden of disease worldwide. It is the basis of epidemiological research and prevention and control to identify the genetic background of respiratory syncytial virus(RSV) epidemic strains. In order to understand the whole genome sequence,molecular structure and genetic variation of HRSV Lanzhou isolate RSV B/LZ11/12,the whole genome was amplified,cloned and sequenced. The phylogenetic tree was constructed with reference sequences of other RSV strains in GenBank. Genetic evolution and similarity analysis were carried out. The amino acids of major proteins F,G and SH were compared and analyzed. In addition,the N-glycosylation sites of F protein were analyzed. The results showed that the nucleotide homology between B/LZ11/12 and other B subtype RSV strains was over 99%. The genome length of B/LZ11/12 was 15,275 bp,which showed all the structural characteristics of RSV. Genetic evolution analysis showed that B/LZ11/12 belonged to the same branch as B/GZ/13-730,B/England 138/2016 and SC225. The genetic evolution tree of G gene indicated that the strain belonged to BA9 genotype of RSV B subtype. The analysis of major amino acid loci showed that the G gene and F gene of the strain had unique change,that is amino acid substitution of Glu87 Gly and Val243 Ala. Nglycosylation site analysis of F protein showed that the strain contains five common modification sites in RSV F protein,namely 27～30(NITE),70～73(NGTD),116～119(NYTI),120～123(NTTK)and 126～129(NVSI). Similarity analysis showed that highly variable regions of the whole genome were SH and G genes,followed by NS1,NS2 and L genes at the 3′ end. The whole genome sequence of RSV B/LZ11/12 Lanzhou isolate was obtained for the first time and its molecular structure was elucidated.
引文
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[7]Ahmed A,Haider S H,Parveen S,Arshad M,Alsenaidy H A,Baaboud A O,Mobaireek K F,AlSaadi M M,Alsenaidy A M,SullenderW.Cocirculation of 72bp duplication group A and 60bp duplication group B respiratory syncytial virus(RSV)strains in Riyadh,Saudi Arabia during 2014[J/OL].PLoS One,2016,11(11):e0166145.
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[11]Eshaghi A,Duvvuri V R,Lai R,Nadarajah J T,Li A,Patel S N,Low D E,Gubbay JB.Genetic variability of human respiratory syncytial virus A strains circulating in Ontario:a novel genotype with a 72nucleotide G gene duplication[J/OL].PLoS One,2012,7(3):e32807.
[12]Lee W J,Kim Y J,Kim D W,Lee H S,Lee H Y,Kim K.Complete genome sequence of human respiratory syncytial virus genotype A with a 72-nucleotide duplication in the attachment protein G gene[J].J Virol.2012,86(24):13810-13811.
[13]Prifert C,Streng A,Krempl C D,Liese J,Weissbrich B.Novel respiratory syncytial virus a genotype,Germany,2011-2012[J].Emerg Infect Dis,2013,19(6):1029-1030.
[14]Auksornkitti V,Kamprasert N,Thongkomplew S,Suwannakarn K,Theamboonlers A,Samransamruajkij R,Poovorawan Y.Molecular characterization of human respiratory syncytial virus,2010-2011:identification of genotype ON1 and a new subgroup B genotype in Thailand[J].Arch Virol,2014,159(3):499-507.
[15]Hirano E,Kobayashi M,Tsukagoshi H,Yoshida L M,Kuroda M,Noda M,Ishioka T,Kozawa K,Ishii H,Yoshida A,Oishi K,Ryo A,Kimura H.Molecular evolution of human respiratory syncytial virus attachment glycoprotein(G)gene of new genotype ON1 and ancestor NA1[J].Infect Genet Evol,2014,28:183-191.
[16]Esposito S,Piralla A,Zampiero A,Bianchini S,Di Pietro G,Scala A,Pinzani R,Fossali E,Baldanti F,Principi N.Characteristics and their clinical relevance of respiratory syncytial virus types and genotypes circulating in northern Italy in five consecutive winter seasons[J/OL].PLoS One,2015,10(6):e0129369.
[17]Kim Y J,Kim D W,Lee W J,Yun M R,Lee H Y,Lee H S,Jung H D,Kim K.Rapid replacement of human respiratory syncytial virus A with the ON1genotype having 72 nucleotide duplication in G gene[J].Infect Genet Evol,2014,26:103-112.
[18]Sullender W M.Respiratory syncytial virus genetic and antigenic diversity[J].Clin.Microbiol.Rev,2000,13:1-15.
[2]Yorita K L,Holman R C,Steiner C A,Effler P V,Miyamura J,Forbes S,Anderson L J,Balaraman V.Severe bronchiolitis and respiratory syncytial virus among young children in Hawaii[J].Pediatr Infect Dis J,2007,26(12):1081-1088.
[3]Branche A R,Falsey A R.Respiratory syncytial virus infection in older adults:an under-recognized problem[J].Drugs Aging,2015,32(4):261-269.
[4]World Health Organization(WHO),Initiative for Vaccine Research(IVR).Respiratory syncytial virus and parainfluenza virus.Disease burden.Geneva:The Oganization,2009.
[5]Matias G,Taylor R,Haguinet F,Schuck-Paim C,Lustig R,Shinde V.Estimates of mortality attributable to influenza and RSV in the United States during 1997-2009 by influenza type or subtype,age,cause of death,and risk status[J].Influenza Other Respir Viruses,2014,8(5):507-515.
[6]Shaw C A,Ciarlet M,Cooper B W,Dionigi L,Keith P,O′Brien K B,Rafie-Kolpin M,Dormitzer P R.The path to an RSV vaccine[J].Curr Opin Virol,2013,3(3):332-342.
[7]Ahmed A,Haider S H,Parveen S,Arshad M,Alsenaidy H A,Baaboud A O,Mobaireek K F,AlSaadi M M,Alsenaidy A M,SullenderW.Cocirculation of 72bp duplication group A and 60bp duplication group B respiratory syncytial virus(RSV)strains in Riyadh,Saudi Arabia during 2014[J/OL].PLoS One,2016,11(11):e0166145.
[8]Trento A,Casas I,Calderon A,Garcia-Garcia M L,Calvo C,Perez-Brena P,Meleto J A.Ten years of global evolution of the human respiratory syncytial virus BA genetype with a 60-nucleotide duplication in the Gprotein gene[J].Virol,2010,84(15),7500-7512.
[9]Munywoki P K,Ohuma E O,Ngama M,Bauni E,Scott J A,Nokes D J.Severe lower respiratory tract infection in early infancy and pneumonia hospitalizations among children,Kenya[J].Emerg Infect Dis,2013,19(2):223-239.
[10]Auksornkitti V,Kamprasert N,Thongkomplew S,Suwannakarn K,Theamboonlers A,Samransamruajkij R,Poovorawan Y.Molecular characterization of human respiratory syncytial virus,2010-20-11:identification of genetype ON1 and a new subgroup B genetype in Thailand[J].Arch.Virol,2013,Epub ahead of print.
[11]Eshaghi A,Duvvuri V R,Lai R,Nadarajah J T,Li A,Patel S N,Low D E,Gubbay JB.Genetic variability of human respiratory syncytial virus A strains circulating in Ontario:a novel genotype with a 72nucleotide G gene duplication[J/OL].PLoS One,2012,7(3):e32807.
[12]Lee W J,Kim Y J,Kim D W,Lee H S,Lee H Y,Kim K.Complete genome sequence of human respiratory syncytial virus genotype A with a 72-nucleotide duplication in the attachment protein G gene[J].J Virol.2012,86(24):13810-13811.
[13]Prifert C,Streng A,Krempl C D,Liese J,Weissbrich B.Novel respiratory syncytial virus a genotype,Germany,2011-2012[J].Emerg Infect Dis,2013,19(6):1029-1030.
[14]Auksornkitti V,Kamprasert N,Thongkomplew S,Suwannakarn K,Theamboonlers A,Samransamruajkij R,Poovorawan Y.Molecular characterization of human respiratory syncytial virus,2010-2011:identification of genotype ON1 and a new subgroup B genotype in Thailand[J].Arch Virol,2014,159(3):499-507.
[15]Hirano E,Kobayashi M,Tsukagoshi H,Yoshida L M,Kuroda M,Noda M,Ishioka T,Kozawa K,Ishii H,Yoshida A,Oishi K,Ryo A,Kimura H.Molecular evolution of human respiratory syncytial virus attachment glycoprotein(G)gene of new genotype ON1 and ancestor NA1[J].Infect Genet Evol,2014,28:183-191.
[16]Esposito S,Piralla A,Zampiero A,Bianchini S,Di Pietro G,Scala A,Pinzani R,Fossali E,Baldanti F,Principi N.Characteristics and their clinical relevance of respiratory syncytial virus types and genotypes circulating in northern Italy in five consecutive winter seasons[J/OL].PLoS One,2015,10(6):e0129369.
[17]Kim Y J,Kim D W,Lee W J,Yun M R,Lee H Y,Lee H S,Jung H D,Kim K.Rapid replacement of human respiratory syncytial virus A with the ON1genotype having 72 nucleotide duplication in G gene[J].Infect Genet Evol,2014,26:103-112.
[18]Sullender W M.Respiratory syncytial virus genetic and antigenic diversity[J].Clin.Microbiol.Rev,2000,13:1-15.