五株人冠状病毒NL63的基因型鉴定及S1 domain基因特征分析
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摘要
为阐明我国部分地区人冠状病毒(Human coronavirus,HCoV)NL63基因特征,本研究对2013年陕西省、2018年河南省和湖南省送检的5株HCoV-NL63核酸检测阳性的呼吸道样本进行HCoV-NL63基因型别鉴定及S1 do-main基因特征分析。通过对HCoV-NL63棘突蛋白(Spike glycoprotein,S)基因的S1 domain区域进行基因扩增和序列测定,同时结合GenBank数据库下载的1983～2018年其他国家74条HCoV-NL63代表株序列和2007～2010年中国流行的12条HCoV-NL63代表株序列,构建基因亲缘性关系树,并对S蛋白的S1 domain区域进行核苷酸序列比对分析。结果提示全球HCoV-NL63流行株可划分为A和B两个基因型;A基因型可进一步划分为A0,A1,A2和A3四个基因亚型,其中本研究将GenBank中2008年中国流行的2株HCoV-NL63毒株划分为新基因亚型(A3);B基因型可进一步划分为B0,B1和B2三个基因亚型。A和B基因型的HCoV-NL63代表株序列在地域分布上无明显差异,但A基因型不同基因亚型的HCoV-NL63序列在年代分布上呈现出一定的时间进化趋势。在我国A和B基因型HCoV-NL63均已被检测到,但主要以A基因型流行为主,且主要集中在A1和A2基因亚型。不同基因型HCoV-NL63序列在S1 domain区域核苷酸和氨基酸序列上存在特征性差异。本研究通过对五株HCoV-NL63基因型鉴定及S1 domain基因特征分析,初步阐明了我国部分地区流行的HCoV-NL63基因型/基因亚型分布情况及基因特征,丰富了我国本土流行的HCoV-NL63基因数据库,为我国HCoV-NL63分子流行病学研究及分子检测和监测技术的改进和验证提供了基础基因数据。
To elucidate the genetic characteristics of human coronavirus(HCoV)NL63 in some areas of China,five HCoV-NL63 nucleic acid positive clinical specimens were collected in Shaanxi Province in 2013,Henan Province and Hunan Province in 2018 to analyze HCoV-NL63 genotyping,genotype distribution and S1 domain genetic characteristics. The gene amplification and sequence determination of the S1 domain region of the HCoV-NL63 spike glycoprotein(S) gene were performed. The phylogenetic analyses were conducted with five sequences obtained in this study,combined with the sequences of 74 international HCoV-NL63 representative strains during 1983～2018 and 12 Chinese HCoV-NL63 representative strain during 2007～2010 from the GenBank database. And the nucleotide and amino acid sequences of the S1 domain were also analyzed. The results suggested that the global HCoV-NL63 epidemic strains were divided into two genotypes(A and B). Genotype A was further divided into four subgenotypes(A0,A1,A2,and A3),and two Chinese HCoV-NL63 sequences in 2008 obtained from GenBank were clustered as a new subgenotype(A3);genotype B can be further divided into three subgenotypes(B0,B1,and B2). There was no significant difference in the geographical distribution between genotype A and B,but the sequences of genotype A showed a chronological evolution trend. Both genotype A and B of HCoV-NL63 were found in China,genotype A is the predominant genotype of HCoV-NL63 in China at present,mainly concentrating on subgenotype A1 and A2. There were characteristic differences in nucleotide and amino acid sequences in the S1 domain region among different genotypes of HCoV-NL63.Through genotype identification and the analyses of S1 domain genetic characteristics of five HCoV-NL63 strains in this study,the distribution and genetic characteristics of the genotypes/subgenotypes of HCoV-NL63 in some areas of China were preliminarily clarified,which enriched the HCoV-NL63 gene database and provided basic genetic data for molecular epidemiological research and the improvement and validation of molecular detection techniques in China.
To elucidate the genetic characteristics of human coronavirus(HCoV)NL63 in some areas of China,five HCoV-NL63 nucleic acid positive clinical specimens were collected in Shaanxi Province in 2013,Henan Province and Hunan Province in 2018 to analyze HCoV-NL63 genotyping,genotype distribution and S1 domain genetic characteristics. The gene amplification and sequence determination of the S1 domain region of the HCoV-NL63 spike glycoprotein(S) gene were performed. The phylogenetic analyses were conducted with five sequences obtained in this study,combined with the sequences of 74 international HCoV-NL63 representative strains during 1983～2018 and 12 Chinese HCoV-NL63 representative strain during 2007～2010 from the GenBank database. And the nucleotide and amino acid sequences of the S1 domain were also analyzed. The results suggested that the global HCoV-NL63 epidemic strains were divided into two genotypes(A and B). Genotype A was further divided into four subgenotypes(A0,A1,A2,and A3),and two Chinese HCoV-NL63 sequences in 2008 obtained from GenBank were clustered as a new subgenotype(A3);genotype B can be further divided into three subgenotypes(B0,B1,and B2). There was no significant difference in the geographical distribution between genotype A and B,but the sequences of genotype A showed a chronological evolution trend. Both genotype A and B of HCoV-NL63 were found in China,genotype A is the predominant genotype of HCoV-NL63 in China at present,mainly concentrating on subgenotype A1 and A2. There were characteristic differences in nucleotide and amino acid sequences in the S1 domain region among different genotypes of HCoV-NL63.Through genotype identification and the analyses of S1 domain genetic characteristics of five HCoV-NL63 strains in this study,the distribution and genetic characteristics of the genotypes/subgenotypes of HCoV-NL63 in some areas of China were preliminarily clarified,which enriched the HCoV-NL63 gene database and provided basic genetic data for molecular epidemiological research and the improvement and validation of molecular detection techniques in China.
引文
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[9] Weiss S R,Navasmartin S. Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syn-drome coronavirus[J]. Microbiol Mol Biol Rev,2005,69(4):635-664.
[10]Pyrc K,Jebbink M F,Berkhout B,van der Hoek L. Ge-nome structure and transcriptional regulation of human coronavirus NL63[J]. Virol J,2004,1(1):7.
[11]Pyrc K,Dijkman R,Deng L,Jebbink M F,Ross H A,Berkhout B,van der Hoek L. Mosaic structure of human coronavirus NL63,one thousand years of evolution[J].J Mol Biol,2006,364(5):964-973.
[12]Al-Khannaq M N,Ng K T,Oong X Y,Pang Y K,Takebe Y,Chook J B,Hanafi N S,Kamarulzaman A,Tee K K. Diversity and evolutionary histories of human coronaviruses NL63 and 229E associated with acute up-per respiratory tract symptoms in Kuala Lumpur,Malay-sia[J]. Am J Trop Med Hyg,2016,94(5):1058-1064.
[13]Kiyuka P K,Agoti C N,Munywoki P K,Njeru R,Bett A,Otieno J R,Otieno G P,Kamau E,Clark T G,van der Hoek L,Kellam P,Nokes D J,Cotten M. Human coronavirus NL63 molecular epidemiology and evolution-ary patterns in rural Coastal Kenya[J]. J Infect Dis,2018,217(11):1728-1739.
[14]Fouchier R A,Hartwig N G,Bestebroer T M,Niemey-er B,de Jong J C,Simon J H,Osterhaus A D. A previ-ously undescribed coronavirus associated with respiratory disease in humans[J]. P Natl Acad Sci,2004,101(16):6212-6216.
[15]Arden K E,Nissen M D,Sloots T P,Mackay I M.New human coronavirus,HCoV-NL63,associated with severe lower respiratory tract disease in Australia[J]. J Med Virol,2010,75(3):455–462.
[16]Bastien N,Anderson K,Hart L,Van Caeseele P,Brandt K,Milley D,Hatchette T,Weiss E C,Li Y.Human coronavirus NL63 infection in Canada[J]. J In-fect Dis,2005,191(4):503-506.
[17]Mourez T,Dina J,van der Hoek L,Gouarin S,Pe-titjean J,Brouard J,Freymuth F. Human coronavirus NL63,France[J]. Emerg Infect Dis,2005,11(8):1225-1229.
[18]Mo?s E,Vijgen L,Keyaerts E,Zlateva K,Li S,Maes P,Pyrc K,Berkhout B,van der Hoek L,Van Ranst M. A novel pancoronavirus RT-PCR assay:frequent de-tection of human coronavirus NL63 in children hospital-ized with respiratory tract infections in Belgium[J].BMC Infect Dis,2005,5(1):6.
[19]Suzuki A,Okamoto M,Ohmi A,Watanabe O,Mi-yabayashi S,Nishimura H. Detection of human coronavi-rus-NL63 in children in Japan[J]. Pediatr Infect Dis J,2005,24(7):645-646.
[20]Esper F,Weibel C,Ferguson D,Landry M L,Kahn J S. Evidence of a novel human coronavirus that is associ-ated with respiratory tract disease in infants and young children[J]. J Infect Dis,2005,191(4):492-498.
[21]朱汝南,钱渊,赵林清,邓洁,王芳,廖斌.从北京地区急性呼吸道感染患儿标本中检测到新型冠状病毒NL63基因[J].中华儿科杂志,2006,44(3):202-205.
[22]Chiu S S,Chan K H,Chu K W,Kwan S W,Guan Y,Poon L L,Peiris J S. Human coronavirus NL63 infec-tion and other coronavirus infections in children hospital-ized with acute respiratory disease in Hong Kong,China[J]. Clin Infect Dis,2005,40(12):1721-1729.
[23]钱艺,谢正德,任丽丽,刘春艳,肖艳,徐保平,杨燕,钱素云,耿荣,申昆玲. 2007~2015年北京地区儿童急性下呼吸道感染人冠状病毒检测及临床分析[J].中华儿科杂志,2015,53(9):707-711.
[24]张素粉. 2010~2015年广州地区HCoV流行病学特征及OC43亚型变异分析[D].中山大学,2016.
[25]张雪,赖植发,陈润莉,何珊茹,吴缃琦,余强.深圳地区婴幼儿人冠状病毒NL63和HKU1的检测与分析[J].现代预防医学,2012,39(10):2534-2535.
[26]吴小青.福州地区急性呼吸道感染患儿HCoV-HKU1、HCoV-OC43、RSV感染的检测及分析[D].福建医科大学,2008.
[27]杨懿婧,胡芸文. 2009~2016年上海人冠状病毒OC43分子流行病学研究[J].中华预防医学杂志,2018,52(1):55-61.
[28]单淑琴,夏科君,干冬梅,华玲玲,黄静.急性下呼吸道感染患儿人冠状病毒感染状况研究[J].中华医院感染学杂志,2017,27(12):2813-2816.
[29]肖秋艳,周利利,夏秋玲,任洛,邓昱,谢晓虹,王莉佳,黄爱龙,符州,罗建,罗征秀,刘恩梅.重庆地区呼吸道感染住院患儿人类冠状病毒流行情况和临床特征[J].中华实用儿科临床杂志,2014,29(10):732-736.
[30]陆柔剑,谭文杰,阮力.新型人冠状病毒HCoV-NL63和HCoV-HKU1的研究进展[J].生物技术通讯,2008,19(1):93-96.
[31]Dominguez S R,Sims G E,Wentworth D E,Halpin R A,Robinson C C,Town C D,Holmes K V. Genomic analysis of 16 Colorado human NL63 coronaviruses iden-tifies a new genotype,high sequence diversity in the N-terminal domain of the spike gene and evidence of recom-bination[J]. J Gen Virol,2012,93(Pt_11):2387-2398.
[32]Lin H X,Feng Y,Wong G,Wang L,Li B,Zhao X,Li Y,Smaill F,Zhang C. Identification of residues in the receptor-binding domain(RBD)of the spike protein of human coronavirus NL63 that are critical for the RBD-ACE2 receptor interaction[J]. J Gen Virol,2008,89(4):1015-1024.
[33]Lin H X,Feng Y,Tu X,Zhao X,Hsieh C H,Griffin L,Junop M,Zhang C. Characterization of the spike pro-tein of human coronavirus NL63 in receptor binding and pseudotype virus entry[J]. Virus Res,2011,160(1-2):283-293.
[2] Tyrrell D A J,Bynoe M L. Cultivation of a novel type of common-cold virus in organ cultures[J]. Br Med J,1965,1(5448):1467-1470.
[3] Mcintosh K,Becker W B,Chanock R M. Growth in suckling-mouse brain of“IBV-like”viruses from pa-tients with upper respiratory tract disease[J]. Proc Natl Acad Sci USA,1967,58(6):2268-2273.
[4] Zhong N S,Zheng B J,Li Y M,Poon,Xie Z H,Li P H,Tan S Y,Chang Q,Xie J P,Liu X Q,Xu J,Li D X,Yuen K Y,Peiris,Guan Y. Epidemiology and cause of severe acute respiratory syndrome(SARS)in Guang-dong,People′s Republic of China,in February,2003[J]. Lancet(London,England). 2003,362(9393):1353-1358.
[5] van der Hoek L,Pyrc K,Jebbink M F,Vermeulen-Oost W,Berkhout R J,Wolthers K C,Wertheim-van Dillen P M,Kaandorp J,Spaargaren J,Berkhout B.Identification of a new human coronavirus[J]. Nat Med,2004,10(4):368-373.
[6] Woo P C,Lau S K,Chu C M,Chan K H,Tsoi H W,Huang Y,Wong B H,Poon R W,Cai J J,Luk W K,Poon L L,Wong S S,Guan Y,Peiris J S,Yuen K Y.Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia[J]. J Virol,2005,79(2):884-895.
[7] Madani T A,Azhar E I,Hashem A M. Evidence for camel-to-human transmission of MERS coronavirus[J].New Engl J Med,2014,371(14):1359-1360.
[8] Dijkman R,Jebbink M F,El Idrissi N B,Pyrc K,Mull-er M A,Kuijpers T W,Zaaijer H L,van der Hoek L.Human coronavirus NL63 and 229E seroconversion in children[J]. J Clin Microbiol,2008,46(7):2368-2373.
[9] Weiss S R,Navasmartin S. Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syn-drome coronavirus[J]. Microbiol Mol Biol Rev,2005,69(4):635-664.
[10]Pyrc K,Jebbink M F,Berkhout B,van der Hoek L. Ge-nome structure and transcriptional regulation of human coronavirus NL63[J]. Virol J,2004,1(1):7.
[11]Pyrc K,Dijkman R,Deng L,Jebbink M F,Ross H A,Berkhout B,van der Hoek L. Mosaic structure of human coronavirus NL63,one thousand years of evolution[J].J Mol Biol,2006,364(5):964-973.
[12]Al-Khannaq M N,Ng K T,Oong X Y,Pang Y K,Takebe Y,Chook J B,Hanafi N S,Kamarulzaman A,Tee K K. Diversity and evolutionary histories of human coronaviruses NL63 and 229E associated with acute up-per respiratory tract symptoms in Kuala Lumpur,Malay-sia[J]. Am J Trop Med Hyg,2016,94(5):1058-1064.
[13]Kiyuka P K,Agoti C N,Munywoki P K,Njeru R,Bett A,Otieno J R,Otieno G P,Kamau E,Clark T G,van der Hoek L,Kellam P,Nokes D J,Cotten M. Human coronavirus NL63 molecular epidemiology and evolution-ary patterns in rural Coastal Kenya[J]. J Infect Dis,2018,217(11):1728-1739.
[14]Fouchier R A,Hartwig N G,Bestebroer T M,Niemey-er B,de Jong J C,Simon J H,Osterhaus A D. A previ-ously undescribed coronavirus associated with respiratory disease in humans[J]. P Natl Acad Sci,2004,101(16):6212-6216.
[15]Arden K E,Nissen M D,Sloots T P,Mackay I M.New human coronavirus,HCoV-NL63,associated with severe lower respiratory tract disease in Australia[J]. J Med Virol,2010,75(3):455–462.
[16]Bastien N,Anderson K,Hart L,Van Caeseele P,Brandt K,Milley D,Hatchette T,Weiss E C,Li Y.Human coronavirus NL63 infection in Canada[J]. J In-fect Dis,2005,191(4):503-506.
[17]Mourez T,Dina J,van der Hoek L,Gouarin S,Pe-titjean J,Brouard J,Freymuth F. Human coronavirus NL63,France[J]. Emerg Infect Dis,2005,11(8):1225-1229.
[18]Mo?s E,Vijgen L,Keyaerts E,Zlateva K,Li S,Maes P,Pyrc K,Berkhout B,van der Hoek L,Van Ranst M. A novel pancoronavirus RT-PCR assay:frequent de-tection of human coronavirus NL63 in children hospital-ized with respiratory tract infections in Belgium[J].BMC Infect Dis,2005,5(1):6.
[19]Suzuki A,Okamoto M,Ohmi A,Watanabe O,Mi-yabayashi S,Nishimura H. Detection of human coronavi-rus-NL63 in children in Japan[J]. Pediatr Infect Dis J,2005,24(7):645-646.
[20]Esper F,Weibel C,Ferguson D,Landry M L,Kahn J S. Evidence of a novel human coronavirus that is associ-ated with respiratory tract disease in infants and young children[J]. J Infect Dis,2005,191(4):492-498.
[21]朱汝南,钱渊,赵林清,邓洁,王芳,廖斌.从北京地区急性呼吸道感染患儿标本中检测到新型冠状病毒NL63基因[J].中华儿科杂志,2006,44(3):202-205.
[22]Chiu S S,Chan K H,Chu K W,Kwan S W,Guan Y,Poon L L,Peiris J S. Human coronavirus NL63 infec-tion and other coronavirus infections in children hospital-ized with acute respiratory disease in Hong Kong,China[J]. Clin Infect Dis,2005,40(12):1721-1729.
[23]钱艺,谢正德,任丽丽,刘春艳,肖艳,徐保平,杨燕,钱素云,耿荣,申昆玲. 2007~2015年北京地区儿童急性下呼吸道感染人冠状病毒检测及临床分析[J].中华儿科杂志,2015,53(9):707-711.
[24]张素粉. 2010~2015年广州地区HCoV流行病学特征及OC43亚型变异分析[D].中山大学,2016.
[25]张雪,赖植发,陈润莉,何珊茹,吴缃琦,余强.深圳地区婴幼儿人冠状病毒NL63和HKU1的检测与分析[J].现代预防医学,2012,39(10):2534-2535.
[26]吴小青.福州地区急性呼吸道感染患儿HCoV-HKU1、HCoV-OC43、RSV感染的检测及分析[D].福建医科大学,2008.
[27]杨懿婧,胡芸文. 2009~2016年上海人冠状病毒OC43分子流行病学研究[J].中华预防医学杂志,2018,52(1):55-61.
[28]单淑琴,夏科君,干冬梅,华玲玲,黄静.急性下呼吸道感染患儿人冠状病毒感染状况研究[J].中华医院感染学杂志,2017,27(12):2813-2816.
[29]肖秋艳,周利利,夏秋玲,任洛,邓昱,谢晓虹,王莉佳,黄爱龙,符州,罗建,罗征秀,刘恩梅.重庆地区呼吸道感染住院患儿人类冠状病毒流行情况和临床特征[J].中华实用儿科临床杂志,2014,29(10):732-736.
[30]陆柔剑,谭文杰,阮力.新型人冠状病毒HCoV-NL63和HCoV-HKU1的研究进展[J].生物技术通讯,2008,19(1):93-96.
[31]Dominguez S R,Sims G E,Wentworth D E,Halpin R A,Robinson C C,Town C D,Holmes K V. Genomic analysis of 16 Colorado human NL63 coronaviruses iden-tifies a new genotype,high sequence diversity in the N-terminal domain of the spike gene and evidence of recom-bination[J]. J Gen Virol,2012,93(Pt_11):2387-2398.
[32]Lin H X,Feng Y,Wong G,Wang L,Li B,Zhao X,Li Y,Smaill F,Zhang C. Identification of residues in the receptor-binding domain(RBD)of the spike protein of human coronavirus NL63 that are critical for the RBD-ACE2 receptor interaction[J]. J Gen Virol,2008,89(4):1015-1024.
[33]Lin H X,Feng Y,Tu X,Zhao X,Hsieh C H,Griffin L,Junop M,Zhang C. Characterization of the spike pro-tein of human coronavirus NL63 in receptor binding and pseudotype virus entry[J]. Virus Res,2011,160(1-2):283-293.