人冠状病毒HKU1 N和S蛋白基因序列及进化分析
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摘要
目的了解人冠状病毒HCoV-HKU1在我国福州急性重症下呼吸道感染患儿中的感染情况以及病毒N基因和S基因的系统进化特征。方法通过RT-PCR法对2007年11月至2015年1月因呼吸道感染住进医院儿科重症监护病房的266例小儿鼻咽抽取物标本进行4种人冠状病毒的检测和人冠状病毒HCoV-HKU1的确认检测,测序并BLAST比对分析8份人冠状病毒阳性产物。结果在266例标本中检出2例HCoV-HKU1感染阳性病例,检出率为0.75%(2/266)。这两例患儿一例诊断为支气管炎、一例诊断为急性重症肺炎。通过检测都发现混合感染了人副流感病毒3型(HPIV-3)。对这两株病毒FZ90和FZ96的N和S蛋白基因进行序列测定、拼接和系统进化分析;结果表明这2株HCoV-HKU1都属于HKU1基因型A。结论人冠状病毒HCoV-HKU1可能是儿童下呼吸道感染中较为重要的一种病原,混合感染导致儿童病情加重。
The aim of this study was to characterize the N and Sprotein encoding genes of human coronaviruses(HCoVHKU1)in children with respiratory tract infection in Fuzhou,Fujian,China.Nasopharyngeal aspirates were collected from children with respiratory tract disease from Nov 2007 to Jan 2015.A total of 266 clinical samples were tested for HCoVs using reverse-transcription polymerase chain reaction(RT-PCR).The positive products were sequenced and compared with those in GenBank by Blast.The positive samples were then tested for HCoV-HKU1 using RT-PCR method.HCoV-HKU1 were detected in 2 patients(0.75%)out of the 266 children.One of children who were positive for HCoV-HKU1 was diagnosed as bronchitis,and the other was serious acute pneumonia.Co-infections with human parainfluenza virus type 3(HPIV-3)were found in the two HCoV-HKU1 positive samples.We compared the N and S gene sequence of the 2 HCoV-HKU1 isolates in Fuzhou,China to other HCoV-HKU1 isolates documented in the GenBank database by using MEGA software version 6.06 and the neighbor-joining method.Phylogenetic analyses revealed that the 2 strains of HCoV-HKU1 were genotype A.HCoV-HKU1 infections were associated with upper and lower respiratory illness in children.
The aim of this study was to characterize the N and Sprotein encoding genes of human coronaviruses(HCoVHKU1)in children with respiratory tract infection in Fuzhou,Fujian,China.Nasopharyngeal aspirates were collected from children with respiratory tract disease from Nov 2007 to Jan 2015.A total of 266 clinical samples were tested for HCoVs using reverse-transcription polymerase chain reaction(RT-PCR).The positive products were sequenced and compared with those in GenBank by Blast.The positive samples were then tested for HCoV-HKU1 using RT-PCR method.HCoV-HKU1 were detected in 2 patients(0.75%)out of the 266 children.One of children who were positive for HCoV-HKU1 was diagnosed as bronchitis,and the other was serious acute pneumonia.Co-infections with human parainfluenza virus type 3(HPIV-3)were found in the two HCoV-HKU1 positive samples.We compared the N and S gene sequence of the 2 HCoV-HKU1 isolates in Fuzhou,China to other HCoV-HKU1 isolates documented in the GenBank database by using MEGA software version 6.06 and the neighbor-joining method.Phylogenetic analyses revealed that the 2 strains of HCoV-HKU1 were genotype A.HCoV-HKU1 infections were associated with upper and lower respiratory illness in children.
引文
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[18]Surjit M,Kumar R,Mishra RN.The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation[J].J Virol,2005,79(17):11476-11486.DOI:10.1128/JVI.79.17.11476-11486.2005
[19]Soonnarong R,Thongpan I,Payungporn S,et al.Molecular epidemiology and characterization of human coronavirus in Thailand,2012-2013[J].Springer Plus,2016,5(1420):1-8.DOI:10.1186/s40064-016-3101-9
[20]AI-Khannaq MN,Ng KT,Oong XY,et al.Molecular epidemiology and evolutionary histories of human coronavirus OC43and HKU1among patients with upper tract infections in Kuala Lumpur,Malaysia[J].Virol J,2010,13(33):1-12.DOI:10.1186/s12985-016-0488-4
[21]Cui LJ,Zhang C,Zhang T,et al.Human coronavirus HCoVNL63and HCoV-HKU1in hospitalized children with acute respiratory infections in Beijing,China[J].Adv Virol,2011,2011:129134.DOI:10.1155/2011/129134
[22]段希洁,陆柔剑,俞晓燕,等.北京地区成人呼吸道感染患者中HCoV-HKU1感染的初步分析与基因型确定[J].生物技术通讯,2013,24(2):205-208.DOI:10.3969/j.issn.1009-0002.2013.02.014
[23]Lee WJ,Chung YS,Yoon HS,et al.Prevalence and molecular epidemiology of human coronavirus HKU1in patients with acute respiratory illness[J].J Med Virol,2013,85:309-314.
[2]Drosten C,Gunther S,Preiser W,et al.Identification of a novel coronavirus in patients with severe acute respiratory syndrome[J].N Engl J Med,2003,348:1967-1976.DOI:10.1056/NEJMoa030747
[3]Van der Hoek L,Pyrc K,Jebbink MF,et al.Identification of a new human coronavirus[J].Nat Med,2004,10:368-373.DOI:10.1038/nm1024
[4]Sloots TP,McErlean P,Speicher DJ,et al.Evidence of human coronavirus HKU1and human bocavirus in Australian children[J].J Clin Virol,2006,35(1):99-102.DOI:10.1016/j.jev.2005.09.008
[5]Lau SK,Woo PC,Yip CC,et al.Coronavirus HKU1and other coronavirus infections in Hong Kong[J].J Clin Microbiol,2006,44:2063-2071.DOI:10.1128/JCM.02614-05
[6]Vabret A,Dina J,Gouarin S,et al.Detection of the new human coronavirus HKU1:a report of 6cases[J].Clin Infect Dis,2006,42:634-639.DOI:10.1086/500136
[7]Virus taxonomy,classification and nomenclature of viruses.Ninth report of the International Committee on Taxonomy of Viruses[Z].San Diego,CA:Academic Press,2012.
[8]Lai MC,Cavanagh D.The molecular biology of coronaviruses[J].Adv virus Res,1997,48:1-100.
[9]Woo PCY,Lau SKP,Tsoi HW,et al.Clinical and molecular epidemiological features of coronavirus HKU1-associated community-acquired pneumonia[J].J Infect Dis,2005,192(11):1898-1907.DOI:10.1086/497151
[10]Woo PCY,Lau SK,Yip CC,et al.Comparative analysis of 22coronavirus HKU1genomes reveals a novel genotype and evidence of natural recombination in coronavirus HKU1[J].J Virol,2006,80(14):7136-7145.DOI:10.1128/JVI.00509-06
[11]修文琼,郑奎城,吴冰珊,等.福州地区重症呼吸道感染患儿中4种人冠状病毒的检测与分析[J].中华实验和临床病毒学杂志,2017,31(5):429-433.DOI:10.3760/cma.j.issn.1003-9279.2017.05.011
[12]陆柔剑,张陵林,谭文杰,等.人冠状病毒HCoV-NL63和HCoV-HKU1常规RT-PCR与实时荧光定量RT-PCR检测方法的建立及应用比较[J].病毒学报,2008,24(4):305-311.DOI:CNKI:SUN:BDXB.0.2008-04-013
[13]段招军,黄灿平,瞿小旺,等.中国内陆发现HCoV-HKU1感染及N和S蛋白基因序列及进化分析[J].病毒学报,2006,22(4):241-247.DOI:10.13242/j.enki.bingduxuebao.001727
[14]Dominguez SR,Shrivastava S,Berglund A,et al.Isolation,propagation,genome analysis and epidemiology of HKU1betacoronaviruses[J].J Gen Virol,2014,95:836-848.DOI:10.1099/vir.0.059832-0
[15]Corman VM,Muller MA,Costabel U,et al.Assays for laboratory confirmation of novel human coronavirus(hCoV-EMC)infections[J].Euro Surveill,2012,17(49):1-9.
[16]Lai MM,Cavanagh D.The molecular biology of coronaviruses[J].Adv Virus Res,1997,48:1-100.
[17]侯云德.分子病毒学[M].北京:学苑出版社,1990:381-394.
[18]Surjit M,Kumar R,Mishra RN.The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation[J].J Virol,2005,79(17):11476-11486.DOI:10.1128/JVI.79.17.11476-11486.2005
[19]Soonnarong R,Thongpan I,Payungporn S,et al.Molecular epidemiology and characterization of human coronavirus in Thailand,2012-2013[J].Springer Plus,2016,5(1420):1-8.DOI:10.1186/s40064-016-3101-9
[20]AI-Khannaq MN,Ng KT,Oong XY,et al.Molecular epidemiology and evolutionary histories of human coronavirus OC43and HKU1among patients with upper tract infections in Kuala Lumpur,Malaysia[J].Virol J,2010,13(33):1-12.DOI:10.1186/s12985-016-0488-4
[21]Cui LJ,Zhang C,Zhang T,et al.Human coronavirus HCoVNL63and HCoV-HKU1in hospitalized children with acute respiratory infections in Beijing,China[J].Adv Virol,2011,2011:129134.DOI:10.1155/2011/129134
[22]段希洁,陆柔剑,俞晓燕,等.北京地区成人呼吸道感染患者中HCoV-HKU1感染的初步分析与基因型确定[J].生物技术通讯,2013,24(2):205-208.DOI:10.3969/j.issn.1009-0002.2013.02.014
[23]Lee WJ,Chung YS,Yoon HS,et al.Prevalence and molecular epidemiology of human coronavirus HKU1in patients with acute respiratory illness[J].J Med Virol,2013,85:309-314.