2016年广州市H1N1流感病毒变异情况分析
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摘要
目的对2016年广州市15株流感病毒8个蛋白的氨基酸位点进行变异分析,了解病毒耐药性和致病性的变化,为后续病毒研究、寻找变异规律提供科学依据。方法从患者咽拭子中提取病毒,采用RT-PCR方法对病毒进行鉴定,应用二代测序技术测定15个分离株的全基因组序列,通过MAGA6.0软件与参考株pdm2009(A/California/07/2009(H1N1))进行比对,分析氨基酸变异情况。结果 15个H1N1流感病毒分离株与参考株pdm2009(A/California/07/2009(H1N1))进行比对发现,8个蛋白片段的变异程度不高,HA蛋白受体结合位点未发现变异,NA蛋白在与耐药性有关的氨基酸位点均未发现变异,表明对神经氨酸酶抑制剂药物敏感,并发现影响抗原性、病毒感染细胞能力和病毒基因组的复制和转录有关的变异。结论 2016年广州市H1N1流感病毒为保守毒株,未发现新的耐药病毒株,发现病毒致病力增强了,因此可能导致流感病毒的局部爆发。
Objective The mutation analysis of the amino acid sites of 8 proteins of 15 influenza virus viruses in Guangzhou in 2016 was conducted to understand the changes of virus resistance and susceptibility,which provided a scientific basis for the follow-up virus research and the search for variation trends. Methods Virus was extracted from the patient′s throat swab and RT-PCR method was used to identify the virus. The whole genome sequence of 15 isolates was determined by second-generation sequencing technology. The MAGA6.0 software and reference strain pdm2009(A/California/07/2009)(H1 N1))were used to perform an alignment to analyze the amino acid variation. Results The comparison of 15 H1 N1 influenza virus isolates with the reference strain pdm 2009(A/California/07/2009(H1 N1)) revealed that the degree of variation of the eight protein fragments was not high,and no mutation was found in the HA protein receptor binding site. No mutations were found in the amino acid sites associated with drug resistance,indicating sensitivity to the neuraminidase inhibitor drug,and mutations related to antigenicity,viral infection,and replication and transcription of the viral genome were found. Conclusion In 2016,the H1 N1 influenza virus in Guangzhou was a conserved strain. No new drug-resistant strains were found,and the virus virulence was found to be enhanced,which might lead to a local outbreak of influenza virus.
Objective The mutation analysis of the amino acid sites of 8 proteins of 15 influenza virus viruses in Guangzhou in 2016 was conducted to understand the changes of virus resistance and susceptibility,which provided a scientific basis for the follow-up virus research and the search for variation trends. Methods Virus was extracted from the patient′s throat swab and RT-PCR method was used to identify the virus. The whole genome sequence of 15 isolates was determined by second-generation sequencing technology. The MAGA6.0 software and reference strain pdm2009(A/California/07/2009)(H1 N1))were used to perform an alignment to analyze the amino acid variation. Results The comparison of 15 H1 N1 influenza virus isolates with the reference strain pdm 2009(A/California/07/2009(H1 N1)) revealed that the degree of variation of the eight protein fragments was not high,and no mutation was found in the HA protein receptor binding site. No mutations were found in the amino acid sites associated with drug resistance,indicating sensitivity to the neuraminidase inhibitor drug,and mutations related to antigenicity,viral infection,and replication and transcription of the viral genome were found. Conclusion In 2016,the H1 N1 influenza virus in Guangzhou was a conserved strain. No new drug-resistant strains were found,and the virus virulence was found to be enhanced,which might lead to a local outbreak of influenza virus.
引文
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[23]Wang C,Zhang Y,Wu B,et al.Evolutionary characterization of the pandemic H1N1/2009 influenza virus in humans based on nonstructural genes[J].PLoS One,2013,8(2):e56201.2019-01-02
[2]Webster RG,Bean WJ,German OT,et al.Evolution and ecology of influenza-A viruses[J].Microbiol Rev,1992,56(1):152-179.
[3]Matrosovich M,Tuzikov A,Bovin N,et al.Early alterations of the receptor-binding properties of H1,H2,and H3 avian influenza virus hemagglutinins after their introduction into mammals[J].J Virol,2000,74(18):8501-8512.
[4]Caglioti C,Selleri M,Rozera G,et al.In-depth analysis of HAand NS1 genes in A(H1N1)pdm09 infected patients[J].PLoSOne,2016,11(5):e0155661.
[5]Liu Y,Childs RA,Matrosovich T,et al.Altered receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1)2009 influenza virus[J].J Virol,2010,84(22):12069-12074.
[6]Chutinimitkul S,Herfst S,Steel J,et al.Virulence-associated substitution D222G in the hemagglutinin of 2009 pandemic influenza A(H1N1)virus affects receptor binding[J].J Virol,2010,84(22):11802-11813.
[7]Colman PM,Hoyne PA,Lawrence MC.Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase[J].J Virol,1993,67(6):2972-2980.
[8]Colman PM.Influenza virus neuraminidase:structure,antibodies,and inhibitors[J].Protein Sci,l994,3(10):1687-1696.
[9]Saito R,Sakai T,Sate I,et al.Frequency of amantadine-resistant inf uenza A viruses during two seasons featuring cocireulation of H1N1 and H3N2[J].J Clin Microbiol,2003,41(5):2164-2165.
[10]Shirashi K,Mitamura K,Sakai-Tagawa Y,et al.High frequency of resistant viruses harboring different mutation in amantadinetreated children with influenza[J].J Infect Dis,2003,188(1):57-61.
[11]Colman PM,Varghese JN,Laver WG.Structure of the catalytic and antigenic sites in influenza virus neuraminidase[J].Nature,1983,303(5912):41-44.
[12]田疆,周经跤,陈艺韵,等.甲型H1N1流感病毒神经氨酸酶基因遗传进化分析[J].中山大学学报:医学科学版,2010,31(2):207-212.
[13]黄平,俞守义,柯同文.逐步预测和统计学筛选人H5N1毒株神经氨酸酶蛋白B细胞表位[J].科学通报,2008,53(22):2748-2753.
[14]Naffakh N,Tomoiu A,Rameix-Welti MA,et al.Host restriction of avian influenza viruses at the level of the ribonucleoproteins[J].Annu Rev Microbiol,2008,62:403-424.
[15]Danzy S,Studdard LR,Manicassamy B,et al.Mutations to PB2and NP proteins of an avian influenza virus combine to confer efficient growth in primary human respiratory cells[J].J Virol,2014,88(22):13436-13446.
[16]Sun Y,Xu Q,Shen Y,et al.Naturally occurring mutations in the PA gene are key contributors to increased virulence of pandemic H1N1/09 influenza virus in mice[J].J Virol,2014,88(8):4600-4604.
[17]Ping J,Keleta L,Forbes NE,et al.Genomic and protein structural maps of adaptive evolution of human influenza A virus to increased virulence in the mouse[J].PLoS One,2011,6(6):e21740.
[18]Peiris JS,de Jong MD,Guan Y.Avian influenza virus(H5N1):a threat to human health[J].Clin Microbiol Rev,2007,20(2):243-267.
[19]Gabriel G,Abram M,Keiner B,et al.Differential polymerase activity in avian and mammalian cells determines host range of influenza virus[J].J Virol,2007,81(17):9601-9604.
[20]Hatta M,Gao E,Halfmann P,et al.Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses[J].Science,2001,293(5536):1840-1842.
[21]Labadie K,Dos Santos Afonso E,Rameix-Welti MA,et al.Hostrange determinants on the PB2 protein of influenza A viruses controlthe interaction between the viral polymerase andnucleoprotein in human cells[J].Virology,2007,362(2):271-282.
[22]Subbarao EK,London W,Murphy BR.A single amino acid in the PB2 gene of influenza A virus is a determinant of host range[J].J Virol,1993,67(4):1761-1764.
[23]Wang C,Zhang Y,Wu B,et al.Evolutionary characterization of the pandemic H1N1/2009 influenza virus in humans based on nonstructural genes[J].PLoS One,2013,8(2):e56201.2019-01-02