一株类禽型H1N1猪流感病毒的进化分析与分子特征
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摘要
2017年12月,上海市一养殖场饲养的猪出现咳嗽、呼吸困难、发热及迅速转归等病症。将猪鼻拭子接种鸡胚进行病毒分离,对血凝试验阳性样品在SPF鸡胚上进一步纯化、增殖,分离到1株猪流感病毒(A/swine/Shanghai/1205/2017)。采用RT-PCR对分离毒株进行全基因组扩增测序,利用DNAstar软件,对测序基因片段进行整个阅读框的核苷酸序列同源性比对分析,并用MEGA 6绘制遗传进化树并分析氨基酸位点。结果显示:分离毒株为H1N1亚型,其8个基因片段均属于类禽型H1N1进化分支,没有出现不同基因型流感病毒片段之间的重组;分离株HA蛋白的裂解位点序列为PSIQSR↓G,具有典型低致病性流感病毒的分子特征。本毒株的分离鉴定为分析我国大陆地区的猪流感流行状况和分子特征提供了参考。
In December 2017,pigs in a farm of Shanghai City appeared a series of symptoms,including cough,dyspnea and fever,and the diseased pigs recovered rapidly. Then the nasal swab samples collected from pigs were inoculated into SPF chicken embryos for virus isolation,after further purifying and proliferating the positive samples detected by haemagglutination assay(HA),one strain of swine influenza virus(A/swine/Shanghai/1205/2017)was isolated. The whole genome of the isolate was amplified and sequenced by RT-PCR,the nucleotide sequence homology of the whole reading frame was analyzed by the DNAstar software,and then the genetic evolution tree was designed using MEGA 6 to analyze the amino acid sites. The results showed that the isolated strain was identifi ed as H1 N1 subtype influenza virus,and its eight gene fragments were classifi ed into evolutionary branch of avian-like H1 N1,with no any recombination among influenza virus fragments of various genotypes. The cleavage site sequence of its HA protein was PSIQSR ↓ G,indicating the typical molecular characteristics of low pathogenicity influenza virus. Therefore,the isolation and identification of H1 N1 strain would provide some reference for analyzing the epidemic situation and molecular characteristics of swine influenza in Mainland China.
In December 2017,pigs in a farm of Shanghai City appeared a series of symptoms,including cough,dyspnea and fever,and the diseased pigs recovered rapidly. Then the nasal swab samples collected from pigs were inoculated into SPF chicken embryos for virus isolation,after further purifying and proliferating the positive samples detected by haemagglutination assay(HA),one strain of swine influenza virus(A/swine/Shanghai/1205/2017)was isolated. The whole genome of the isolate was amplified and sequenced by RT-PCR,the nucleotide sequence homology of the whole reading frame was analyzed by the DNAstar software,and then the genetic evolution tree was designed using MEGA 6 to analyze the amino acid sites. The results showed that the isolated strain was identifi ed as H1 N1 subtype influenza virus,and its eight gene fragments were classifi ed into evolutionary branch of avian-like H1 N1,with no any recombination among influenza virus fragments of various genotypes. The cleavage site sequence of its HA protein was PSIQSR ↓ G,indicating the typical molecular characteristics of low pathogenicity influenza virus. Therefore,the isolation and identification of H1 N1 strain would provide some reference for analyzing the epidemic situation and molecular characteristics of swine influenza in Mainland China.
引文
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[3]KYRIAKIS C S,BROWN I H,FONI E,et al.Virological surveillance and preliminary antigenic characterization of influenza viruses in pigs in five European countries from 2006 to 2008[J].Zoonoses&public health,2011,58(2):93-101.
[4]GA?LLE S,LARSEN L E,DüRRWALD R,et al.European surveillance network for influenza in pigs:surveillance programs,diagnostic tools and swine influenza virus subtypes identified in 14 European countries from 2010 to 2013[J].Plos one,2014,9(12):e115815.
[5]BI Y,FU G,CHEN J,et al.Novel swine influenza virus reassortants in pigs,China[J].Emerging infectious diseases,2010,16(7):1162-1164.
[6]YU H,ZHANG P C,ZHOU Y J,et al.Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 infl uenza viruses from pigs in China[J].Biochemical&biophysical research communications,2009,386(2):280-283.
[7]VINCENT A,AWADA L,BROWN I,et al.Review of infl uenza A virus in swine worldwide:a call for increased surveillance and research[J].Zoonoses&public health,2014,61(1):4-17.
[8]XIE J F,ZHANG Y H,ZHAO L,et al.Emergence of Eurasian avian-like swine influenza A(H1N1)virus from an adult case in Fujian Province,China[J].Virologica sinica,2018,33(3):282-286.
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[11]INOUE E,WANG X,OSAWA Y,et al.Full genomic amplification and subtyping of influenza A virus using a single set of universal primers[J].Microbiology&immunology,2010,54(3):129-134.
[12]HOFFMANN E,STECH J,GUAN Y,et al.Universal primer set for the full-length amplifi cation of all infl uenza A viruses[J].Archives of virology,2001,146(12):2275-2289.
[13]MANABU I,KIMIHITO I,REIKO Y,et al.Predicting the antigenic structure of the pandemic(H1N1)2009infl uenza virus hemagglutinin[J].Plos one,2010,5(1):e8553.
[14]GRAAF M D,FOUCHIER R A M.Role of receptor binding specifi city in infl uenza A virus transmission and pathogenesis[J].The EMBO journal,2014,33(8):823-841.
[15]GLASER L,STEVENS J,ZAMARIN D,et al.ASingle amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity[J].Journal of virology,2005,79(17):11533-11536.
[16]TUMPEY T M,MAINES T R,VAN HOEVEN N,et al.A two-amino acid change in the hemagglutinin of the1918 infl uenza virus abolishes transmission[J].Science,2007,315(5812):655-659.
[17]IGARASHI M,ITO K,KIDA H,et al.Genetically destined potentials for N-linked glycosylation of infl uenza virus hemagglutinin[J].Virology,2008,376(2):323-329.
[18]SCHNELL J R,CHOU J J.Structure and mechanism of the M2 proton channel of infl uenza A virus[J].Nature,2008,451(7178):591-595.
[19]CZUDAI-MATWICH V,OTTE A,MATROSOVICH M,et al.PB2 Mutations D701N and S714R promote adaptation of an influenza h5n1 virus to a mammalian host[J].Journal of virology,2014,88(16):8735-8742.
[20]LIU S,ZHU W,FENG Z,et al.Substitution of D701Nin the PB2 protein could enhance the viral replication and pathogenicity of Eurasian avian-like H1N1 swine influenza viruses[J].Emerging microbes&infections,2018,7(1):75.
[21]SHOPE R E.Swine infl uenza[J].Journal of experimental medicine,1931,54(3):373-385.
[22]苏惠娟,李鹏,阳姹,等.欧亚禽系H1N1猪流感病毒在中国的流行和遗传进化[J].中国科学(生命科学),2018,48(12):1287-1294.
[23]VIJAYKRISHNA D,SMITH G J D,PYBUS O G,et al.Long-term evolution and transmission dynamics of swine infl uenza A virus[J].Nature,2011,473(7348):519-522.
[24]QI X,PAN Y,QIN Y,et al.Molecular characterization of avian-like H1N1 swine infl uenza a viruses isolated in Eastern China,2011[J].Virologica sinica,2012,27(5):292-298.