2017年山东16株H9N2亚型禽流感病毒的分离鉴定及其HA基因序列分析
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摘要
为了解山东省H9N2亚型禽流感的流行情况, 2017年从山东省不同地区分离并鉴定16株H9N2亚型禽流感病毒(AIV)株,并对其血凝素(HA)基因进行扩增、克隆和测序,将所得序列进行同源性和遗传进化分析。结果显示,2017年分离的16株病毒之间HA基因的核苷酸序列同源性为91.2%～99.8%,氨基酸同源性为93.6%～100.0%。系统进化树显示,16株病毒均属于H9.4.2.5分支,表明H9.4.2.5分支毒株仍是山东省H9N2亚型AIV主要流行株。重要氨基酸位点研究显示,16株病毒的HA蛋白的裂解位点仍是K/RSSR↓GLF,符合低致病性AIV的特征;16株病毒的234受体结合位点多为L,除198位受体结合位点存在变异外,其他受体结合位点均保守;16株病毒的潜在糖基化位点有7个～8个,其中7个位点较保守,而218位糖基化位点均缺失,其中3株病毒分别在145位、206位和285位新增糖基化位点。总之,山东省分离的H9N2亚型AIV在分子生物学上发生了部分新的遗传进化,但与临床上病毒的致病性与流行规律的关系还有待于进一步研究。
To understand the epidemic situation of H9N2 subtype avian influenza virus in Shandong province,16 strains of H9N2 subtype avian influenza viruses were isolated and identified from different parts of Shandong province in 2017.Hemagglutinin(HA)genes were amplified,cloned and sequenced,and the homology and genetic evolution of the sequences were analyzed.The results showed that the nucleotide homologies of HA genes of 16 strains isolated in 2017 ranged from 91.2%to 99.8%,and the amino acid homologies ranged from 93.6%to 100.0%.Phylogenetic tree showed that all strains belonged to H9.4.2.5 branch,indicating that the branch was still the main epidemic one in our province.The important amino acid sites showed that the cleavage site of HA protein of 16 strains was still K/RSSR↓GLF,which still accorded with the characteristics of low pathogenic bird flu.The 234 receptor binding sites were mostly L,and the other receptor binding sites were conservative except for the 198 receptor binding sites which had some variations.There were 7-8 potential glycosylation sites,of which 7 sites were conservative,while 218 glycosylation sites were missing,and 3 strains were added to 145,206 and 285 glycosylation sites,respectively.In conclusion,the genetic evolution of H9N2 subtype AIV has occurred in molecular biology,but the relationship with the pathogenicity and epidemic law of the clinical virus remains to be further studied,thus laying the foundation for the prevention and control of H9N2.
To understand the epidemic situation of H9N2 subtype avian influenza virus in Shandong province,16 strains of H9N2 subtype avian influenza viruses were isolated and identified from different parts of Shandong province in 2017.Hemagglutinin(HA)genes were amplified,cloned and sequenced,and the homology and genetic evolution of the sequences were analyzed.The results showed that the nucleotide homologies of HA genes of 16 strains isolated in 2017 ranged from 91.2%to 99.8%,and the amino acid homologies ranged from 93.6%to 100.0%.Phylogenetic tree showed that all strains belonged to H9.4.2.5 branch,indicating that the branch was still the main epidemic one in our province.The important amino acid sites showed that the cleavage site of HA protein of 16 strains was still K/RSSR↓GLF,which still accorded with the characteristics of low pathogenic bird flu.The 234 receptor binding sites were mostly L,and the other receptor binding sites were conservative except for the 198 receptor binding sites which had some variations.There were 7-8 potential glycosylation sites,of which 7 sites were conservative,while 218 glycosylation sites were missing,and 3 strains were added to 145,206 and 285 glycosylation sites,respectively.In conclusion,the genetic evolution of H9N2 subtype AIV has occurred in molecular biology,but the relationship with the pathogenicity and epidemic law of the clinical virus remains to be further studied,thus laying the foundation for the prevention and control of H9N2.
引文
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[2] 毕英佐.H9N2亚型禽流感的流行现状和防制措施[J].中国家禽,2009,31(11):33-34.
[3] 李树纯,李心海,仲书官,等.H9N2亚型禽流感病毒基因组的遗传进化分析[J].病毒学报,2012,28(1):7-14.
[4] 丛彦龙,钟颖,孙艺学,等.H9N2亚型禽流感病毒流行病学及其疫苗的研究进展[J].中国兽医学报,2017,37(2):386-392.
[5] 刁有祥,丁家波.低致病性禽流感病毒对养禽业的危害[J].中国家禽,2006(6):1-3.
[6] Maclachlan N J,Dubovi J E.兽医病毒学[M].孔宪刚,刘胜旺,译.4版.北京:中国农业出版社,2015:375.
[7] 刘金华,甘孟候.中国禽病学[M].2版.北京:中国农业出版社,2016:109.
[8] 顾敏,彭大新,刘秀梵.我国H9N2亚型禽流感病毒的流行和进化特点[J].生命科学,2015,27(5):531-538.
[9] 孟芳,徐怀英,张伟,等.近20年中国部分地区鸡源H9N2亚型禽流感病毒HA基因遗传演化及其变异频率[J].微生物学报,2016,56(1):35-43.
[10] 刘金华,吴清民,陈福勇,等.鸡源株与人源株H9N2流感病毒血凝素受体结合位点氨基酸比较与分析[J].中国预防兽医学报,2003(6):17-19.
[11] Jiang W M,Liu S,Hou G Y,et al.Chinese and global distribution of h9 subtype avian influenza viruses[J].PLoS One,2013,7(12):e52671.
[12] huo S,Bi Y H,Wong G,et al.Epidemiology,evolution,and recent outbreaks of avian influenza virus in China[J].J Virol,2015,89(17):8671-8676.
[13] Chen H L,Subbarao K,Swayned D,et al.Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as a pandemic vaccine candidate[J].Vaccine,2003,21(17):1974-1979.
[14] Sun Y P,Liu J H.H9N2 influenza virus in China:a cause of concern[J].Protein Cell,2015,6(1):18-25.
[15] 陈陆,郑鹿平,赵军,等.H9N2亚型禽流感病毒HA蛋白S145N变异株致病性及抗原特性[J].畜牧兽医学报,2012,43(1):82-89.
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