新疆南疆驴源H3N8亚型EIVHA基因序列分析
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摘要
为明确新疆南疆驴是否感染马流感病毒(Equine influenza virus,EIV)及驴源EIV HA基因特征,根据GenBank登录的EIV基因序列,设计合成1对引物,采集新疆南疆地区2个驴屠宰点33份驴肺组织样品,采用RT-PCR扩增驴源EIV HA基因片段,并对扩增产物进行测序与序列分析。结果表明,中国新疆南疆驴源EIV A/donkey/Xinjiang/1/2015(H3N8)株HA基因长1 704bp,编码567个氨基酸。同源性分析结果显示,与国内外H3N8亚型EIV参考毒株核苷酸和氨基酸同源性分别为88.9%~99.9%和88.3%~99.8%,与国内毒株和哈萨克斯坦毒株在一个进化分支内。与GenBank数据库中唯一1个驴源毒株A/donkey/Xinjiang/5/2007(H3N8)的比对结果显示,2个毒株裂解位点、糖基化位点、抗原位点和受体结合位点氨基酸完全一致,只是错位2个氨基酸。确定新疆南疆存在驴感染EIV,可能为国内疫情的延续或周边国家传入。
In order to clarify whether donkey was infected by EIV in South Xinjiang and the characteristics of HA gene of EIV from donkey,apair of primers was designed and synthesized according to the sequence of HA gene from GenBank.The HA gene fragment was amplified by RT-PCR for 33 samples of donkey lungs from two donkey slaughter houses in South Xinjiang.Then the electrophoresis analysis,gel recovery,sequencing and sequence analysis were utilized.The results showed that HA gene of A/donkey/Xinjiang/1/2015(H3N8)strain was 1 704 bp in length,encoding 567 amino acids.The homology of the nucleotide sequence and the amino acid sequence with H3N8 subtype of EIV reference strains from domestic and foreign was 88.9%-99.9% and 88.3%-99.8%,respectively.The strain,the Chinese strains and Kazakhstan strain were in same evolutionary branching.Amino acids of cleavage site,glycosylation site,glycosylation site and receptor binding site were completely consistent with A/donkey/Xinjiang/5/2007(H3N8)that the only one strain from donkey in the GenBank database,but two amino acids were misplaced.There were donkeys infected with EIV in South Xinjiang and what may be continuation of the domestic epidemic or introduced from the neighboring countries.
In order to clarify whether donkey was infected by EIV in South Xinjiang and the characteristics of HA gene of EIV from donkey,apair of primers was designed and synthesized according to the sequence of HA gene from GenBank.The HA gene fragment was amplified by RT-PCR for 33 samples of donkey lungs from two donkey slaughter houses in South Xinjiang.Then the electrophoresis analysis,gel recovery,sequencing and sequence analysis were utilized.The results showed that HA gene of A/donkey/Xinjiang/1/2015(H3N8)strain was 1 704 bp in length,encoding 567 amino acids.The homology of the nucleotide sequence and the amino acid sequence with H3N8 subtype of EIV reference strains from domestic and foreign was 88.9%-99.9% and 88.3%-99.8%,respectively.The strain,the Chinese strains and Kazakhstan strain were in same evolutionary branching.Amino acids of cleavage site,glycosylation site,glycosylation site and receptor binding site were completely consistent with A/donkey/Xinjiang/5/2007(H3N8)that the only one strain from donkey in the GenBank database,but two amino acids were misplaced.There were donkeys infected with EIV in South Xinjiang and what may be continuation of the domestic epidemic or introduced from the neighboring countries.
引文
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[15]GARCIA-SASTRE A.The neuramindase of bat influenza viruses is not a neuraminidase[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(46):18635-18636.
[2]韦建华,何奇松,冯淑萍,等.广西马流感病毒(A/Equine/Guangxi/1/09)NA基因的克隆与序列分析[J].南方农业学报,2015,46(5):895-899.WEI J H,HE Q S,FENG SH P,et al.Cloning and sequence analysis on NA gene of equine influenza virus(A/Equine/Guangxi/1/09)in Guangxi[J].Journal of Southern Agriculture,2015,46(5):895-899(in Chinese with English abstract).
[3]CRAWFORD P C,DUBOVI E J,ASTLEMAN W L,et al.Transmission of equine influenza virus to dogs[J].Science,2005,310(5747):482-485.
[4]TU J G,ZHOU H B,JIANG T ZH,et al.Isolation and molecular characterization of equine H3N8influenza viruses form pigs in China[J].Archives of Virology,2009,154(5):887-890.
[5]何晶,马伟,孙建华,等.H3N8亚型马流感病毒HA1基因原核表达载体的构建及表达[J].新疆农业大学学报,2013,36(1):12-15.HE J,MA W,SUN J H,et al.Construction of prokaryotic expression vector of H3N8subtype equine influenza virus HA1gene of and its expression[J].Journal of Xinjiang Agricultural University,2013,36(1):12-15(in Chinese with English abstract).
[6]FOUCHIER R A M,MUNSTE V,WALLENSTEN A,et al.Characterization of a novel influenza A virus hemagglutinin subtype(H16)obtained from black-headed gulls[J].Journal of Virology,2005,79(5):2814-2822.
[7]GUO Y,WANG M,YOSHIHRO K,et al.Characterization of a new Avain-like influenza virus from horses in China[J].Virology,1992,188(1):245-255.
[8]殷震,刘景华.动物病毒学(第二版)[M].北京:科学出版社,1997:704-728.YIN ZH,LIU J H.Animal Virology[M].2nd Edition.Beijing:Science Press,1997:704-728(in Chinese).
[9]蒋桃珍.H3N8亚型马流感病毒的致病性研究及灭活疫苗的研制[D].武汉:华中农业大学,2009.JIANG T ZH.Pathogenicity of equine influenza virus H3N8subtype and development of inactivated vaccine against equine influenza[D].Wuhan:Huazhong Agricultural University,2009(in Chinese with English abstract).
[10]YAMANAKA T,NIWA H,TSUJIMURA K,et al.Epidemic of equine influenza among vaccinated racehorses in Japan in 2007[J].Journal of Veterinary Medical Science,2008,70(6):623-625.
[11]BRYANT N,RASH A,LEWIS N,et al.Australian equine influenza:vaccine protection in the UK[J].Veterinary Record,2008,162(15):491-492.
[12]郭巍,闫研,王英原,等.新疆地区一株马流感病毒的分离及鉴定[J].中国预防兽医学报,2008,30(8):584-586.GUO W,YAN Y,WANG Y Y,et al.Isolation and identification of equine influenza virus in Xinjiang[J].Chinese Journal of Preventive Veterinary Medicine,2008,30(8):584-586(in Chinese with English abstract).
[13]TO K K W,TSANG A K L,CHAN J F W,et al.Emergence in China of human disease due to avian influenza A(H10N8)-Cause for concern?[J].Journal of Infection,2014,68(3):205-215.
[14]CHAN J F,TO K K,TSE H,et al.Interspecies transmission and emergence of novel virues:lesons from bats and birds[J].Trends in Microbiology,2013,21(10):544-555.
[15]GARCIA-SASTRE A.The neuramindase of bat influenza viruses is not a neuraminidase[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(46):18635-18636.