【作者】 李燕；

【导师】 崔保安； 李新生；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 禽流感是一种由A型流感病毒(Avian Influenza,AIV)引起的以禽类感染为主的传染性疾病,根据其致病性的不同可分为高致病性禽流感和低致病性禽流感。低致病性禽流感引发呼吸道症状及产蛋率下降,其中H9亚型禽流感病毒是影响我国养禽业的主要低致病性病毒亚型。近年来河南多家鸡场在进行H9亚型禽流感病毒免疫后,疑似病例仍不断发生,给养鸡业造成了一定的经济损失,给鸡群的防疫安排带来了相当的困难。本研究从河南郑州、南阳、焦作等不同地区采集180份疑似病料,通过接种9-11日龄SPF鸡胚尿囊腔,收集尿囊液,进行血凝试验、血凝抑制试验初步筛选到10份H9亚型禽流感病毒。根据流感病毒命名的标准体系分别命名为A/chicken/Luohe/01/2009(H9 Subtype)、A/chicken/Luohe/02/2010(H9 Subtype)、A/chicken/Xinxiang/04/2009(H9 Subtype)、A/chicken/Xingyang/18/2009(H9 Subtype)、A/chicken/Xinzheng/22/2009(H9 Subtype)、A/duck/Luohe/37/2009(H9 Subtype)、A/chicken/Kaifeng/44/2009(H9 Subtype)、A/chicken/Zhengzhou/45/2009(H9 Subtype)、A/chicken/ Zhengzhou /131/2010(H9 Subtype)、A/chicken/Xinxiang/132/2010(H9 Subtype),分别简称为LH01、LH02、XX04、XY18、XZ22、LH37、KF44、ZZ45、ZZ131、XX132。对其中4株H9亚型禽流感病毒进行理化特性、血凝特性、EID50、MDT、ICPI试验等生物特性研究。所得LH01、XZ22、ZZ131和XX132 4个分离株生物学特性符合禽流感病的基本特性,其EID50结果分别为10-8.68、10-8.5、10-9.5、10-7.83 EID50/0.1ml; MDT值分别为85.6 h、107.2 h、117.6 h、110h;ICPI值分别为17/80=0.2125、13/80=0.1625、14/80=0.175、11/80=0.1375。为了解这4株H9亚型禽流感病毒分离株的分子流行特性,在全基因组序列测定的基础上,比较分析了核苷酸及推导氨基酸全序列之间及与GenBank中登陆的H9亚型代表毒株基因序列之间的同源性,并分析HA及NA的推导氨基酸蛋白裂解位点、受体结合位点及潜在的糖基化位点和红细胞吸附位点等的变异状况及其系统进化地位。这四个毒株的PB2、PB1、PA、HA、NP、NA、M、NS基因的开放阅读框分别由2280、2274、2151、1683、1497、1401、759、864个碱基组成,分别编码759、757、716、560、498、466、252/97、217/121氨基酸残基。这4株病毒HA上的裂解位点序列均为RSSR↓GLF,具有典型的低致病性禽流感病毒的特征,NA茎部均缺失62、63、64位的I、T、E3个氨基酸残基。同源性分析显示XZ22和XX132这两个毒株的8个基因的同源性都最高,都在99%以上,基本处于同一进化分支上;这四个分离株与1997-2009年间大部分中国大陆分离株亲缘关系很近,而且NA、NP和三个聚合酶基因与部分不同亚型病毒间同源性也不低。系统进化关系显示这4个分离毒株的两个表面蛋白基因HA、NA和非结构基因NS均属于类Y280群;而两个内部基因NP、M和聚合酶PA基因可以分为两个群,LH01属于类Y280群,而XZ22、ZZ131和XX132均属于类G1群;余下两个聚合酶基因PB1和PB2也大致分为两个群,LH01和ZZ131属于类Y280群,XZ22和XX132属于类G1群。这些结果为河南H9亚型禽流感病毒的分子流行情况、鉴别诊断及防治提供技术依据。更多还原

【Abstract】 Avian Influenza(AI) was an infection disease caused by type A avian influenza virus(AIV), and it can be divided into highly pathogenic and low pathogenic according to their pathogenicity. A variety of disease syndromes range from sub-clinical to mild upper respiratory disease to loss of egg production caused by low pathogenic avian influenza virus.H9 subtype avian influenza virus was the main subtype viruses of low pathogenicity which affecting our poultry industry.In recent years a number of farms were suspected to infecte AIV after immuning the H9 subtype vaccine in Henan Province. AI caused amount of loss in economic and brought difficulties in the epidemic.180 samples were collected from different regions of Zhengzhou, Nanyang, Jiaozuo etal in Henan Province and viruses were isolated by carring out in embryonated eggs. Viruses were isolated by inoculated in 9-11 day embryonated eggs via the allantoic cavity and collected the allantoic fluid and identified by HA and HI tests.The result suggested that ten H9 subtype avian influenza were isolated. According to the standard body of influenza virus named them as respectively: A/chicken/Luohe/01/2009(H9 Subtype)、A/chicken/Luohe/02/2010(H9 Subtype)、A/chicken/Xinxiang/04/2009(H9 Subtype)、A/chicken/Xingyang/18/2009(H9 Subtype)、A/chicken/Xinzheng/22/2009(H9 Subtype)、A/duck/Luohe/37/2009(H9 Subtype)、A/chicken/Kaifeng/44/2009(H9 Subtype)、A/chicken/Zhengzhou/45/2009(H9 Subtype)、A/chicken/ Zhengzhou /131/2010(H9 Subtype)、A/chicken/Xinxiang/132/2010(H9 Subtype),abbreviated for LH01、LH02、XX04、XY18、XZ22、LH37、KF44、ZZ45、ZZ131、XX132. Which four H9 subtype avian influenza viruses for studying on biological characteristics of physical and chemical properties, hemagglutination , EID50, MDT, ICPI. The results were that their physical and chemical properties meet the basic characteristics of the avian influenza viruse. Their results of EID50 were 10-8.68, 10-8.5,10-9.5, 10-7.83 EID50/0.1ml;Their results of MDT were 85.6 h,107.2 h,117.6 h,110h;Their results of ICPI were17/80=0.2125, 13/80=0.1625,14/80=0.175,11/80=0.1375.In order to understand the molecular epidemiology of H9 subtype avian influenza in Henan Province, we have been sequencing whole genome nucleotide sequence of four isolated strains. Then comparated and analyzed nucleotide and deduced amino acid sequences of these four H9 subtype of avian influenza virus to the representatives of H9 subtype strains in GenBank , and analyzed HA and NA of the deduced amino acid cleavage site, receptor binding sites and potential glycosylation sites and HB sites with analysis software to learn the variation and the phylogenetic position . The date indicated that the open reading frame of PB2, PB1, PA, HA, NP, NA, M, NS gene of these viruses were constitute of 2280, 2274, 2151, 1683, 1497, 1401, 759, 864bp, and separately coded 759,757,716,560,498,466,252/97,217/121 amino acids.The HA cleavage site sequence of these four viruses both were RSSR↓GLF, which was a typical characteristics of low-pathogenic avian influenza virus.The stalks of NA were lacked of I、T、E amino acid residues in sites of 62,63,64. Homology analysis showed that the eight fragments gene of two strains XX132 ,XZ22 were highly homologous to more than 99%, and their evolutionary relations were also in the same evolutionary branch. The four isolated viruses were closed to most of the isolates in China’s mainland in the 1997-2009 years, and homology of their NA, NP and three polymerase genes were close related to partial sequence of different subtypes of avian influenza virus. Phylogenetic relationships showed that the two surface protein genes and non-structural genes belong to the G1 group; and two internal genes NP, M and PA polymerase genes can be divided into two groups, of which LH01 belong to the Y280 group ,or XZ22, ZZ131 and XX132 belong to the Y280 group; and two polymerase genes PB1 and PB2 are broadly divided into two groups, of which LH01 and ZZ131 belong to the Y280group , or XZ22 and XX132 belong to the G1 group. These results provided the technical foundation for the molecular epidemiology, differential diagnosis and prevention research of H9 subtype avian influenza viruse in Henan.更多还原