禽流感(Avian influenza,AI)为国际兽医局(OIE)规定的A类传染病,严重危害养禽业的健康发展.该研究共分为三部分.试验1,从山东省和河北省不同地区共分离到8株高致病性AIV,通过血清学试验、HA基因扩增和国家流感中心检测(1株),鉴定为H5N1亚型AIV.通过半数鸡胚致死量(ELD50)试验,发现所有分离毒株的ELD50都在10-7以上.结果表明在山东省爆发的高致病性禽流感,主要是由H5N1亚型AIV引起的.试验2,将其中7株分离病毒接种12日龄SPF鸡胚,收取病毒尿囊液,根据已发表的H5N1亚型AIV HA基因序列设计了一对特异性引物P1和P2,用酚-SDS法提取病毒基因组RNA,以此为模板,通过一步法反转录PCR(RT-PCR)分别扩增了7株分离毒株的HA基因全长编码区.测序结果表明,HA基因长约1660个核苷酸,有一个完整的开放式阅读框架,编码550个氨基酸,包括信号肽、HA1和HA2三部分.同源性分析表明,该试验分离毒株和大陆其他分离毒株(包括香港)的核苷酸同源性达到96％,都同属于欧亚种系.遗传发生树分析表明分离毒株基本分为2个谱系,同时裂解点处基因序列分析表明该核苷酸含有强毒株裂解位点的特征性序列,从而从分子水平证明了该病毒分离株为高致病力毒株.试验3,将其中7株H5N1亚型AIV,分别接种6~8周龄的雌性昆明鼠,在接种后15天内,连续观察小鼠的死亡及发病情况.结果发现,河北毒株可以导致小白鼠死亡,接种济南毒株和寿光毒株小白鼠出现发病症状,接种郓城毒株也出现了轻微症状,接种其他分离毒株小白鼠未出现明显症状.此试验结果说明,大陆分离的H5N1亚型AIV毒株也能感染哺乳动物,导致动物发病甚至死亡,这就提醒我们加强对H5N1亚型AIV的检测,谨防其感染人类.关键词:禽流感病毒;血凝素;H5N1;RT-PCR;小鼠致病性ABSTRACT Avian influenza (AI) is one of the A type of infectious diseases described by the OIE which can cause considerable economic losses to poultry industry .The AI genus of H5N1 subtype belong to highly pathogenic avian influenza (HPAI), which widely epidemiced in china and other countries in recently years caused prodigious economic expense. Especially the 97 HongKong"Avian Influenza affair"induced 18 person s infection and 6 person s death.There are also many death in Vietnam, Thailand and other countries caused by the AI at the beginning of 2004. The affairs warned people that H5N1 subtypes AIV has serious threatened to the lives of human being.Hemagglutinin (HA) is the main pathogenic gene, and it is also the target immune antigen of chicken which undertaking the inducing of antibody against AI,to execute humoral immunity,which play important role in the anti-infection immunity importantly. This disquisition is divided into three parts. The first is isolation and identification:Eight highly pathogenic influenza viruses were isolated from differentarea in Shandong province and Hebei province during 2001to 2002, and they were identified to be H5N1subtypethroughtheserologyexamination,HAgenetic proliferation and the identification of the center of thecountry avian influenza. The trial of embryo lethal dose 50 (ELD50) showed that the outbreaking of HPAIV in Shandong province and Hebei province mostly due to H5N1 subtypes. The second is the judgement at the genetic level:12-day-old SPF chicken embryo was inoculated with various concentration of AIV (H5N1 subtype), and the allantoic fluid was obtained. The genomic RNAs of these viruses were extracted by phenol-SDS. The purified genomic RNAs were used as templates for cDNA. According to the published sequence of H5N1HAgenes, a pair of specific primers P1 and P2 was synthesized. The whole genetic encoding area of the 7-isolated strain of HA was proliferated by the method of one step RT-PCR.It demonstrated that the entire coding sequences of HA genes were 1660 nucleotides with a single open reading frame,encoding a polypeptide of 550 amino acids including the signal peptide, HA1 and HA2 .The results of HAgenes homology analysis showed that the homology of all virus was 96％,which belong to Europe and Asia lineages .The result of phylogenic tree showed that the viruses isolated in Shandong province and Hebei province was classified two pedigree, and the deduced analysis of HA genes splitting site suggested that these nucleotides contain characteristic sequence of splitting site of high pathogenic AI strain. The third is the duplication of the disease: the 6-8 weeks Kunming mice were inoculatedwith 7 strain AIV .In the following 15 days, only Hebei strain virus can lead the mice death ,and some others viruses causedtrivial syndrome .It showed that the H5N1 subtype of AIV isolated in Shandong province and Hebei province can make mammal infection, so we should enhance theinspection of H5N1 subtype AIV to avoid the people infection.Key words:avian influenza(AI); Hemagglutinin(HA);H5N1; RT-PCR;Pathogenicity to mice.