【作者】 陈琳；

【导师】 吴德峰；

【作者基本信息】 福建农林大学 ， 兽医（专业学位）， 2015， 硕士

【摘要】 禽流感是由禽流感病毒(Avian influenza virus, AIV)感染禽类后引起的一种疾病综合征,包括呼吸道、消化道、产蛋量下降或免疫抑制等疾病,可分为高致病性禽流感、低致病性禽流感和无致病性禽流感三类。虽然H9N2亚型禽流感病毒属于低致病性禽流感,但疫病在我国广泛流行,且还发生了多起感染人的事件。由于H9N2禽流感病毒具有频繁变异与重组的特性,不仅给养殖业造成了巨大的损失,同时也严重威胁着人类的生命安全。预防和控制禽流感最有效的手段就是接种疫苗,目前国内比较常见的用来制备H9N2亚型禽流感的疫苗株主要是A/Chicken/Shanghai/F/98(F株)与A/Chicken/Shanghai/6/96等。这些毒株均是08年之前分离的,与现在流行株的抗原性差异非常大,因而疫苗急需升级换代。本试验从2013年在浙江、安徽、安徽等地活禽市场所采样品中分离获得8个H9 AIV分离株。这些H9N2禽流感毒株通过10倍稀释,分别经接种鸡胚尿囊腔接种,最后取尿囊液血凝效价最高的进行下一轮的纯化。三次纯化后,收集最高稀释度和血凝效价的鸡胚尿囊液,存放于-80度冰箱。通过鸡胚尿囊RNA的提取后,经过反转录、PCR等获得所需要的目的基因片段(其中PB2、 PB1、PA、HA、NP基因分成两段扩增)。PCR产物割胶回收并纯化,并送上海美吉公司进行测序。序列测定完成后用DNAstar软件中的SeqMan程序进行序列拼接、用MegAlign程序对拼接后的序列序列进行同源性分析。并用Mega6软件绘制进化树,分析不同年份、不同地域的H9N2亚型AIV流行毒株的遗传进化。结果表明：这8株分离株它们之间的HA基因的同源性为95.7-100%,氨基酸的同源性为96.4-99.8%,NA的核苷酸同源性在93.7%-99.8%之间,氨基酸的的同源性为93.1%-99.8%,NS的核苷酸同源性为95.3%-100%,M的核苷酸同源性为98.2%-100%、NP的核苷酸同源性为95.3%-100%、PA的核苷酸同源性为97.2%-100%、PB1的核苷酸同源性为96.1%-100%、PB2的核苷酸同源性为96.1%-100%。遗传进化分析结果表明,这8株H9N2亚型禽流感病毒株的HA、NA、 NS、M、NP、PA、PB1、PB2基因均属于同一亚系分支,具有较高的同一性。但这些H9N2 AIV与之前的疫苗毒(F株)的HA基因的遗传关系较远,不在同一分支上。结论：现在的H9N2 AIV流行株在抗原性方面较为一致,但与过去毒株的抗原性相差甚远；这些H9N2 AIV流行株的获得,为新的H9 AIV疫苗研制奠定了毒株的基础。更多还原

【Abstract】 Avian Influenza is caused by the avian influenza virus, which can infect poultry and cause many diseases, including respiratory tract, enteron, egg production decline or immunosuppression disease. Avian Influenza can be divided into three categories, including highly pathogenic avian influenza,low pathogenic avian influenza and none pathogenic avian influenza. Though H9N2 subtype avian influenza virus is low pathogenic, it is popular in the whole country and can infect people. Due to the personalities of frequent mutation and recombination, H9N2 AIV not only causes huge losses to farming, but also serious threats to human. The most effective means of prevention and control of avian influenza is vaccination. The current domestic common used for the preparation of H9N2 subtype avian influenza vaccine strain were mainly A/Chicken/Shanghai/F/98 and A/Chicken/Shanghai/6/96 and so on. These strains were isolated before 2008, Whose antigenicity were significantly different from those of the popular stains. Thus, the vaccine virus is urgently needed upgrading.In this study, eight H9 AIV isolates were isolatedfrom the live poultry markets in Zhejiang, Anhui and Hunan provinces in the year of 2013.10 fold dilution of these H9N2 avian influenza strains were inoculated with chicken embryonated eggs respectively.Then the highest of the hemagglutination titer from the highest dilution was selected into the next round of purification. After three purification, the highest dilution and hemagglutination titer of allantoic fluid was collected and stored at -80 degree freezer.After the extraction of RNA with allantoic fluid, reverse transcription and PCR were performedin which PB2, PB1, PA, HA, NP genes were amplified by divided in two parts and the rest of the genes arewhole fragements.The purified PCR products were sent to be sequenced in Shanghai Majorbio Co., Ltd.The sequences were matched by the SeqMan software (DNAstar) and homology analysis were performed by MegAlign.Phylogenetic tree was drawed by using Mega6 in order to analyze the genetic evolution of the subtype AIV pandemic strains of H9N2 in different years and regions.The results demonstrated that:the nucleotide homology of HA among the eight strains is 95.5-100% and its amino acid homology is 96.4-99.6%, NA nucleotide homology is 95.3%-100% and its amino acid homology is 93.1%-99.8%, NS nucleotide homology is 94.1%-94.1%, M nucleotide homology of 98.2%-100%, NP nucleotide homology is 95.3%-100%, PA nucleotide homology is 97.2%-100%, PB1 nucleotide homology is 96.1%-100%, PB2 nucleotide homology is 96.1%-100%. Genetic evolution analysis results show that the HA, NA, NS, M, NP, PA, PB1 and PB2 gene of eight strains of H9N2 subtype avian influenza viruses, all belonged to the same sublineagewith high identities..However, the distance between these H9N2 AIVs and vaccine strains were far away and they were not in the same branch.conclusion:The present H9N2 AIV epidemic strains were much more similar with respect to antigenicity, but were far different from those in the past; The acquisitions of H9N2 AIV epidemic strains were a solid foundation for the new vaccine development of H9N2 AIV.更多还原