广西H3亚型禽流感病毒生态进化及检测技术的研究

发布时间：2018-03-06 13:48

本文选题：H3亚型禽流感病毒　切入点：分离鉴定　出处：《广西师范大学》2015年硕士论文　论文类型：学位论文

【摘要】：禽流感(Avian influenza, AI)是一种世界范围性的禽类传染病,给养禽业造成巨大的经济损失,对人类也存在潜在的威胁。H3亚型禽流感病毒(Avian influenza virus, AIV)为低致病性AIV,不仅可以感染禽类,而且在猪、马、犬等哺乳动物体内也可检测到。为了解广西地区H3亚型AIV的生态流行趋势,掌握其遗传进化特点以及与不同宿主(猪、马、犬和人)的H3亚型流感病毒之间的传播关系,同时建立针对H3亚型AIV的核酸检测技术来达到及早诊断和防控的目的,进行了如下研究。本研究从2009～2014年活禽市场采集样品中,通过血清学试验初步筛选出主要以H3亚型AIV混合感染的毒株,并应用鸡胚终点稀释法及血清中和法对病毒进行了分离和纯化。为了进一步确认其基因型,对分离株HA和NA基因进行了克隆及序列测定,发现其具有H3N2、H3N6和H3N8三种基因型。对其中14株分离株进行了生物学特性试验,结果表明这14株分离株均为低致病性禽流感,且均具有优先选择于禽源SA α-2,3-Gal受体结合的特性,不具有感染人的潜在能力。通过对14株H3亚型AIV分离株的八个基因片段(HA、NA、PB2、PB1、PA、NP、NS和M)进行克隆、序列测定和分析,表明14株分离株HA基因裂解位点均只有一个碱性氨基酸,符合低致病性禽流感的分子特征；与GenBank中不同宿主的H3亚型流感病毒及与分离株的八个基因片段同源性最高的毒株进行各片段核苷酸序列同源性比较,并构建各个基因组分子遗传进化树。遗传进化分析表明14株分离株各基因组均属于欧亚谱系的禽源进化分支,相邻年份的分离株各基因组核苷酸同源性较高,进化树分布位置相对集中,可能来源于相同的祖先；分离年份间隔较大的毒株,各基因组核苷酸序列同源性相对较低,亲缘关系较远,具有明显的时间差异性,上述结果表明H3亚型AIV可能处于不断进化中；同一分离株的八个基因片段来源较为复杂,可能是由不同亚型AIV发生基因重排而来；14株分离株与猪源、人源、马源、犬源H3亚型流感病毒各基因组核苷酸序列同源性较低,亲缘关系较远,具有明显的种属差异性。本研究针对H3亚型AIV建立了两种核酸检测方法：1)多重RT-PCR方法,包括H3N2亚型AIV双重RT-PCR和H3N2亚型与M基因三重RT-PCR。2)实时荧光定量RT-PCR方法,包括H3N2亚型AIV双重实时荧光定量RT-PCR和H3N8亚型AIV一步法实时荧光定量RT-PCR。该方法都实现了一管多检的目的,节省了时间与成本,同时也提高了检测的准确性,为临床样品的检测及H3亚型AIV的防控提供了技术支撑。
[Abstract]:Avian influenza virus (AII) is a worldwide avian infectious disease, which causes enormous economic losses to the poultry industry and has a potential threat to humans. Avian influenza virus (AIV) is a low pathogenic avian influenza virus, which can not only infect birds. In order to understand the ecological epidemic trend of H3 subtype AIV in Guangxi region, to understand its genetic and evolutionary characteristics and with different hosts (pig, horse, horse), The relationship between the transmission of H3 subtype influenza virus in dogs and humans, and the establishment of nucleic acid detection for H3 subtype AIV for early diagnosis and prevention and control, were studied as follows. This study was carried out from the live poultry market from 2009 to 2014. In order to confirm the genotypes of H3 subtype AIV, the virus was isolated and purified by serological test, and the virus was isolated and purified by chicken embryo end point dilution method and serum neutralization method. The HA and na genes of the isolates were cloned and sequenced. It was found that they had three genotypes of H _ 3N _ 2, H _ 3N _ 6 and H _ 3N _ 8. The biological characteristics of 14 of them were tested. The results showed that the 14 isolates were all low pathogenic avian influenza. All of them were preferentially selected for the binding of SA 伪 -2N 3-Gal receptor, and did not have the potential to infect human beings. Eight gene fragments of 14 H3 subtype AIV isolates were cloned, sequenced and analyzed. The results showed that there was only one basic amino acid in HA gene cleavage site of 14 isolates, which was consistent with the molecular characteristics of low pathogenic avian influenza. The nucleotide sequence homology was compared with H3 subtype influenza virus in different hosts of GenBank and the isolates with the highest homology of eight gene fragments. The genetic evolution analysis showed that all the genomes of 14 isolates belonged to the avian evolutionary branch of Eurasian lineage, and the nucleotide homology of each genome in adjacent years was high. The distribution of the evolutionary tree is relatively concentrated, which may come from the same ancestor. The nucleotide sequence homology of each genome is relatively low, and the genetic relationship is far away. These results suggest that H3 subtype AIV may be in continuous evolution, and that the origin of eight gene fragments of the same isolate is more complex, which may be the result of gene rearrangement from different subtypes of AIV to 14 isolated strains and porcine, human and horse sources. The nucleotide sequence homology of H3 subtype influenza virus in dogs was low and the genetic relationship was far away. In this study, two nucleic acid detection methods for H3 subtype AIV were established. Including H3N2 subtype AIV double RT-PCR and H3N2 subtype and M gene triple RT-PCR.2) real-time fluorescence quantitative RT-PCR method, including H3N2 subtype AIV dual real-time fluorescence quantitative RT-PCR and H3N8 AIV one-step real-time fluorescence quantitative quantitative RT-PCR. It can save time and cost, improve the accuracy of detection, and provide technical support for the detection of clinical samples and the prevention and control of H3 subtype AIV.
【学位授予单位】：广西师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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