火鸡的流感病毒唾液酸受体分布特点及其在流感病毒生态系统中的位置和作用

发布时间：2018-01-08 19:11

本文关键词：火鸡的流感病毒唾液酸受体分布特点及其在流感病毒生态系统中的位置和作用　出处：《广西医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：背景和目的：周期性的流感疫情是可以预测的,相比之下,如果大流行出现一种新型流感病毒亚型,则整个人群对其具有很少或缺乏免疫力,是不可预测的事件。大部分新出现的流感病毒亚型都是由禽流感病毒突变而来,目前研究的热点是,流感病毒是如何从其“自然基因库”的水禽类动物进化为具备跨种属障碍感染人。流感病毒外表面有两种糖蛋白,其中一种叫血凝素(Hemagglutinin,HA),它们有助于流感病毒与宿主细胞外表面的糖链特异性接触吸附、结合并进入细胞内的作用。大部分的禽流感病毒与唾液酸(Sialic Acid, SA) a2-3Gal受体结合,人流感病毒与SAa2-6Gal受体结合,这种差异直接导致了禽流感病毒很难跨种属传播到人群。甲型流感病毒易发生变异,特别是膜表面的HA抗原,流感病毒发生抗原变异主要有两种方式,一种是主要引起点突变的抗原漂移,驱使病毒发生抗原漂移的主要原因,是宿主对病毒表面抗原的免疫压力所导致的适应性突变；另一种方式主要是病毒一个或多个病毒基因片段RNA被置换的抗原转换,导致新重配病毒株的产生。基因重配学说认为流感病毒变异亚型大流行,是通过人和禽流感病毒基因重配而来。由于猪呼吸道上皮细胞表面同时存在禽类受体(SAa2-3Gal)和人流感病毒受体(SAa2-6Gal),因此,猪对禽类流感病毒和人流感病毒都具有一定的易感性,如果发生两种病毒同时感染猪的事件,那么禽流感病毒与人流感病毒在猪体内感染、复制的同时可能发生了基因重配,使禽流感病毒获得与人呼吸道SAα2-6Gal受体结合的能力。但是最近几年来,香港、东南亚等地陆续发生了多例禽流感病毒(包括HSN1,H9N2等亚型流感病毒)直接由禽类动物传染给人的事件,随后发现陆地禽类动物也同时具有两种流感病毒受体。火鸡是一类在欧美国家常见的陆地禽类动物,1998年后在北美多次从火鸡体内分离到禽流感病毒和猪流感病毒重组的病毒,提示火鸡可能是一种重要的流感病毒中间宿主,但到目前为止,对火鸡体内的唾液酸受体分布,尚未清楚.。本研究利用凝集素特异性结合SA受体的方法来确定火鸡体内SA受体的分布情况,并用禽流感病毒和人流感病毒结合实验来验证此结果,探讨火鸡在流感病毒生态中的位置及在新型流感病毒大爆发中的作用。方法：(1)采用亲和组化染色法,火鸡组织切片分别滴加生物素标记凝集素MAA-Ⅱ(特异性标记SAa2-3Gal受体)和SNA(特异性标记SAa2-6Gal受体),检测火鸡气管、支气管、次级支气管、副支气管以及直肠组织的SAa2-3Gal和SAa2-6Gal受体的表达。(2)利用与SAa2-3Gal受体结合的鸭源性禽流感病毒株(H9N1)和与SAa2-6Gal受体结合的人流感病毒株(H1N1),分别在鸡胚和MDCK通过接种传代法扩增病毒,纯化后与荧光素Alexa488结合。然后滴加标记了荧光素Alexa488的H9N1禽流感病毒和H1N1人流感病毒于火鸡气管、支气管、次级支气管、副支气管以及直肠的组织切片上,荧光显微镜下观察拍照并分析结果。结果：(1)火鸡呼吸道和消化道SAa2-3Gal的受体分布特点：禽类SAa2-3Gal受体主要分布在火鸡的气管、支气管、次级支气管和副支气管四个呼吸道解剖部位中的大部分区域,亲和组化分析呈强阳性分布,而在直肠内表达较少并呈弱阳性分布,SAa2-3Gal受体在上述各部位的分布差异有统计学意义。(2)火鸡呼吸道和消化道SAa2-6Gal受体分布特点：火鸡体内的SAα2-6Gal受体分布在呼吸道的气管、支气管和次级支气管大部分区域,亲和组化分析呈弱阳性分布,而在呼吸道的的终末端副支气管分布极少,在直肠呈极少量分布,SAα2-6Gal受体在上述各部位的分布差异有统计学意义。(3)SAα2-3Gal受体和SAα2-6Gal受体各解剖部位分布比较：禽类SAα2-3Gal受体在火鸡呼吸道的气管、支气管、次级支气管以及副支气管四个解剖部位中的分布明显高于人SAα2-6Gal受体,而在消化道的直肠组织中火鸡SAα2-6Gal受体与SAα2-3Gal受体分布无明显差异。(4)利用荧光素Alexa488标记的H9N1禽流感病毒以及H1N1人流感病毒与火鸡呼吸道及消化道结合特点：H9N1禽流感病毒与火鸡呼吸道的气管、支气管和次级支气管及副支气管的上皮细胞的大部分区域结合,而H1N1人流感病毒仅少量的与火鸡呼吸道的气管、支气管和次级支气管的上皮细胞结合。H9N1与H1N1流感病毒与火鸡的直肠组织上皮细胞都未见荧光结合。荧光素标记病毒结合实验结果基本与凝集素亲和组化所做出的火鸡呼吸道和消化道的SAα2-3Gal受体和SAα2-6Gal受体的分布特点相一致。结论：(1)火鸡呼吸道上皮细胞均有流感病毒SAα2-3Gal受体和SAα2-6Gal受体,以SAα2-3Gal受体为主。在气管、支气管、次级支气管及副支气管四个解剖部位,SAα2-3Gal受体均呈高密度分布,火鸡SAα2-6Gal受体少量分布,而且仅在气管、支气管、次级支气管三个解剖部位有分布。两种SA受体在直肠组织中均分布较少；(2)火鸡呼吸道组织对H9N1流感病毒敏感,气管、支气管、次级支气管及副支气管组织均较易结合H9N1禽流感病毒,支持SAα2-3Gal受体的分布特点；(3)H1N1人流感病毒主要结合火鸡上段呼吸道的气管、支气管、次级支气管组织,且少量分布,与SAα2-6Gal受体的分布特点基本吻合；(4)火鸡呼吸道上段均含有SAα2-3Gal受体和SAα2-6Gal受体,并且禽流感病毒和人流感病毒均能结合在呼吸道上段,提示火鸡也是一种甲型流感病毒“基因混合器”,可能有助于病毒基因重配,有形成大流行株的可能。
[Abstract]:Background and purpose: a seasonal influenza epidemic situation can be predicted, in contrast, if the emergence of a new pandemic influenza virus subtype, the entire population has little or lack of immunity to it, is unpredictable events. Most of the new influenza virus subtype of avian influenza virus is the mutation. The focus of current research is that the flu virus is palmipeds animal evolution from their "natural gene pool" to have cross the species barrier to infect people. There are two types of influenza virus surface glycoprotein, which is called a hemagglutinin (Hemagglutinin, HA), they help sugar chain specific adsorption on influenza virus and contact the outer surface of the host cell, and combined with the role of intracellular entry. Avian influenza virus and most of the sialic acid (Sialic Acid, SA) a2-3Gal receptor binding, binding to human influenza virus and SAa2-6Gal receptor, this difference directly guide It is difficult for the avian influenza virus to spread across species to the population.
Influenza A virus to mutate, especially HA membrane surface antigen, influenza virus antigenic variation occurs mainly in two ways, one is the main starting point mutation induced antigenic drift, driven mainly due to virus antigen drift, mutation free of host adaptation pressure disease virus surface antigen caused by another; a virus is the main way of one or more virus gene fragments are converted to RNA replacement antigen, leading to new reassortant virus strains. The reassortant influenza virus subtype mutation theory that a pandemic, by human and avian influenza virus gene reassortment. Due to the surface of pig airway epithelial cells also exist in poultry receptor (SAa2-3Gal) and human influenza virus receptor (SAa2-6Gal), therefore, the pig has a certain susceptibility to avian influenza virus and human influenza virus, if two kinds of virus infected pigs incident, Then the avian influenza virus and human influenza virus infection in pigs, replication may occur at the same time the gene reassortment, the avian influenza virus acquired the ability to combine with human respiratory SA alpha 2-6Gal receptor. But in recent years, Hongkong, Southeast Asia and other places have occurred in many cases of avian influenza virus (including HSN1, H9N2 etc. influenza virus) directly infect humans events from birds to animal, then found in terrestrial poultry animal also has two kinds of influenza virus receptor. Turkey is a kind of terrestrial poultry animal common in Europe and the United States, 1998 in North America many times from Turkey isolated avian influenza virus and swine influenza virus recombinant virus, suggesting that Turkey is one of the most important intermediate host of influenza virus, but so far, the distribution of sialic acid receptors in Turkey, is not clear. This study method with the lectin binding specificity of SA receptor To identify the distribution of SA receptor in Turkey and verify the results by combining avian influenza virus with human influenza virus, we discuss the position of Turkey in the influenza virus ecology and its role in the outbreak of a new influenza virus.
Methods: (1) using affinity histochemical staining, Turkey tissue sections were dropping biotinylated lectin II (MAA- specific marker SAa2-3Gal receptor) and SNA (a specific marker for SAa2-6Gal receptor), detection of Turkey trachea, bronchus, secondary bronchi, the expression of SAa2-3Gal and SAa2-6Gal receptor in rectal tissue and bronchial side. (2) the use of duck derived avian influenza virus binds to the SAa2-3Gal receptor (H9N1) and human flu virus binds to the SAa2-6Gal receptor (H1N1), respectively in the chick embryo and MDCK by inoculating passage amplification of virus, purified with fluorescein Alexa488. Then add the marker of H9N1 avian influenza virus and H1N1 the human influenza virus Alexa488 in Turkey fluorescein trachea, bronchus, secondary bronchi, bronchial and rectal side on tissue sections were observed under fluorescence microscope and photographed the analysis results.
Results: (1) receptor distribution characteristics of Turkey respiratory and digestive tract SAa2-3Gal: avian SAa2-3Gal receptor mainly located in Turkey's trachea, bronchus, bronchus and four secondary side bronchial airway anatomy in most area, affinity group analysis showed strong positive expression in the rectum and distribution, and less positive distribution. There were statistically significant differences in the distribution of the various parts of the SAa2-3Gal receptor. (2) Turkey respiratory and digestive tract SAa2-6Gal receptor distribution in Turkey: SA alpha 2-6Gal receptor distribution in respiratory trachea, tracheal and bronchial branches in most regions of the secondary, affinity group analysis showed weak positive distribution, and in the end side of respiratory tract bronchial distribution rarely, is a very small amount of the distribution in the rectum, there were statistically significant differences in the distribution of the various parts of the SA alpha receptor. 2-6Gal (3) SA and SA receptor alpha 2-3Gal alpha 2-6Gal The receptor anatomic site distribution: trachea, avian SA alpha 2-3Gal receptor in Turkey respiratory bronchi, bronchial and secondary distribution parabronchi four anatomic sites are significantly higher than that in human SA alpha 2-6Gal receptor, and SA receptor SA and Turkey alpha 2-6Gal alpha 2-3Gal receptor distribution had no obvious difference in rectal tissue of digestive tract (. 4) using fluorescein labeled Alexa488 H9N1 avian influenza virus and human influenza virus H1N1 and Turkey respiratory and digestive tract: H9N1 combined with the characteristics of avian influenza virus and Turkey respiratory trachea, bronchi and secondary bronchi in most regions and side bronchial epithelial cells with H1N1, and only a small amount of human influenza virus and respiratory tract in Turkey there was no fluorescence with the trachea, bronchi and secondary bronchi epithelial cells with.H9N1 and H1N1 influenza virus and Turkey rectal tissue epithelial cells were labeled. The results of virus binding test were basically consistent with the distribution characteristics of SA alpha 2-3Gal receptor and SA alpha 2-6Gal receptor in Turkey respiratory tract and alimentary tract made by lectin affinity histochemistry.
Conclusion: (1) Turkey had respiratory epithelial cells of influenza virus SA alpha 2-3Gal receptor and SA receptor alpha 2-6Gal, alpha SA to 2-3Gal receptor. In the trachea, bronchus, bronchus and four secondary side bronchial anatomy, SA alpha 2-3Gal receptor showed a high density distribution, Turkey SA alpha 2-6Gal receptor and only a small amount of the distribution. In the trachea, bronchus, secondary bronchus three anatomic site distribution. Two SA receptors are distributed less in rectal tissues; (2) Turkey respiratory tissue sensitivity to H9N1 influenza virus, trachea, bronchus, bronchus and trachea tissue secondary collateral branch were more easily with the H9N1 avian influenza virus, the distribution characteristics of SA alpha 2-3Gal support receptor; (3) H1N1 human influenza virus with upper respiratory tract mainly Turkey trachea, bronchus, secondary bronchial tissue, and a small amount of the distribution, basically consistent with the distribution of SA alpha 2-6Gal receptor; (4) the upper respiratory tract of Turkey Both of them contain SA alpha 2-3Gal receptor and SA alpha 2-6Gal receptor, and avian influenza virus and human influenza virus can bind to the upper respiratory tract, suggesting Turkey is also a "gene mixer" of influenza A virus, which may help virus gene redistribution and have the possibility of forming a pandemic strain.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：S852.65;R373.13

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