H5N1亚型禽流感病毒以巨胞饮作用侵入细胞的研究
发布时间：2018-10-30 10:07
【摘要】：禽流感(Avian Influenza)是由A型流感病毒(Influenza A Virus)引起的禽类传染病。根据HA和NA的抗原性,禽流感病毒分为16个HA亚型和9个NA亚型。根据毒力差异可分为高致病性禽流感病毒、低致病性禽流感病毒。自1997年高致病性禽流感(HPAI)H5N1亚型爆发以来,该病毒造成禽类的大量死亡,并且能够感染人类。如何有效地防治H5N1高致病性禽流感病毒成为研究的重点和热点。为启动复制,病毒须将自身基因组运输到宿主细胞内。少数病毒与细胞膜直接融合将自身基因组释放至细胞(例如HIV),而大部分病毒则通过内吞作用进入细胞。由于细胞存在多种内吞途径,每种病毒采用的具体的内吞途径也不尽相同。研究表明,流感病毒侵入细胞是通过HA蛋白与细胞表面的唾液酸受体结合,经过网格蛋白和小窝蛋白介导的侵入途径进入细胞,而Dynamin蛋白是网格蛋白和小窝蛋白介导内吞的关键蛋白。本研究为了研究病毒内吞作用的侵入途径,采用AIV A/duck/Guangxi/35/2001(DK/35)(H5N1)、rDK/35(Q226L/G228S)(H5N1)、rDK35/HA-A160T(H5N1)作为主要研究用毒株,加入Dynamin蛋白的抑制剂Dynasore,通过间接免疫荧光观察病毒侵入情况。结果表明,在胎牛血清存在的情况下,流感病毒能够采用不依赖于Dynamin的侵入方式,即流感病毒能够采用不依赖于网格蛋白和小窝蛋白介导的侵入方式侵入细胞。本研究进一步采用巨胞饮作用抑制剂5-(N-乙基-N-异丙基)阿米洛利EIPA,观察病毒侵入情况,结果表明,巨胞饮作用在不依赖于网格蛋白和小窝蛋白介导的侵入体外培养的细胞中起到一定作用。此外,去除细胞表面的唾液酸受体,观察病毒侵入情况。结果表明,唾液酸受体在病毒不依赖于Dynamin的侵入方式中起着一定作用。同样,采用不同受体结合性病毒感染A549时,巨胞饮作用同样发挥一定作用。在Dynasore存在的情况下,采用DK/35感染DF1、MDCK,结果表明不同种源细胞中,巨胞饮在病毒侵入时发挥一定作用。本实验所验证的巨胞饮在流感病毒侵入细胞时可能发挥的作用,为流感病毒的感染机理的研究提供了实验依据,对于后续的流感病毒的防控提供了一定的思路。
[Abstract]:Avian influenza (Avian Influenza) is a avian infectious disease caused by influenza A virus (Influenza A Virus). According to the antigenicity of HA and NA, avian influenza virus is divided into 16 HA subtypes and 9 NA subtypes. According to virulence difference can be divided into highly pathogenic avian influenza virus, low-pathogenic avian influenza virus. Since the outbreak of the highly pathogenic avian influenza (HPAI) H5N1 subtype in 1997, the virus has caused a large number of deaths in poultry and can infect humans. How to effectively prevent and cure H5N1 highly pathogenic avian influenza virus has become the focus and hotspot of research. To initiate replication, the virus must transport its own genome into the host cell. A few viruses are directly fused with cell membranes to release their genomes into cells (such as HIV), while most viruses enter cells through endocytosis. Due to the existence of multiple endocytosis pathways, the specific endocytosis pathways used by each virus are different. Studies have shown that influenza virus invades cells through a sialic acid receptor binding to the HA protein, and enters the cell via a griddle protein and nest protein mediated invasion pathway. Dynamin protein is the key protein of endocytosis mediated by grid protein and fossa protein. In this study, AIV A/duck/Guangxi/35/2001 (DK/35) (H5N1), rDK/35 (Q226L/G228S) (H5N1), rDK35/HA-A160T (H5N1) were used to study the invasion pathway of virus endocytosis. Dynasore, an inhibitor of Dynamin protein, was used to observe the invasion of the virus by indirect immunofluorescence. The results showed that in the presence of fetal bovine serum, influenza virus could invade the cells without Dynamin, that is, the influenza virus could invade the cells in a manner independent of grid protein and nest protein. In this study, 5- (N-ethyl-N-isopropyl) amiloride EIPA, was further used to observe the invasion of the virus. Megacyte plays a role in the invasion of cells independent of grid protein and fossa protein in vitro. In addition, the sialic acid receptor on the cell surface was removed and virus invasion was observed. The results showed that sialic acid receptors play a role in the viral invasion independent of Dynamin. Similarly, when A549 was infected with different receptor-binding viruses, giant cell drink also played a role. In the presence of Dynasore, the results of DF1,MDCK, infection with DK/35 showed that megakyne played a role in virus invasion in different provenance cells. The possible role of giant cell drink in the invasion of influenza virus provides experimental basis for the study of the infection mechanism of influenza virus and provides some ideas for the prevention and control of subsequent influenza virus.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
，
本文编号：2299697
[Abstract]:Avian influenza (Avian Influenza) is a avian infectious disease caused by influenza A virus (Influenza A Virus). According to the antigenicity of HA and NA, avian influenza virus is divided into 16 HA subtypes and 9 NA subtypes. According to virulence difference can be divided into highly pathogenic avian influenza virus, low-pathogenic avian influenza virus. Since the outbreak of the highly pathogenic avian influenza (HPAI) H5N1 subtype in 1997, the virus has caused a large number of deaths in poultry and can infect humans. How to effectively prevent and cure H5N1 highly pathogenic avian influenza virus has become the focus and hotspot of research. To initiate replication, the virus must transport its own genome into the host cell. A few viruses are directly fused with cell membranes to release their genomes into cells (such as HIV), while most viruses enter cells through endocytosis. Due to the existence of multiple endocytosis pathways, the specific endocytosis pathways used by each virus are different. Studies have shown that influenza virus invades cells through a sialic acid receptor binding to the HA protein, and enters the cell via a griddle protein and nest protein mediated invasion pathway. Dynamin protein is the key protein of endocytosis mediated by grid protein and fossa protein. In this study, AIV A/duck/Guangxi/35/2001 (DK/35) (H5N1), rDK/35 (Q226L/G228S) (H5N1), rDK35/HA-A160T (H5N1) were used to study the invasion pathway of virus endocytosis. Dynasore, an inhibitor of Dynamin protein, was used to observe the invasion of the virus by indirect immunofluorescence. The results showed that in the presence of fetal bovine serum, influenza virus could invade the cells without Dynamin, that is, the influenza virus could invade the cells in a manner independent of grid protein and nest protein. In this study, 5- (N-ethyl-N-isopropyl) amiloride EIPA, was further used to observe the invasion of the virus. Megacyte plays a role in the invasion of cells independent of grid protein and fossa protein in vitro. In addition, the sialic acid receptor on the cell surface was removed and virus invasion was observed. The results showed that sialic acid receptors play a role in the viral invasion independent of Dynamin. Similarly, when A549 was infected with different receptor-binding viruses, giant cell drink also played a role. In the presence of Dynasore, the results of DF1,MDCK, infection with DK/35 showed that megakyne played a role in virus invasion in different provenance cells. The possible role of giant cell drink in the invasion of influenza virus provides experimental basis for the study of the infection mechanism of influenza virus and provides some ideas for the prevention and control of subsequent influenza virus.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
，
本文编号：2299697
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