【作者】 李建丽；

【导师】 毕丁仁；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 博士

【摘要】 禽流感(Avian Influenza,AI)是由禽流感病毒(Avian Influenza Virus,AIV)引起的一种禽类感染和/或疾病综合征,被世界动物卫生组织(OIE)和我国定为A类传染病。自1878年首次在意大利暴发以来,已在世界各地广泛流行。近年来,AI出现了新特点,尤其是高致病性禽流感病毒(HPAIV)通过抗原漂移和抗原转变,已获得了感染人并造成死亡的能力,这赋予了HPAIV更重要的公共卫生学意义。目前,AIV致病机制还不甚清楚,可能是多基因共同作用的结果。其中,非结构蛋白(Non-structural protein,NS1)只存在于病毒感染的细胞内,是在细胞水平上的调节AIV致病性的关键因子。NS1蛋白主要是通过其RNA结合区和效应区与宿主内的许多蛋白相互作用来实现的,但这些研究大多数是采用人流感及其适应性细胞来研究的,而利用AIV及其天然宿主的研究还未见报道。因此,深入开展HPAIV的NS1蛋白与其天然宿主蛋白之间的相互作用研究,可进一步揭示NS1蛋白的功能及其调节HPAIV致病力的机制。因此,本研究利用HPAIV及SPF鸡为实验材料,运用组织病理学及蛋白质相互作用技术,研究了SPF鸡感染AIV的细胞凋亡、NS1蛋白的分布及NSl蛋白的相互作用蛋白,取得了如下成果:1 H9N2亚型AIV NSl基因的遗传进化分析利用RT-PCR方法,扩增了1998-2005年间分离的9株H9N2亚型AIV的NS1基因,序列分析表明,9株AIV NS1基因完整的阅读框均为654bp,编码217个氨基酸,其核苷酸及其推导氨基酸同源性分别为95.4%-99.8%和93.6%-100%;9株病毒的NS1蛋白的C-端均缺失了13个aa,已成为中国大陆分离株(H9N2亚型)的一个分子标记。进化分析表明,所有分离株都属于基因A群,形成了一个独立分支,且均起源于中国最早的分离株(Ck/BJ/1/94株),说明H9N2亚型AIV的NS1基因随着流行时间出现了分化。另外,除Ck/HN/A3/98株外其余8株属于Ck/SH/F/98-like,这说明了Ck/SH/F/98-like株的H9N2亚型AIV在中国大陆鸡群中广泛存在。2 SPF鸡感染高致病性禽流感病毒(H5N1亚型)细胞凋亡的研究用末端转移酶介导的缺口末端标记法(TdT-mediated X-dUTP nick end labling,TUNEL)研究了SPF鸡人工感染HPAIV(H5N1亚型)的细胞凋亡,结果表明,HPAIV(H5N1亚型)能诱导各脏器不同程度的细胞凋亡,以胸腺、法氏囊、胰脏、肾脏的细胞凋亡最严重,凋亡细胞主要为血管内皮细胞、腺上皮细胞、软骨细胞及部分淋巴细胞。本研究表明,HPAIV可诱导本动物发生快速的细胞凋亡。3 HPAIV(H5N1亚型)NS1蛋白在感染鸡组织中分布研究利用NS1蛋白单克隆抗体,建立了检测NS1蛋白的免疫组化法(SABC法),并研究了HPAIV感染30-31h后NS1蛋白在鸡各脏器中的分布,结果表明,NS1蛋白主要分布于心脏、胰脏、肾脏及小脑颗粒层;阳性细胞主要为各组织器官的小或微血管内皮细胞、心肌细胞、肾小管上皮细胞、腺上皮细胞、部分淋巴细胞及坏死灶周围的细胞,说明了心肌、胰腺、肾脏、小脑及血管内皮细胞可能是HPAIV的主要靶器官。结合细胞凋亡的研究结果,可推测,心血管系统在HPAIV的复制和扩散中起重要作用。同时也证明了NS1蛋白与细胞凋亡有相关性。4利用酵母双杂交系统筛选NS1蛋白相互作用的宿主蛋白本研究利用SMART技术首次构建了HPAIV感染鸡脾脏cDNA文库,然后利用酵母双杂交系统从该cDNA文库中初步筛选到了4个能与NS1蛋白相互作用的蛋白,序列测定和Blast分析表明,4个基因分别为鸡源ISG12-2、IRF2、PPM1F及AIV的M蛋白,其中ISG12-2和IRF2基因含完整阅读框。推测,NS1蛋白可能是通过调节干扰素的产生、自身磷酸化及调节病毒复制等方式来发挥其调节AIV致病力的功能。这些蛋白的获得,进一步解释了NS1蛋白在调节HPAIV致病性方面的作用,同时也为HPAIV感染人的机制提供了有意义的参考。5利用细菌双杂交系统和GST融合蛋白沉降技术验证NS1蛋白与ISG12-2和IRF2蛋白的相互作用本研究利用细菌双杂交系统和GST融合蛋白沉降技术进一步验证NS1蛋白分别与ISG12-2及IRF2蛋白的相互作用,结果表明,在体内和体外,NS1蛋白与ISG12-2及IRF2蛋白间存在相互作用。NS1蛋白与ISG12-2和IRF2的相互作用,可能抑制某些IFN表达相关通路,拮抗IFN的表达,降低机体的抗病毒状态,利于AIV的复制和扩散。更多还原

【Abstract】 Avian influenza(AI) is a fatal syndrome of avian caused by avian influenza virus (AIV),characterized either by the general or respiratory disease,which was defined as the type A infectious disease by OIE.Since the first breakout in Italy in 1887,AIVs have occured worldwide in avian-raising countries.For the past few years,some new characters of AIV have appeared,especially for highly pathogenic avian influenza viruses(HPAIV).Antigen shift or drift has appeared easily for HPAIV,which can make the HPAIV obtain the capability to infection and make the human death,such as in HongKong and Southeast Asia.So HPAIV is more important for the public hygieology.At present,the pathogenic mechanism of AIV in chicken has been still not clear, which maybe the common results of more than one gene.Hemagglutinin(HA)、Neuraminidase(NA) and Non-structural protein(NS1)were more important in the all genes of AIV.HA and NA are the key factors for the subtype、antigen variation and pathogenic regulation during influenza virus infection.It has been reported that the NS1 protein,a non-structural protein of the type A influenza virus,can interact with the host cell proteins in vitro via its RNA-binding domain and effector domain,By the protein-protein interaction,NS1 protein can shutoff the host cellular protein synthesis, antagonize the alpha/beta interferon(IFN-α/β) system,induce apoptosis and regulate the viral protein expression.So NS1 protein is an important virulence factor which decides a great damage to host cells on the cell level.Those researches about NS1 Protein were mostly carried out using the human influenza virus and its suitable cell line,but not the avian influenza virus and its natural host(chicken).To elucidate the interaction between NS1 protein and natural host(chicken) protein is not only significant to understand the function of NS1 protein and how to regulate the virulence,but also helpful for preventing and curing the infection of AIV.In present study,the histopathology method and protein-protein interaction technology were applied to study on the pathology of H5NI AIV strain on both cellular and protein level in order to document the reason why the chicken is dead shortly.The results were reported as follow:1.Genetic analysis of the NS1 Genes of H9N2 Influenza VirusesThe nonstructural genes(NS1) of nine H9N2 subtype influenza viruses isolated from diseased chickens on different farms during1998-2005 were amplified by RT-PCR and completely sequenced.The nucleotide and deduced amino acid sequences of NS1 genes of these isolates were analysis and compared.The results showed that NS1 gene of all H9N2 isolates contained 654 bp and encoded 217 amino acids.The homologies of the nucleotide and deduced amino acid of the isolates were 95.4%-99.8%and 93.6%-100%, respectively.Comparison of the amino acid sequences of NS1 proteins of these isolates with other H9N2 viruses demonstrated that NS1 proteins of the nine strains had a deletion of 13 amino acid residues at the carboxy terminus,which may be the molecule mark of the isolates in China’s mainland.Phylogenetic analysis revealed that the NS1 genes of these isolates fell into the same lineage and belonged to allele A.The other eight isolates belonged to the Ck/SH/F/98-1ike lineage except Ck/HN/A3/98 strain,but all the isolates derived from Ck/BJ/1/94 strain firstly isolated from China’s mainland in 1994.The result indicated that H9N2 subtype AIV appear differentiation following the time and the viruses belonging to Ck/SH/F/98-like acquired an epidemiological advantage in chicken population in China’s mainland.2.Study on the apoptosis of SPF chicken challenged with HPAIV(H5N1 subtype)The study on the apoptosis of dead SPF chicken challenged with HPAIV(H5N1 subtype) was carried out using the TdT-mediated X-dUTP nick end labling method (TUNEL) The results documented that HPAIV could induce the different apoptosis in different organs or tissues.The apoptosis in Thymus、Bursa of Fabricius、Pancreas and Kindey was more severe than the other organs or tissues.The apoptosis cells included the vascular endothelial cells,glandular epithelium cartilage corpuscle and lympholeukocyte. According to the researches about the apoptosis,it was deduced that the apoptosis, shortly happened to the chicken incubated with HPAIV,maybe the mechanism which causes the chicken death quickly.3.Study on the distribution of NS1 protein in the different organs of infected chickenThe monoclonal antibody of NS1 protein was applied to develop the immunohistochemistry method(SABC method) in order to detect the NS1 protein.The study on the distribution of NS1 protein was detected in the different organs of chicken infected with HPAIV after 30-31hours.The main organs which can detect the positive signal of NS1 protein was the heart、pancreas、kidney and cerebellum granular layer.The positive signals were stronger than the others.The positive cells were involved in small or microvascular endotheliocyte、myocardial cell、renal tubular epithelial cell、glandular epithelium and epithelium.The results illustrated that the heart、pancreas、kidney cerebellum and microvascular endotheliocyte may be the main target organs in which HPAIV can duplicated effectively and product many effective virion.In this present study,It was presumed that the cardiovascular system was important for the duplication and diffusion of AIV.At the same time,the obvious associativity between the NS1 protein and apotosis was proved. 4.Screening and identification of host protein interacted with NS1 protein using the yeast two hybrid systemIn present study,spleen cDNA library of chicken incubated with HPAIV(H5N1 subtype) was firstly constructed using the switching mechanism at 5′end of RNA transcript technology.Four proteins which can interact with NS1 protein were screened from this cDNA library using the yeast two hybrid system(Y2H).These proteins included the chicken interferon-stimulated gene12-2(ISG12-2),interferon regulatory factor 2(IRF2),Protein phosphatase 1F(PPM1F) and M protein of AIV.The interaction between NS1 protein and ISG12-2 and IRF2 was identified,both in vitro and in vivo,by bacteria two-hybrid system and GST fusion proteins sedimentation technology, respectively.It was deduced that some signal pathway associated with IFN expression may be inhibited by the protein-protein interaction,which would affect the expression of IFN and degress the IFN-mediated antiviral responses.The results would explain the function of NS1 protein and how to regulate the pathogenicity.These studies also will give some reference to the mechanism which HPAIV infects human being.5.Identification of the interaction between NS1 protein and ISG12-2 or IRF2 using the bacterial two hybrid system and GST pull-down testIn this study,the interactons between NS1 protein with ISG12-2 and IRF2 protein were further identified using the bacterial two hybrid system and GST pull-down test,respectively.The results showed that NS1 protein interacted with ISG12-2 and IRF2 protein in vivo and in vitro.It was presumed that some pathways associated with the expressing of interferon were blocked and the expressing of interferon was inhibited by the interactions,which degraded the antivirus immune response and was beneficial for the duplication and diffusion of AIV.更多还原