H9N2亚型流感病毒浙江株的分子特征及Toll样受体介导的免疫损伤机制研究

发布时间：2018-05-12 11:30

本文选题：H9N2亚型流感病毒 + 蛋白数据分析库　；参考：《浙江大学》2013年博士论文

【摘要】：1998年,香港地区报告首起人感染H9N2亚型禽流感病毒(简称H9N2病毒)病例以后,中国大陆也相继出现了人感染H9N2病毒病例。有研究表明,H9N2病毒在我国禽类从业人群中的感染率高达15%左右,并引起急性呼吸道感染症状。当前,H9N2病毒在我国家禽中持续传播,并出现了大规模基因重组,H9N2病毒已经成为人类呼吸道感染的重要病原之一,并作为一个潜在的流感大流行候选病毒而引起了全球的重视。本研究对2011年100份来自浙江省家禽粪便样本进行了病毒分离,对获得的3株H9N2病毒进行全基因序列测定和进化分析；应用生物信息学软件,对网络数据库中H9N2病毒蛋白信息,进行了收集处理,构建一个实用、快速和高效的,能用于H9N2病毒生物学特征分析的蛋白数据分析库；分别在小鼠和人细胞水平上建立了感染模型,通过对Toll样受体(TLR)、NF-κB和IL-6表达水平之间的相互关系进行分析,初步探讨H9N2病毒对人类的致病机制。首先,在2011年2月至6月期间,对来源于浙江省活禽市场中健康家禽的粪便样本进行收集,获得100份样本,并集中进行病毒分离,将获得的3株H9N2和9株H1亚型病毒株进行了基因扩增、序列测定和同源性分析。结果表明,浙江省家禽中存在H9N2、H1N2和H1N3病毒,均为欧亚系；H9N2病毒出现了对金刚烷胺类药物耐药的S31N突变位点。病毒进化分析表明,出现了H9N2和H1N2病毒重组现象,提示需加强家禽中流感病毒监测。本研究在中国范围内首次报道了H1N3亚型流感病毒,为H1亚型流感流行病学研究提供了重要资料。应用生物信息学软件,对网络数据库中所有2013株H9N2病毒的蛋白信息,进行了全面收集和重新处理,建立了一个总共包含PB2、PB1、PB1-F2、PA、HA、 NP、NA、M1、M2、NS1和NS2等11个流感病毒蛋白的数据分析库,共包含了8948条序列信息。分析发现,禽类是H9N2病毒的主要自然宿主(占97.47%),主要分离于1997至2012年期间(占96.32%),主要来自亚洲地区(占96.37%)。有78.10%的H9N2病毒HA蛋白234位氨基酸为亮氨酸,具有与人类细胞优先结合的特性；有9.3%的H9N2病毒M2蛋白出现了对金刚烷胺类药物耐药的S31N突变点。该蛋白数据分析库,能快速进行有针对性的病毒生物特征分析,为有效预测H9N2病毒的感染性、致病性和耐药性提供参考。为了评价H9N2病毒对哺乳动物的致病性,本研究首先在小鼠水平上建立了H9N2病毒诱导急性肺损伤的动物模型,对正常小鼠和受病毒感染小鼠的外周血和肺脏中的IL-6、SOD、TLR4、TLR7和NF-κB表达水平进行检测。结果表明,H9N2病毒浙江株A/chicken/Zhejiang/329/2011(Zj329)能感染小鼠,出现典型的急性临床症状。病毒感染小鼠后,小鼠肺组织细胞中TLR4、TLR7和NF-κB mRNA水平发生了明显上调,并引起外周血中炎性因子IL-6大量表达,在感染后第6d,这些参数值都达到最高。外周血中SOD表达水平在病毒感染后的2-6d内发生下降,在第4d达到最低值。进一步在人肺A549细胞水平上建立了H9N2病毒(Zj329)的细胞感染模型,对Zj329病毒感染后细胞中和细胞上清中TLR4、TLR7、NF-κB和IL-6表达水平进行了检测。结果表明,Zj329病毒能有效感染A549细胞,出现典型的细胞病变现象。病毒感染A549细胞后,细胞中TLR4和TLR7mRNA水平都发生了明显上调,在病毒感染后12h达到最高值,同时NF-κB和IL-6表达活性也发生了明显上调,在24h内随时间增加而增强。综上所述,本研究得出以下结论： 1、浙江省家禽中存在H9N2病毒,均为欧亚系。 2、首次发现H9N2和H1N2亚型流感病毒之间出现了重组现象。 3、H9N2病毒浙江株能有效感染小鼠和人A549细胞,发生肺脏损伤和细胞病变,TLR4、TLR7、NF-κB和IL-6介导了H9N2病毒感染哺乳动物而引起的免疫损伤过程。总之,在近年来禽流感病毒感染人类时有发生的情况下,本研究构建的具有实用和快速的H9N2病毒蛋白数据分析库,为有效分析H9N2病毒的感染性、致病性和耐药性提供参考。通过本研究的开展,获得了H9N2病毒浙江株的相关信息,并在中国范围内首次报道了H1N3亚型流感病毒,发表了相关SCI论文3篇,不仅丰富了我国流感病毒的分子流行病学资料,也将为人感染禽流感防治工作的开展提供科学依据。同时,本研究对于阐明H9N2病毒在感染人类呼吸道过程中的致病机制,具有重要意义。
[Abstract]:In 1998, the first human infection of the H9N2 subtype of avian influenza virus (H9N2 virus) was reported in Hongkong area, and the human infection of H9N2 virus has also appeared in the mainland of China. Some studies have shown that the infection rate of H9N2 virus in our poultry workers is about 15%, and the symptoms of acute respiratory infection are cited. At present, the H9N2 virus is in me. H9N2 has become one of the important pathogens of human respiratory infection, and has attracted worldwide attention as a potential influenza pandemic candidate virus.
In this study, 100 avian feces samples from Zhejiang province were isolated in 2011. The total gene sequencing and evolution analysis of 3 H9N2 viruses were carried out. The information of H9N2 virus protein in the network database was collected and processed by using bioinformatics software to construct a practical, fast and efficient way to be used for H9N2 disease. The protein data analysis library of the analysis of toxic biological characteristics; the infection model was established on the level of mice and human cells, and the relationship between the expression level of Toll like receptor (TLR), NF- kappa B and IL-6 was analyzed, and the pathogenic mechanism of H9N2 virus to human was preliminarily discussed.
First, from February 2011 to June, the fecal samples from healthy poultry from the live poultry market in Zhejiang province were collected, 100 samples were collected, and virus isolation was concentrated. 3 strains of H9N2 and 9 H1 subtypes were amplified, sequenced and homologous analysis. The results showed that there were H9N2, H1 in poultry in Zhejiang province. The N2 and H1N3 viruses were all Eurasian, and the H9N2 virus appeared in the S31N mutation site of drug resistance to amantadine. The analysis of virus evolution showed that the recombination of H9N2 and H1N2 viruses appeared, which suggested that the monitoring of influenza virus in poultry should be strengthened. This study reported the first influenza virus of H1N3 subtype for the first time in China, which was the epidemic of H1 subtype influenza. The study of disease studies provides important information.
With the application of bioinformatics software, the protein information of all 2013 H9N2 viruses in the network database was collected and retreated. A data analysis library including 11 influenza viruses, including PB2, PB1, PB1-F2, PA, HA, NP, NA, M1, M2, NS1 and NS2, was set up. The total of 8948 sequences of information were included. The main natural host of the virus (97.47%), mainly from 1997 to 2012 (96.32%), mainly from Asia (96.37%). 78.10% of the H9N2 virus HA protein 234 amino acids are leucine, with the characteristics of the combination of human cells, and 9.3% of the H9N2 virus M2 protein appears to be S31N resistant to the amantadine drug S31N The protein data analysis library can quickly carry out targeted viral biometric analysis to provide a reference for the effective prediction of H9N2 virus infection, pathogenicity and drug resistance.
In order to evaluate the pathogenicity of H9N2 virus to mammals, this study first established an animal model of H9N2 virus induced acute lung injury at the mouse level. The expression of IL-6, SOD, TLR4, TLR7 and NF- kappa B in the peripheral blood and lung of normal mice and infected mice was detected. The results showed that the H9N2 virus Zhejiang strain A/chicken/ was found to be A/chicken/. Zhejiang/329/2011 (Zj329) can infect mice and have typical acute clinical symptoms. After the virus infected mice, the levels of TLR4, TLR7 and NF- kappa B mRNA in the lung tissue of mice were obviously up-regulated, and the inflammatory factor IL-6 was expressed in large numbers in the peripheral blood. The values of these parameters reached the highest level after infection. The SOD expression level in peripheral blood was the highest. It decreased within 2-6d after viral infection and reached the lowest level at 4D.
A cell infection model of H9N2 virus (Zj329) was established on the level of human lung A549 cells, and the expression of TLR4, TLR7, NF- kappa B and IL-6 in the cells and cell supernatant after Zj329 virus infection was detected. The results showed that the Zj329 virus could effectively infect A549 cells and present typical cytopathic phenomena. The virus infected A549 cells after the virus infection. The levels of TLR4 and TLR7mRNA in the cell were obviously up-regulated, and 12h reached the highest value after the virus infection. Meanwhile, the expression of NF- kappa B and IL-6 also increased obviously, and increased with time in 24h.
To sum up, this study draws the following conclusions:
1, there are H9N2 viruses in poultry in Zhejiang Province, all of which are Eurasian.
2, it was the first time that there was a recombination between H9N2 and H1N2 influenza viruses.
3, H9N2 virus Zhejiang strain can effectively infect mice and human A549 cells and have lung injury and cytopathic lesions. TLR4, TLR7, NF- kappa B and IL-6 mediate the immune damage caused by H9N2 virus infection in mammals.
In a word, in the case of the outbreak of avian influenza virus infection in recent years, the practical and rapid data analysis library of H9N2 virus was constructed to provide a reference for the effective analysis of the infection, pathogenicity and drug resistance of the H9N2 virus. Through this study, the related information of the Zhejiang virus of the H9N2 virus was obtained, and in China The H1N3 subtype influenza virus was reported for the first time, and 3 articles related to SCI were published, which not only enriched the molecular epidemiological data of the influenza virus in China, but also provided a scientific basis for the development of the prevention and control of avian influenza. At the same time, this study clarifies the pathogenesis of H9N2 virus in the infection of human respiratory tract. Significance.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R511.7

【参考文献】