荧光素酶标记的重组流感病毒（IAV-Gluc）构建及其呼吸道沉积可视化研究

发布时间：2018-06-27 05:10

本文选题：甲型H1N1流感病毒 + 反向遗传学　；参考：《中国人民解放军军事医学科学院》2017年博士论文

【摘要】：近年来,频繁爆发的甲型流感不仅给人类的生命健康带来了严重威胁,而且给经济造成了巨大损失,甚至影响到了社会稳定。通过气溶胶传播、扩大感染范围是其能引起全球大流行的一个重要因素。2009年爆发的甲型H1N1流感就是因病毒能经气溶胶传播,具有强大的人传人能力,而被世界卫生组织(WHO)将警戒级别提至最高级—6级。该事件进一步引发了人们对流感病毒气溶胶传播机理研究的关注。但是目前对于流感病毒的气溶胶传播机制还不是十分清楚。影响流感病毒气溶胶传播效率的因素有很多,如环境因素、流感病毒自身的关键位点氨基酸序列、宿主呼吸道唾液酸相关的受体类型、体内的抗病毒药物或疫苗免疫水平、在呼吸道的沉积效率等。与研究病毒气溶胶扩散动力学同等重要的是,监测具有气溶胶传播特性的流感病毒在感染宿主过程中呼吸道内载量的动态变化,能为流感病毒的组织嗜性、复制与清除规律、致病与传播分子机制的研究提供基础数据和技术支撑,对于进一步研究流感病毒的气溶胶传播机制具有重大意义。目前,传统的病毒体内沉积研究方法需要大量使用动物,在不同的时间点剖检,不仅费时、费力,而且存在较大的生物安全隐患。而活病毒标记及可视化研究能够有效克服上述不足,可以实时、便捷地反映病毒感染过程。本课题利用反向遗传操作技术,在甲型H1N1流感病毒A/PuertoRico/8/34(PR8)的NA末端插入外源荧光素酶基因Gluc(Gaussia luciferase),构建可视化的重组荧光素酶甲型H1N1流感病毒(IAV-Gluc),为开展流感病毒动物感染研究提供了一种实时、动态、可视化监测呼吸道病毒载量变化的方法。该方法通过检测活体动物呼吸道生物发光情况,快速确定病毒的组织分布及载量情况,实现对动物体内病毒载量连续、实时的动态监测,具有操作简便、结果直观、可快速定性定量等特点。本研究共分四部分:一是完成了IAV-Gluc的构建与鉴定。以A/Puerto Rico/8/34(PR8)毒株为骨架,利用反向遗传学技术成功构建8个双向转录/表达质粒,将8个质粒共转染293T细胞(人胚胎肾细胞),转染上清接种9日龄的SPF鸡胚,进行重组病毒IAV-Gluc拯救。通过凝集试验(HA)、基因扩增、形态学观察、荧光素酶基因表达水平的验证和鉴定,均证实了重组荧光素酶甲型H1N1流感病毒(IAV-Gluc)构建成功,且该病毒能够稳定表达Gluc。二是完成了IAV-Gluc生物学特性研究。本研究从稳定性、致病性、传播能力、增殖能力、NA活性及受体结合特性六个方面对拯救毒株IAV-Gluc的生物学特性进行了分析比较。结果表明,IAV-Gluc能够在P1-P5代稳定表达荧光素酶基因,且在5代间表达无显著差异;致病力约为野生毒PR8的1/1000;在豚鼠间获得了100%的直接接触传播与气溶胶传播能力,与野生毒株一致;在SPF鸡胚上增殖能力约为野生毒的1/20,增殖曲线无明显差异;与野生毒的神经氨酸酶相对活性不存在显著性差异,表明对病毒的释放能力无影响;二者均表现出完全的α2,3唾液酸受体结合能力。上述结果表明获得的IAV-Gluc除致病力减弱外,极大地保留了野生毒株的生物学特性。三是完成了IAV-Gluc在呼吸道内沉积的可视化监测研究。本研究结合体外成像技术,分析了病毒在小鼠体内复制、清除的动态过程。以IAV-Gluc滴鼻感染BALB/c小鼠(5×103EID50/只)后,利用IVIS SPECTRUM体外成像系统对其体内的病毒载量进行连续6天的可视化监测,24h即能在体外检测到生物发光信号,48h后达到峰值。生物发光来源为小鼠肺部和气管,且肺部的生物荧光强度与病毒滴度有极强的正相关线性关系(R2=0.9866);同时,建立了生物发光快速鉴定病毒感染的方法,相比于传统的qRT-PCR技术来说,不仅可以直接评价动物体内活病毒载量,而且在保证准确性前提下,大大缩短了样品处理步骤和实验所需时间。四是完成了不同感染方式下IAV-Gluc在呼吸道内沉积与复制的比较研究。当保证滴鼻与气溶胶暴露两种方式下攻毒的剂量相同(2.43×105 EID50)时,滴鼻时有12.27%的病毒到达豚鼠肺部且呈点状分布,气溶胶暴露时有1.34%的病毒到达豚鼠肺部且呈均匀分布,后者导致病毒在48h内更加有效的复制;从复制效率来看,气溶胶暴露感染组的豚鼠肺部病毒载量持续增高48h后才开始下降,最高载量为原始沉积量的11073倍,而滴鼻感染组的豚鼠肺部病毒载量在24h开始下降,且最高载量仅为原始沉积量的15.4倍。说明气溶胶暴露方式感染更有利于病毒在肺部的复制,比滴鼻方式具有更高的感染效能。本研究成功构建了携带Gaussia Luciferase基因的可视化重组荧光素酶甲型H1N1流感病毒(IAV-Gluc);从稳定性、致病性、传播能力、增殖能力、NA活性及受体结合特性等方面鉴定,结果显示该重组病毒极大地保留了野生毒株的生物学特性;滴鼻感染小鼠后在肺部和气管内可检测到生物发光信号,并且生物发光强度与病毒滴度呈正相关;该病毒适用于对感染动物的快速鉴定研究,在前处理和检测时间上优于传统的qRT-PCR技术,而且能够直接评价体内的活病毒载量;同时,利用可视化病毒进行气溶胶暴露研究首次发现,甲型H1N1流感病毒气溶胶暴露比滴鼻感染在豚鼠肺部增殖持续时间更长,载量更高,分布更均匀,具有更高的感染效能。本研究为进一步评价甲型流感病毒的体内沉积与体外传播规律研究、阐明其致病分子机制和抗病毒药物的筛选及候选疫苗株的研发提供基础,同时为开展其他亚型流感病毒研究提供技术参考。
[Abstract]:In recent years, frequent outbreak of influenza A influenza not only poses a serious threat to human life and health, but also causes great losses to the economy, and even affects social stability. The spread of the infection by aerosol is an important factor in the global pandemic that causes the global pandemic in.2009. The influenza A (H1N1) influenza is caused by the virus. The aerosol transmission, which has powerful human ability, has been raised to the highest level - 6 by the WHO (WHO). This event has further aroused people's attention to the research on the transmission mechanism of influenza virus aerosol. However, the current mechanism of influenza virus transmission is not yet very clear. There are many factors such as the environmental factors, the amino acid sequence of the key loci of the influenza virus itself, the type of receptor related to the saliva acid in the host respiratory tract, the antiviral drugs in the body or the immunization level of the vaccine in the body, the efficiency of the deposition of the respiratory tract, etc.. The dynamic changes in the respiratory load in the respiratory tract of the influenza virus infected by the sol-transmission characteristics can provide basic data and technical support for the tissue eosinophilia of the influenza virus, the law of replication and scavenging, the research of pathogenic and spreading molecular mechanism, and is of great significance to the further study of the mechanism of aerosol transmission of influenza virus. The traditional methods for the study of the virus in vivo need to use a large number of animals. It is not only time-consuming and laborious, but also has a great potential for biological safety at different time points. However, the study of living virus markers and visualization can effectively overcome the above deficiencies and can reflect the process of virus infection in real time and conveniently. As a technique, the exogenous luciferase gene Gluc (Gaussia luciferase) was inserted at the NA terminal of H1N1 influenza A (PR8) virus A/PuertoRico/8/34 (PR8) to construct a visual recombinant luciferase a H1N1 influenza virus (IAV-Gluc). It provides a real-time, dynamic and visual monitoring of the viral load variation of the respiratory virus in the study of influenza virus infection. By detecting the bioluminescence of the respiratory tract of living animals, this method can quickly determine the distribution and load of the virus, and realize the continuous and real-time dynamic monitoring of the viral load in the animal body. It has the characteristics of simple operation, direct result and rapid qualitative and quantitative. The study is divided into four parts: first, the IAV-Gluc is completed. Construction and identification. Using the A/Puerto Rico/8/34 (PR8) strain as the skeleton, 8 bi-directional transcriptional / expression plasmids were successfully constructed by reverse genetics technology. The 8 plasmids were co transfected to 293T cells (human embryonic kidney cells) and transfected into the supernatant to inoculate the SPF chicken embryos of 9 days old. The recombinant virus IAV-Gluc rescue was carried out. By HA, gene amplification and morphological view Verification and identification of luciferase gene expression level confirmed the success of recombinant luciferase a H1N1 influenza virus (IAV-Gluc), and the virus was able to stabilize the expression of Gluc. two to complete the study of the biological characteristics of IAV-Gluc. This study was from stability, pathogenicity, transmission, proliferation, NA activity and receptor binding properties of six. The biological characteristics of the rescue strain IAV-Gluc were analyzed and compared. The results showed that IAV-Gluc could express the luciferase gene steadily in the P1-P5 generation, and there was no significant difference in the expression between the 5 generations; the pathogenicity was about 1/1000 of the wild venom PR8, and 100% of the direct contact transmission and aerosol transmission ability between the guinea pigs and the wild strains were obtained. There was no significant difference in proliferation curve between the SPF chicken embryo and the wild poison 1/20; the relative activity of the wild poisonous neuraminidase had no significant difference, indicating that there was no effect on the release ability of the virus; all two showed the complete alpha 2,3 sialic acid receptor binding ability. The results showed that the obtained IAV-Gluc was the pathogenicity of the virus. The biological characteristics of the wild strains were greatly preserved. Three the visual monitoring of the IAV-Gluc in the respiratory tract was completed. This study combined with in vitro imaging techniques to analyze the dynamic process of replication and clearance of the virus in mice. The IVIS SPECTRUM body was used after the infection of the BALB/ c mice (5 x 103EID50/). The external imaging system visualized the viral load in the body for 6 days. 24h could detect the bioluminescence signal in vitro and peak after 48h. The bioluminescence was derived from the lung and trachea of mice, and the biological fluorescence intensity of the lung was closely related to the virus titer (R2=0.9866); at the same time, the bioluminescence was established. The method of rapid identification of virus infection by luminescence, compared with the traditional qRT-PCR technology, not only can directly evaluate the live virus load in the animal body, but also greatly shorten the sample processing steps and the time required for the experiment under the premise of ensuring the accuracy. Four, the ratio of the deposition and replication of IAV-Gluc in the respiratory tract under different infection modes has been completed. When the dosage of the nose drops and the aerosol exposure were equal (2.43 x 105 EID50), when the nose drops, 12.27% of the virus arrived in the lungs of the guinea pig and showed a spot distribution. When the aerosol was exposed, 1.34% of the virus reached the guinea pig lung and distributed evenly. The latter was more effective in replicating the virus in the 48h; from replication effect. In the aerosol exposure group, the lung virus load of the guinea pigs exposed to the infection group continued to increase after 48h, and the maximum load was 11073 times that of the original deposit, while the virus load of the guinea pig lungs began to decrease at 24h, and the maximum load was only 15.4 times that of the original deposition. Replication of the lungs is more effective than nasal drip. This study successfully constructed a visualized recombinant luciferase a H1N1 influenza virus (IAV-Gluc) carrying the Gaussia Luciferase gene, and identified the recombinant disease from stability, pathogenicity, transmission ability, proliferation, NA activity and receptor binding properties. The biological characteristics of the wild virus are greatly preserved, and the bioluminescence signal can be detected in the lungs and trachea after the nose drops infected mice, and the bioluminescence intensity is positively correlated with the virus titer; the virus is suitable for the rapid identification of infected animals and is superior to the traditional qRT-PCR technology in the preprocessing and detection time. It can be used to evaluate the live virus load in the body directly. At the same time, the study of aerosol exposure by visual virus is the first time to find that the exposure of H1N1 influenza A virus is longer, more load, more uniform and more effective than the infection of nasal drops in the lungs of Guinea pigs. This study is to further evaluate influenza A (a) influenza. The study of the body deposition and in vitro propagation of the virus, clarifies the screening of its pathogenic molecular mechanism and antiviral drugs, and provides the basis for the research and development of candidate vaccine strains, and provides a technical reference for the development of other subtype influenza viruses.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R373

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