狂犬病病毒L蛋白K1685和K1829对病毒致病性和免疫逃避的作用及机制

发布时间：2018-05-14 08:40

本文选题：狂犬病病毒 + L蛋白　；参考：《华中农业大学》2015年博士论文

【摘要】：狂犬病是由狂犬病病毒(RABV)引起的一种古老的人畜共患传染病,全世界每年约有55000人死于狂犬病,主要发生在非洲和亚洲一些发展中国家,我国是狂犬病高发国家之一。RABV为单股负链RNA病毒,其基因组共编码五个结构蛋白,分别是N、P、M、G和L蛋白,其中L蛋白的研究甚少。通过对其它单股负链RNA病毒的L蛋白的相关研究,尤其是与RABV同属于弹状病毒科的水疱性口炎病毒(VSV),表明L蛋白含有K-D-K-E的保守催化四聚体区域,该区域主要在病毒mRNA加帽过程中行使N-7和2’-O甲基转移酶(MTase)的功能,预计会在病毒的致病性及病毒逃逸宿主的先天性免疫中起到重要作用。本研究首先利用在线软件ClustalW2对RABV、VSV、麻疹病毒、仙台病毒、新城疫病毒和尼帕病毒的L蛋白羧基端进行序列比对,结果表明RABV的L蛋白中也存在K-D-K-E四聚体区域,且四个关键氨基酸K、D、K、E是完全保守的。此外,进一步通过比较RABV不同毒株之间的L蛋白中的K-D-K-E四聚体区域,发现该保守区域在不同的RABV毒株间是高度保守的。为了探索该K-D-K-E四聚体区域在RABV的致病性及逃逸宿主先天性免疫方面的作用,我们利用反向操作技术将该保守四聚体区域中的四个关键氨基酸位点K(第1685位)、D(第1797位)、K(第1829位)、E(第1867位)分别进行突变,构建一系列的重组狂犬病病毒(rRABV),并将这些重组病毒与亲本病毒在致病性上进行体外和体内的比较。体外实验表明,D1797和E1867突变的rRABV(rB2c-K1797A和r B2c-K1867A)体外扩增2代时便会快速回复突变为亲本型,而K1685和K1829突变的rRABV(r B2c-K1685A和rB2c-K1829A)的遗传性状则比较稳定,细胞传代扩增至15代仍未发生回复突变,因此,本研究将以rB2c-K1685A和rB2c-K1829A两个突变毒株为主要研究对象。病毒体外生长曲线结果表明两个突变毒株的病毒滴度和在细胞中的扩散能力都要明显低于亲本毒株;在对小鼠的致病性实验中发现,无论是以后肢肌肉注射方式还是脑内注射方式进行感染,rB2c-K1685A和r B2c-K1829A都不能致成年小鼠发病;且其对乳鼠的致病性(体重变化、临床症状、死亡率)显著低于亲本毒株rB2c。为了研究这些突变了K-D-K-E四聚体区域的重组病毒是否对病毒逃逸宿主先天性免疫方面也产生影响,我们将这些突变的重组病毒与亲本病毒在诱导I型干扰素(interferon,IFN)的产生及对其敏感性方面进行了比较,结果表明与亲本病毒相比,rB2c-K1685A和r B2c-K1829A对I型IFN敏感性增加,但其在感染RAW264.7细胞后并未诱导更多的I型IFN的产生。研究表明IFIT1/2等干扰素刺激因子(interferon stimulated gene,ISG)可以对一些2’-O MTase缺陷的病毒复制产生影响,为了研究突变的rRABV是否也受这些ISG的影响,我们分别构建了过表达IFIT1和IFIT2的细胞系,用重组病毒感染这些细胞,结果表明过表达IFIT1并不能明显抑制重组病毒的复制,而过表达IFIT2则能显著抑制重组病毒的复制。为了进一步研究K-D-K-E四聚体区域的突变是否对病毒的免疫原性产生影响,我们将重组病毒和亲本病毒分别免疫小鼠,并于免疫后第2周进行病毒中和抗体(VNA)滴度的测定,发现重组病毒和亲本病毒诱导产生的VNA均值都在10 IU/ml,在免疫后第3周用CVS-24进行攻毒实验,重组病毒rB2c-K1829A可以达到90%的保护率,而rB2c-K1685A和亲本病毒都能达到100%的免疫保护率。以上结果表明K-D-K-E四聚体区域的突变对病毒的免疫原性未有显著影响。综上所述,K-D-K-E四聚体区域的突变对可以显著降低RABV的致病性,使其对I型IFN以及IFIT2更为敏感,但并未显著影响病毒的免疫原性。该研究将对进一步理解L蛋白的K-D-K-E四聚体区域在RABV的致病性和逃逸宿主先天性免疫方面的作用机制奠定基础,同时也为RABV弱毒疫苗的研发提供了新的思路。
[Abstract]:Rabies is an ancient zoonotic infectious disease caused by rabies virus (RABV). About 55000 people die from rabies every year in the world, mainly in Africa and some developing countries in Asia. Our country is one of the high incidence countries of rabies,.RABV is a single strand negative chain RNA virus, and its genome COCODES five structural proteins, which are N, P, M, respectively. The study of G and L proteins, including L protein, is very small. Through the study of the L protein of other single stranded negative chain RNA viruses, especially with RABV belonging to the bullous stomatitis virus (VSV), which belongs to the family of the family of elastic viruses, it is indicated that L protein contains the conserved catalytic four polymer region of K-D-K-E, and this region is mainly made of N-7 and 2 '-O methyl during the virus mRNA adding process. The function of transferase (MTase) is expected to play an important role in the virulence of the virus and the innate immunity of the host of the virus. In this study, the sequence alignment of the L protein carboxyl ends of the L protein of RABV, VSV, measles virus, Sendai virus, Newcastle disease virus and NPV virus was compared with the online software ClustalW2. The results showed that the L protein of RABV was also found to be the same. There is a K-D-K-E four polymer region, and four key amino acids, K, D, K, and E are completely conserved. Furthermore, the conserved region is highly conservative among the different RABV strains by comparing the K-D-K-E four polymer region of the L protein between RABV different strains. In order to explore the K-D-K-E four polymer region in RABV pathogenicity and escape. The host's innate immune function, we use reverse operation technology to transform the four key amino acid sites K (1685th bits), D (1797th), K (1829th), and E (1867th)) in the conservative four polymer region to construct a series of recombinant rabies virus (rRABV), and the virulence of these recombinant viruses to the parent virus. In vitro and in vivo comparison, in vitro experiments showed that D1797 and E1867 mutations of rRABV (rB2c-K1797A and R B2c-K1867A) could quickly return to parental type when the 2 generation of amplification in vitro, and the genetic traits of rRABV (R B2c-K1685A and rB2c-K1829A) of K1685 and K1829 mutations were more stable than that of the 15 generation. Therefore, two mutant strains of rB2c-K1685A and rB2c-K1829A will be studied in this study. The results of the virus in vitro growth curve show that the virus titer of the two mutant strains and the ability to spread in the cells are significantly lower than those of the parent strain; in the pathogenicity test of the mice, it is found that it is the side muscle injection side. RB2c-K1685A and R B2c-K1829A did not cause infection in adult mice, and the pathogenicity (body weight, clinical symptoms, mortality) of the mice was significantly lower than that of the parent strain rB2c. in order to study whether the recombinant virus of the K-D-K-E four polymer region had a congenital immune response to the escape host of the virus. In addition, we compared the production of these mutant recombinant viruses with parental viruses in inducing I type interferon (interferon, IFN) and their sensitivity to their sensitivity. The results showed that compared with parental viruses, rB2c-K1685A and R B2c-K1829A increased the sensitivity to I IFN, but they did not induce more I after infected RAW264.7 cells. The production of type IFN. Studies have shown that IFIT1/2 and interferon stimulated gene (ISG) can affect the replication of some 2 '-O MTase defects. In order to study whether the mutant rRABV is also affected by these ISG, we respectively constructed the cell lines that express IFIT1 and IFIT2, and infect these cells with the recombinant virus. The results showed that overexpression of IFIT1 did not significantly inhibit the replication of recombinant virus, while overexpression of IFIT2 could significantly inhibit the replication of recombinant virus. In order to further study whether the mutation of the K-D-K-E four polymer region has an effect on the immunogenicity of the virus, we immunized mice with the recombinant virus and the parent virus respectively, and second weeks after the immunization. The detection of the titer of virus neutralizing antibody (VNA) showed that the VNA average of the recombinant virus and the parent virus was 10 IU/ml, and CVS-24 was used to attack the virus at third weeks after the immunization. The recombinant virus rB2c-K1829A could reach the 90% protection rate, and the rB2c-K1685A and parent virus could reach 100% of the immune protection rate. The above results showed that K- Mutations in the D-K-E four polymer region have no significant effect on the immunogenicity of the virus. To sum up, the mutation in the K-D-K-E four polymer region can significantly reduce the pathogenicity of RABV, making it more sensitive to I IFN and IFIT2, but does not significantly affect the immunogenicity of the virus. The study will further understand the K-D-K-E four polymer region of the L protein. It laid the foundation for the pathogenicity and the mechanism of escape immunity of RABV, and provided a new idea for the development of RABV attenuated vaccine.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.65

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