E种肠道病毒HY12准种与进化及VP1基因突变对病毒复制的影响

发布时间：2018-03-03 03:20

本文选题：E种肠道病毒　切入点：全长基因组序列　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：牛肠道病毒(Bovine enteroviruses)属于小RNA病毒科肠道病毒属的成员,该病毒主要引起动物的呼吸道和消化道疾病,给养殖业带来极大的危害。2012年,本实验室从吉林省暴发的一种临床上以消化道和呼吸道为主要特征、发病率及死亡率高达50%以上的病牛群分离到国内首株E种肠道病毒HY12毒株。由于牛肠道病毒感染在国内为新发疫病,有关该病毒的生物学特性、致病机理等方面缺乏深入研究。本研究应用RT-PCR方法对HY12毒株的全长基因组序列进行扩增,获得的HY12毒株全长基因组序列为7 469 bp,编码一个2 176个氨基酸的前体多聚蛋白。序列分析显示,HY12毒株的全基因组序列与SL305毒株的亲缘关系最近,为E种肠道病毒。HY12结构蛋白基因(VP1、VP2、VP3和VP4)的核苷酸序列与14/3/96毒株的亲缘关系最近;而5′UTR及3D基因与SL305毒株的亲缘关系最近,表明HY12毒株可能为重组病毒,属于E种肠道病毒的E3基因亚型。与其它牛肠道病毒比较,HY12毒株的VP1和VP4蛋白存在多个特有的氨基酸位点。对同一代次的HY12毒株的序列测定发现,HY12毒株基因组的多位点在同一核苷酸位置出现不同的碱基序列,表明HY12毒株以病毒的准种形式存在。对HY12准种的进化与变异的研究发现,不同代次的HY12传代病毒其VP1和VP2基因序列与HY12病毒的主序列不完全相同,为不同的克隆类型,确定HY12准种存在明显的多样性和复杂性。同时发现,HY12病毒准种在传代过程中其突变率、复杂性随着传代次数的增加逐渐增大,主序列的比例及序列间的同源性随着传代次数的增加逐渐降低的进化规律。生物学特性的比对结果显示,不同代次的HY12毒株的生物学特性存在明显差异。与第2代的HY12病毒比较,第40代次HY12病毒的TCID50增高,蚀斑大小明显增大。对不同代次的HY12主序列分析发现,第40代次VP1基因272位核苷酸的点突变(272 GA),导致其91位氨基酸由精氨酸突变为组氨酸(R91H),该氨基酸的突变使其抗原性及三级结构发生改变。应用分子生物学、反向遗传学、病毒学等技术手段成功构建出E种肠道病毒HY12毒株的全长感染性克隆Dp OK12-HY12,并成功拯救出HY12病毒(r HY12)和建立HY12病毒的反向遗传学平台,并利用该拯救平台,拯救出VP1突变株病毒r HY12(H),证实了VP1蛋白(R91H)的突变导致HY12病毒生物学特性显著改变和病毒复制的增强。与亲本毒HY12和拯救毒r HY12相比,r HY12(H)的病毒滴度增高,蚀斑大小增大,且r HY12(H)病毒的基因组拷贝数和蛋白表达水平显著升高,对3日龄的ICR乳鼠的致病性较强,表明VP1蛋白91位氨基酸由精氨酸突变为组氨酸(R91H)显著改变HY12毒株生物学特性,影响病毒复制及其致病性。该结果为进一步深入研究病毒与宿主之间的相互作用、病毒的复制机理、致病机理及病毒准种的进化机制打下基础。
[Abstract]:Bovine enterovirusesis a member of the family RNA viridae, which causes respiratory and digestive tract diseases in animals and causes great harm to the breeding industry. A clinical outbreak in Jilin Province was characterized by digestive tract and respiratory tract. The first HY12 strain of enterovirus E was isolated from cattle with morbidity and mortality of more than 50%. Because bovine enterovirus infection is a new epidemic disease in China, the biological characteristics of this virus are concerned. In this study, RT-PCR method was used to amplify the full-length genomic sequence of HY12 strain. The full-length genomic sequence of HY12 strain was 7 469 BP, encoding a 2,176 amino acid precursor protein. Sequence analysis showed that the whole genome sequence of HY12 strain was most closely related to SL305 strain. The nucleotide sequences of VP1VP2VP3 and VP4) were most closely related to 14 / 3 / 96 strains of enterovirus, while 5- and 3D genes were most closely related to SL305 strains, suggesting that HY12 strains might be recombinant viruses. Compared with other bovine enterovirus strains, VP1 and VP4 proteins of HY12 have many unique amino acid loci. Sequence analysis of the same generation of HY12 strains revealed that the genome of HY12 strain. Different nucleotide sequences appeared at the same nucleotide site at multiple loci. The results showed that the HY12 strains existed in the form of quasispecies of the virus. The study on the evolution and variation of HY12 quasispecies showed that the sequences of VP1 and VP2 genes in different generations of HY12 subculture viruses were not identical to the main sequences of HY12 viruses, and they were different clone types. At the same time, it was found that the mutation rate and complexity of quasispecies of HY12 increased with the increase of passage times. The evolutionary law that the proportion of the main sequence and the homology among the sequences gradually decrease with the increase of the number of generations. Compared with the second generation of HY12 virus, the TCID50 of the 40th generation of HY12 virus increased and the size of the plaque increased obviously. The main sequence analysis of HY12 of different generations showed that there was a significant difference in the biological characteristics of different generations of HY12 virus strains, and compared with the second generation of HY12 virus, the TCID50 of the 40th generation of HY12 virus increased and the size of plaque increased obviously. The point mutation of 272-nucleotide at the 40th generation of VP1 gene led to the amino acid mutation of 91 amino acids from arginine to histidine R91H. The mutation of this amino acid changed its antigenicity and tertiary structure. The full-length infectious clone DpOK12-HY12 of E species enterovirus HY12 strain was successfully constructed by virology, and the reverse genetic platform of HY12 virus was successfully saved by using DpOK12-HY12) and the reverse genetic platform of HY12 virus was established. It was proved that the mutation of VP1 protein R91H) resulted in a significant change in the biological characteristics of HY12 virus and the enhancement of virus replication. The titer of rHY12H1H) was higher and the size of plaque was larger than that of parent virus HY12 and rescue virus r HY12. Moreover, the genomic copy number and protein expression of r HY12 / H) virus were significantly increased, and the pathogenicity of rHY12 / HV was stronger in ICR suckling mice at 3 days of age, indicating that the amino acid mutation of VP1 protein 91 from arginine to histidine R91H significantly changed the biological characteristics of HY12 strain. The results laid a foundation for further study on the interaction between virus and host, the mechanism of virus replication, the mechanism of pathogenicity and the evolution mechanism of virus quasispecies.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65

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