肠道病毒D68型（EV-D68）属于小RNA病毒科肠道病毒属D种肠道病毒（EV-D）。EV-D68原本是一种罕见的呼吸道病毒，近年来却在全球范围内暴发流行并引起神经系统症状。2014年在美国的暴发是最为严重的一次。对于近年来世界各地EV-D68流行株的进化分析，发现与1962年分离的原型株Fermon相比，所有近年流行的EV-D68毒株都发生了不同程度的变异，本课题组的前期研究发现，对比2014年美国与中国流行株的基因序列，在VP2、VP3、VP1、2A、2B、3D和3’UTR等处中美流行株存在明显差异。这些位点的突变是否引起病毒的复制及致病能力的差异目前还没有相关报道，因此，本研究的目的是通过实验研究验证生物信息学和分子流行病学发现的若干变异，揭示EV-D68基因变异与致病性的关系。

研究不同位点的变异，构建感染性克隆是基础。本研究首先利用聚合酶不完全引物延伸（PIPE）法将2014年我国流行株的基因组分段插入pBR322载体，通过序列测定确定插入片段的正确性。全部基因组连接入载体后，通过体外转录制备病毒的RNA，将RNA转染入RD细胞后，观察细胞病变情况。通过观察细胞病变的出现、Western Blot呈现EV-D68特异性条带和子代病毒的接种确认感染性克隆构建成功。

接下来以此感染性克隆为基础，构建了分布于VP2、VP3、VP1、2A、2B、3D的8个位点和非翻译区3’UTR的序列的不同突变株，大部分突变位点的RNA转染细胞时致细胞病变的能力降低，有些突变使得病毒失去复制能力。只有位于VP2的A220E位点的突变在转染RNA时致细胞病变的能力增强。进一步研究发现，与转染RNA相反，A220E的子代病毒感染细胞的能力较弱，表现为24小时内生长曲线较原型株降低，造成此现象的原因是A220E位点的突变导致病毒吸附细胞的能力较原型株明显降低。

综上所述，本研究构建了 EV-D68的感染性克隆，在此基础上研究了不同位点的突变导致病毒的复制能力降低。在VP2的单一位点突变A220E导致病毒对细胞的吸附能力降低从而影响了病毒感染细胞的能力。

Enterovirus D68 (EV-D68) belongs to the D Enterovirus（EV-D）of Enterovirus family in Picornaviridae. EV-D68, originally a rare kind of respiratory virus, has broken out globally and caused neurological symptoms in recent years. The outbreak in 2014 in the United States was the most serious. For the analysis of the evolution of EV-D68 popular strains worldwide in recent years, EV - D68 was found to have various degrees of mutations, compared to Fermon, a prototype strain isolated in 1962. The previous research of our research group has found that, the gene sequence of the American strain isolated in 2014 compared with that of the Chinese strain, they had significant differences from each other in VP2, VP3, VP1, 2A, 2B, 3D and 3 'UTR, etc. Whether these locus mutation can cause the differences of viral replication and pathogenecity has no relevant reports so far. Therefore, the aim of this study is to verify several variations found by ethods of bioinformatics and molecular epidemiology through the experimental research, revealing the relationship between EV-D68 gene mutations and pathogenicity.

To study the mutations of different sites, it is the basis to construct infectious clone. In this study, the genome of the Chinese popular strain in 2014 was firstly inserted into the pBR322 vector by using the polymerase incomplete primer extension (PIPE) method, and the correctness of insert fragments was determined by sequencing. After the whole genome was inserted into the vector, RNAs were prepared by transcriptional preparation of the virus, and RNA were transfected into RD cells to observe the degree of cytopathic effect. By observing the appearance of cell cytopathic effect, western blot presenting the specific band of EV-D68 and the inoculation of progeny virus, we confirmed the success of infectious clone construction.

Then based on this infectious clone, we constructed the different mutant strains at eight sites in VP2, VP3, VP1, 2A, 2B, 3D and 3 'UTR. Most mutations at these sites could reduce the ability to cause cytopathic effect, after RNAs of the mutant strains were transfcted into cells. Further study showed that, contrary to that of transfected RNAs, the ability of A220E virus supernatant to infect cells was weaker, reflected in that the growth curve of A220E was lower in 24 hours than that of the prototype strain. The reason for this phenomenon is that the locus mutation A220E leads to a significant decrease in the ability of the virus to adsorb cells.

In summary, this study has constructed an infectious clone of EV-D68. And on this basis, we studied the reduction of viral replication capacity caused by the mutations in different sites. The single locus mutation A220E in VP2 caused a decrease in the adsorption capacity of virus to cells, thus affecting the ability of virus to infect cells.