2015年长春市手足口病病原学特征与EV71和CVA16分子分型

发布时间：2018-02-04 09:33

本文关键词： 手足口病病原学 EV71 CVA16 分子分型　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景与目的:手足口病(hand,foot and mouth disease,HFMD)是由人的肠道病毒感染引起的,近年来发病率和死亡率位于我国丙类传染病之首,少数患者可能出现无菌性脑膜炎、神经源性肺水肿以及伴有心肌炎等症状,个别重症的患儿随着病情的恶化,可引起严重并发症,若治疗不及时甚至会导致死亡。目前报道的与手足口病相关的肠道病毒大约有20多种,EV71和CVA16是造成手足口病的主要病原体。随着基因测序技术的飞速发展以及网络数据资源的共享,以核苷酸为基础的分子生物学研究为病毒进化研究提供了方便快捷的方法。本研究对2015年长春市手足口病病原学特征进行了分析,阐明2015年长春市引发手足口病的主要病原体,并对其中分离到的EV71和CVA16进行分子分型与进化研究,进一步阐明其遗传进化规律和特点,为长春市手足口病的预防和控制工作提供一定的参考依据。材料与方法:本研究共收集了2015年长春市手足口病标本36份,将标本接种到RD细胞,进行病毒的分离和鉴定,对出现典型细胞病变效应(CPE)的标本进行病毒RNA的提取,分别采用肠道病毒通用引物、EV71和CVA16特异性引物进行鉴定。并对分离得到的EV71和CVA16,扩增其VP1序列,将PCR产物回收、测序,使用Bioedit与MEGA6.0软件对分离获得的EV71和CVA16的VP1序列进行核苷酸和氨基酸同源性比对分析,构建系统进化发育树。结果与结论:2015年长春市手足口病的发病高峰期主要集中在4-6月份,占全年收集病例的44.4%,与2015年全国手足口病发病高峰期相一致;手足口病患者性别构成无显著性差异;发病年龄主要集中在2-4岁的儿童,占总发病数的75%,其中发病数最多的是3岁组,占33.3%。从2015年长春市36份手足口病标本中共分离获得17株肠道病毒,肠道病毒的阳性分离率为47.2%,阳性标本中有5株EV71病毒(占29.4%),6株CVA16病毒(占35.3%),其它肠道病毒为6株(占35.3%)。在2015年长春市手足口病病原体中除了EV71和CVA16占主要部分,其它肠道病毒在手足口病病原谱中也占据重要的位置。分离获得的5株EV71,其VP1序列相互间核苷酸和氨基酸的序列同源性较高,核苷酸之间的同源性为96.2%-97.7%;氨基酸彼此间的同源性为99.2%-99.5%,5株EV71的VP1序列未发现有明显差别,5株EV71均为C4a亚型,这与近年来中国大陆各省份和地区分离的EV71主要的流行优势株保持一致。分离获得的6株CVA16,其VP1序列相互间核苷酸和氨基酸序列的同源性较高,其核苷酸相互间的同源性为97.4%-99.9%;氨基酸之间的同源性为99.3%-99.9%,6株CVA16的VP1序列未发现有明显差别,6株CVA16均为B2b亚型,这与近年来中国大陆各省份和地区分离的CVA16主要的流行优势株保持一致,没有不同抗原的新亚型的出现,并且较为稳定的核酸序列为疫苗的研制提供了较好的机会。
[Abstract]:Background & objective: hand foot and mouth disease (HFMD) is caused by human enterovirus infection. In recent years, morbidity and mortality are the first among Class C infectious diseases in China, a few patients may have aseptic meningitis, neurogenic pulmonary edema and accompanied with myocarditis and other symptoms. Can cause serious complications, if treatment is not timely or even lead to death. There are about 20 kinds of enterovirus associated with hand, foot and mouth disease reported at present. EV71 and CVA16 are the main pathogens causing HFMD. With the rapid development of gene sequencing technology and the sharing of network data resources. The molecular biology based on nucleotides provides a convenient and rapid method for the study of viral evolution. The etiological characteristics of hand-foot-mouth disease in Changchun in 2015 were analyzed in this study. In 2015, the main pathogens of HFMD in Changchun were elucidated, and the molecular typing and evolution of EV71 and CVA16 isolated from them were studied, and the rules and characteristics of genetic evolution were further elucidated. Materials and methods: in 2015, 36 specimens of hand, foot and mouth disease were collected and inoculated into Rd cells in Changchun. The virus was isolated and identified, and the virus RNA was extracted from the specimens with typical cytopathic effect. The common primers of enterovirus were used respectively. EV71 and CVA16 specific primers were used to identify and amplify the VP1 sequence of EV71 and CVA16. The PCR products were recovered and sequenced. Bioedit and MEGA6.0 software were used to analyze the nucleotide and amino acid homology of VP1 sequences of EV71 and CVA16. Results and conclusion: in 2015, the peak period of HFMD in Changchun was mainly in April-June, accounting for 44.4% of the cases collected in the whole year. It was consistent with the peak period of hand, foot and mouth disease in China in 2015. There was no significant difference in gender composition in patients with hand, foot and mouth disease. The age of onset was mainly in children aged 2 to 4 years, accounting for 75% of the total incidence, and the most common incidence was in the group of 3 years old. In 2015, 17 strains of enterovirus were isolated from 36 specimens of hand, foot and mouth disease in Changchun, and the positive isolation rate of enterovirus was 47.2%. Among the positive specimens, there were 5 strains of EV71 virus (29. 4%) and 6 strains of CVA16 virus (35. 3%). In 2015, EV71 and CVA16 were the main pathogens of HFMD in Changchun. Other enterovirus play an important role in the pathogeny of HFMD. The VP1 sequences of 5 strains EV71were highly homologous to each other in nucleotide and amino acid sequences. The homology between nucleotides was 96.2-97.7; The homology of amino acids was 99.2-99.5. The VP1 sequences of 5 EV71 strains showed no significant difference. The 5 strains of EV71 were all C4a subtypes. This was consistent with the main dominant strains of EV71 isolated from provinces and regions in mainland China in recent years. 6 CVA16 strains were isolated. The homology of nucleotide and amino acid sequence between VP1 sequence and amino acid sequence was high, and the homology of nucleotide sequence was 97.4- 99.9. The homology of amino acids was 99.3-99.9. There was no significant difference in the VP1 sequence of 6 CVA16 strains. The six CVA16 strains were all B2b subtypes. This is consistent with the main dominant strains of CVA16 isolated from provinces and regions in mainland China in recent years, and there are no new subtypes of different antigens. And relatively stable nucleic acid sequence provides a good opportunity for vaccine development.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.1

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