2011年在新疆地区循环的C组人肠道病毒的血清型分布及基因特征研究

发布时间：2018-06-29 17:32

  本文选题：C组人肠道病毒 + 柯萨奇病毒A1型　； 参考：《中国疾病预防控制中心》2013年硕士论文

【摘要】：研究背景：人肠道病毒(EV)属于小RNA病毒目、小RNA病毒科、肠道病毒属的成员,按照其VP1区核苷酸的同源性,被分为EV-A、EV-B、EV-C和EV-D共4组,目前已发现119种病毒,其中有64种被早期的中和试验所定型。但是由于EV型别众多,新型别的鉴定受到特异性肠道病毒抗血清供应的限制,同时中和试验费时繁琐,病毒发生抗原漂移、重组等都会影响中和鉴定结果,无法快速对血清型别作出鉴定,尤其无法应对越来越多新型或变异的EV在人类的循环。因VP1区是其重要的蛋白编码区,包含重要的抗原决定簇,所以也是其基因定型的靶基因。近年来新发现的EV型别均是以VP1区的核苷酸序列为依据的基因定型,结果与血清学分型十分吻合,是国际上公认的新型HEV分型的金标准之一。大多数EV的感染是无症状的或只引起轻微症状,如非特异性发热、皮疹、或轻微上呼吸道症状(普通感冒)；但有时也可以引起广泛多样的临床疾病,包括急性出血性结膜炎、无菌性脑膜炎、急性弛缓性麻痹、手足口病、心肌炎和新生儿败血症等。由于EV的型别众多,可引起多种疾病,近年来越来越受到各国疾病控制部门的重视。我国地域辽阔,人肠道病毒种类多,常年都有疫情发生。新疆维吾尔自治区地处亚欧大陆的正中心,其EV的流行特点和型别特征等相关研究报道较少。本研究对2011年新疆地区循环的EV-C的血清型分布,以及进一步的分子流行病学进行了研究。研究目的：通过对2011年新疆地区人群中循环的HEV-C血清型鉴定及基因特征分析,初步阐明新疆地区HEV-C的血清型和基因型别分布,丰富我国EV-C的毒株库,进而掌握在新疆地区乃至中国大陆循环的EV-C的基本情况,为EV所引起相关传染病诊断、预防和控制提供科学基础。研究方法：按照标准方法对粪便标本进行病毒分离,对阳性分离物进行RNA提取,VP1编码区RT-PCR, PCR产物的核苷酸序列测定,构建系统发生树并进行基因特征的研究。基于全长VP1区的遗传进化距离,选取17株EV-C作为代表株进行全基因组序列测定并进行序列特征分析。对首次发现两株致RD细胞病变的CVA1,进行与原型株在P1区差异位点比较,对CVA1-8-124进行连续传代并比较其中4个代数毒株在P1区差异位点并对这4个代数毒株进行温度敏感性实验和对CVA1新疆分离株的病毒受体进行初步探索。主要结果：2011年新疆地区共分离到32株8种血清型的EV-C,分别是2株CVA1,6株CVA11,1株CVA13,7株CVA17,4株CVA20,7株CVA24,2株EV-C96和3株EV-C99。8种EV-C分布在南疆地区的4个地区,其中以喀什地区的EV-C种类最多,除了CVA1只能在RD细胞上复制之外,其余的7种EV-C都能同时在RD和HEp-2细胞上复制；基于全长VPl区的系统发生树发现EV-C新疆分离株不仅与GenBank上的序列遗传进化距离较远,而且除了CVA1之外,新疆分离株之间的遗传进化距离也较远,表明2011年在新疆地区循环的8种EV-C均存在新的基因型或基因亚型,另外发现不仅EV-C新疆分离株与GenBank上的序列的遗传进化距离较远,而且GenBank上的序列之间的遗传进化距离也较远,在核苷酸水平上其最高值都已经超过普遍接受的EV基因定型的临界值：0.25,个别病毒甚至超过了0.30；在氨基酸水平上CVA13, CVA20和CVA24三种EV-C的遗传进化距离的最大值超过普遍接受的EV定型的临界值：0.15,从这一点看出EV-C要远比EV-A和EV-B更复杂,建议考虑对EV-C的分子分型的标准进行适当调整,以更好地对EV-C进行基因特征研究；基于全长VP1区遗传进化距离,选取17株作为代表株进行全基因组序列测定,并进行核苷酸序列特征分析。通过对原型株和新疆分离株的重组分析,发现CVA1与CVA22在5'UTR, P2和P3区存在重组,CVA11、CVA17和CVA20三种病毒相互重组最为普遍,首次发现两株EV-C99存在型间重组,并找到确切的供体信息,是与CVA13新疆分离株在3D区发生重组。本研究中首次发现两株能在RD细胞上复制的CVA1,通过与原型株和对其中一株(CVA1-8-124)进行连续传代,对其中4个代次的毒株在P1区进行氨基酸差异位点比较,一共找到6个比较有意义的氨基酸差异位点,作为我们下一步进行反向遗传学研究优先定点突变的位点,对CVA1-8-124株连续传代的4个代次的毒株进行温度敏感性实验,结果证实都为温度不敏感株；对CVA1新疆分离株的病毒受体进行初步探索,发现CVA1可以利用一个新的未知的病毒受体在RD细胞上复制,对于CVA1所使用的病毒受体需要继续进行研究。结论：EV-C在南疆地区具有广泛的分布,而且细胞培养分离病毒的研究结果没有发现已有的研究表明的EV-C对HEp-2细胞更敏感的结论；基于全长VP1区遗传进化距离表明2011年新疆地区循环的8种EV-C均存在新的基因型或基因亚型,EV-C的基因复杂程度要高于EV-A和EV-B；对于17株EV-C代表株的重组分析,再次支持EV-C同EV-A和EV-B一样存在广泛的重组,并首次发现EV-C99存在型间重组,重组是HEV中非常普遍的现象；对于首次分离到的致RD细胞病变的CVA1的病毒受体进行初步探索的结果表明其可能利用一个新的未知的病毒受体在RD细胞上复制,对于CVA1受体需要继续进行研究。
[Abstract]:Background: human enterovirus (EV) belongs to the order of small RNA virus, small RNA virus family and enterovirus. According to its VP1 nucleotide nucleotide homology, it is divided into 4 groups, EV-A, EV-B, EV-C and EV-D, and 64 of them have been identified by early neutralization tests. However, the new identification is due to the large number of EV types. The restriction of the antiserum supply of the specific enterovirus, the time-consuming and cumbersome of the neutralization test, the antigen drifting and regrouping of the virus will affect the results of neutralization identification, and can not be used to identify the serological types quickly. In particular, more and more new or mutated EV can not be dealt with in the human cycle. The VP1 region is an important protein coding area, including The important antigen determinant is the target gene of its gene stereotype. In recent years, the newly discovered EV types are all the gene stereotypes based on the nucleotide sequence of the VP1 region. The results are very consistent with the serotype of serum, which is one of the internationally recognized gold standards for the new type of HEV classification. Most of the EV infection is asymptomatic or only minor. Symptoms such as non specific fever, rash, or mild upper respiratory symptoms (common cold); but sometimes it can also cause a wide variety of clinical diseases, including acute hemorrhagic conjunctivitis, aseptic meningitis, acute flaccid paralysis, hand foot and mouth disease, myocarditis and neonatal septicemia. Many diseases can be caused by the number of types of EV. In recent years, more and more attention has been paid to the disease control department of various countries. China has a vast territory, many kinds of human enteroviruses and an epidemic situation all year long. The Xinjiang Uygur Autonomous Region is located in the center of the Eurasian continent, and the prevalence and type characteristics of EV are seldom reported. This study on the circulation of the serum of EV-C in the 2011 in Xinjiang Type distribution and further molecular epidemiology study. Objective: through the analysis of HEV-C serotype identification and gene characteristics of circulating HEV-C in the population of 2011, the distribution of serotypes and genotypes of HEV-C in Xinjiang region was clarified, and the virus library of EV-C in China was enriched, and then in Xinjiang region and even in China. The basic situation of EV-C in the land cycle provides a scientific basis for the diagnosis, prevention and control of related infectious diseases caused by EV. Research methods: virus isolation of fecal specimens, RNA extraction of positive isolates according to standard methods, nucleotide sequence determination of RT-PCR, PCR products in VP1 coding region, construction of phylogenetic tree and genetic characteristics Based on the genetic distance of the full length VP1 region, 17 EV-C strains were selected as the representative strains to carry out complete genome sequencing and sequence characteristics analysis. For the first time, two strains of RD cell lesions were found to be compared with the prototype strain in the P1 region, and the CVA1-8-124 was continuously passaged and 4 of the algebraic strains were compared in the P1 region. The temperature sensitivity test of the 4 algebraic strains and the preliminary exploration of the virus receptors of the CVA1 Xinjiang isolates were carried out. The main results were as follows: in 2011, 32 strains of serotype EV-C were isolated in Xinjiang region, which were 2 strains of CVA1,6 strain CVA11,1 strain CVA13,7 strain CVA20,7 strain CVA24,2 strain EV-C96 and 3 EV-C99.8 species EV-C score. In 4 regions of Southern Xinjiang, among them, the most EV-C species in the Kashi region, in addition to the replication of CVA1 only on RD cells, the other 7 kinds of EV-C can be replicated on RD and HEp-2 cells simultaneously. The phylogenetic tree based on the full length VPl region found that the EV-C isolated from the EV-C is not only far away from the sequence genetic evolution on GenBank, but also in addition to the sequence of GenBank. In addition, the genetic evolution distance between Xinjiang isolates was also far from CVA1, indicating that the 8 kinds of EV-C circulating in the Xinjiang region had a new genotype or gene subtype in 2011. In addition, it was found that not only the genetic evolution distance between the EV-C Xinjiang isolates and the GenBank sequences was far away, but also the genetic evolution distance between the sequences on the GenBank was also far away. At nucleotide levels, the highest values have exceeded the commonly accepted critical values of EV gene stereotypes: 0.25, individual viruses are even more than 0.30; at amino acid levels, the maximum genetic distance of the three EV-C, CVA13, CVA20 and CVA24, exceeds the generally accepted critical value of EV stereotypes: 0.15, from this point, EV-C is far more than EV-. A and EV-B are more complex. It is suggested that the standard of molecular typing of EV-C should be adjusted so as to better study the genetic characteristics of EV-C. Based on the genetic distance of the whole VP1 region, 17 strains are selected as representative strains to carry out the whole genome sequencing, and the nucleotide sequence characteristic analysis is carried out. The weight of the prototype strain and the Xinjiang isolate is weighed. The group analysis showed that CVA1 and CVA22 were reorganized in 5'UTR, P2 and P3 regions. The three viruses of CVA11, CVA17 and CVA20 were most common, and two strains of EV-C99 existed for the first time, and the exact donor information was found. It was reorganized in 3D region with the CVA13 Xinjiang isolate. In this study, two strains could be replicated on the RD cells for the first time. By continuous generation of a prototype plant and one of them (CVA1-8-124), a total of 6 significant amino acid differences were found by comparing the amino acid difference sites in the P1 region between the 4 generations of the strains. As the next step, we carried out the reverse genetics to study the site of the first point mutation and the continuous generation of the CVA1-8-124 strain. The temperature sensitivity test of the 4 generations of the 4 generations proved to be a temperature insensitive strain; a preliminary exploration of the virus receptor of the CVA1 Xinjiang isolate found that CVA1 could be replicated on RD cells with a new unknown virus receptor, and that the virus receptor used by CVA1 should continue to be studied. Conclusion: EV-C is in the south. The area of Xinjiang is widely distributed, and the results of cell culture and isolation of virus have not found the conclusion that EV-C is more sensitive to HEp-2 cells. Based on the full length VP1 region, the genetic evolution distance of the 8 kinds of EV-C circulatory in Xinjiang region all have a new genotype or a gene subtype in 2011, and the genetic complexity of EV-C is required. It is higher than EV-A and EV-B; for the recombinant analysis of 17 EV-C strains, EV-C has a wide restructure as EV-A and EV-B, and it is the first time to find that EV-C99 exists intertype recombination. Recombination is a very common phenomenon in HEV; the results of the initial exploration of the virus receptor of CVA1 for the first isolated RD cell lesion show that it can be found. A new unknown viral receptor can be used to replicate on RD cells, and CVA1 receptors need further research.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.5
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本文编号：2082853