發(fā)布時間：2018-06-29 17:32

  本文選題：C組人腸道病毒 + 柯薩奇病毒A1型�。� 參考：《中國疾病預防控制中心》2013年碩士論文

【摘要】：研究背景：人腸道病毒(EV)屬于小RNA病毒目、小RNA病毒科、腸道病毒屬的成員,按照其VP1區(qū)核苷酸的同源性,被分為EV-A、EV-B、EV-C和EV-D共4組,目前已發(fā)現(xiàn)119種病毒,其中有64種被早期的中和試驗所定型。但是由于EV型別眾多,新型別的鑒定受到特異性腸道病毒抗血清供應的限制,同時中和試驗費時繁瑣,病毒發(fā)生抗原漂移、重組等都會影響中和鑒定結果,無法快速對血清型別作出鑒定,尤其無法應對越來越多新型或變異的EV在人類的循環(huán)。因VP1區(qū)是其重要的蛋白編碼區(qū),包含重要的抗原決定簇,所以也是其基因定型的靶基因。近年來新發(fā)現(xiàn)的EV型別均是以VP1區(qū)的核苷酸序列為依據(jù)的基因定型,結果與血清學分型十分吻合,是國際上公認的新型HEV分型的金標準之一。大多數(shù)EV的感染是無癥狀的或只引起輕微癥狀,如非特異性發(fā)熱、皮疹、或輕微上呼吸道癥狀(普通感冒)；但有時也可以引起廣泛多樣的臨床疾病,包括急性出血性結膜炎、無菌性腦膜炎、急性弛緩性麻痹、手足口病、心肌炎和新生兒敗血癥等。由于EV的型別眾多,可引起多種疾病,近年來越來越受到各國疾病控制部門的重視。我國地域遼闊,人腸道病毒種類多,常年都有疫情發(fā)生。新疆維吾爾自治區(qū)地處亞歐大陸的正中心,其EV的流行特點和型別特征等相關研究報道較少。本研究對2011年新疆地區(qū)循環(huán)的EV-C的血清型分布,以及進一步的分子流行病學進行了研究。研究目的：通過對2011年新疆地區(qū)人群中循環(huán)的HEV-C血清型鑒定及基因特征分析,初步闡明新疆地區(qū)HEV-C的血清型和基因型別分布,豐富我國EV-C的毒株庫,進而掌握在新疆地區(qū)乃至中國大陸循環(huán)的EV-C的基本情況,為EV所引起相關傳染病診斷、預防和控制提供科學基礎。研究方法：按照標準方法對糞便標本進行病毒分離,對陽性分離物進行RNA提取,VP1編碼區(qū)RT-PCR, PCR產(chǎn)物的核苷酸序列測定,構建系統(tǒng)發(fā)生樹并進行基因特征的研究�；谌LVP1區(qū)的遺傳進化距離,選取17株EV-C作為代表株進行全基因組序列測定并進行序列特征分析。對首次發(fā)現(xiàn)兩株致RD細胞病變的CVA1,進行與原型株在P1區(qū)差異位點比較,對CVA1-8-124進行連續(xù)傳代并比較其中4個代數(shù)毒株在P1區(qū)差異位點并對這4個代數(shù)毒株進行溫度敏感性實驗和對CVA1新疆分離株的病毒受體進行初步探索。主要結果：2011年新疆地區(qū)共分離到32株8種血清型的EV-C,分別是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種EV-C分布在南疆地區(qū)的4個地區(qū),其中以喀什地區(qū)的EV-C種類最多,除了CVA1只能在RD細胞上復制之外,其余的7種EV-C都能同時在RD和HEp-2細胞上復制；基于全長VPl區(qū)的系統(tǒng)發(fā)生樹發(fā)現(xiàn)EV-C新疆分離株不僅與GenBank上的序列遺傳進化距離較遠,而且除了CVA1之外,新疆分離株之間的遺傳進化距離也較遠,表明2011年在新疆地區(qū)循環(huán)的8種EV-C均存在新的基因型或基因亞型,另外發(fā)現(xiàn)不僅EV-C新疆分離株與GenBank上的序列的遺傳進化距離較遠,而且GenBank上的序列之間的遺傳進化距離也較遠,在核苷酸水平上其最高值都已經(jīng)超過普遍接受的EV基因定型的臨界值：0.25,個別病毒甚至超過了0.30；在氨基酸水平上CVA13, CVA20和CVA24三種EV-C的遺傳進化距離的最大值超過普遍接受的EV定型的臨界值：0.15,從這一點看出EV-C要遠比EV-A和EV-B更復雜,建議考慮對EV-C的分子分型的標準進行適當調(diào)整,以更好地對EV-C進行基因特征研究；基于全長VP1區(qū)遺傳進化距離,選取17株作為代表株進行全基因組序列測定,并進行核苷酸序列特征分析。通過對原型株和新疆分離株的重組分析,發(fā)現(xiàn)CVA1與CVA22在5'UTR, P2和P3區(qū)存在重組,CVA11、CVA17和CVA20三種病毒相互重組最為普遍,首次發(fā)現(xiàn)兩株EV-C99存在型間重組,并找到確切的供體信息,是與CVA13新疆分離株在3D區(qū)發(fā)生重組。本研究中首次發(fā)現(xiàn)兩株能在RD細胞上復制的CVA1,通過與原型株和對其中一株(CVA1-8-124)進行連續(xù)傳代,對其中4個代次的毒株在P1區(qū)進行氨基酸差異位點比較,一共找到6個比較有意義的氨基酸差異位點,作為我們下一步進行反向遺傳學研究優(yōu)先定點突變的位點,對CVA1-8-124株連續(xù)傳代的4個代次的毒株進行溫度敏感性實驗,結果證實都為溫度不敏感株；對CVA1新疆分離株的病毒受體進行初步探索,發(fā)現(xiàn)CVA1可以利用一個新的未知的病毒受體在RD細胞上復制,對于CVA1所使用的病毒受體需要繼續(xù)進行研究。結論：EV-C在南疆地區(qū)具有廣泛的分布,而且細胞培養(yǎng)分離病毒的研究結果沒有發(fā)現(xiàn)已有的研究表明的EV-C對HEp-2細胞更敏感的結論；基于全長VP1區(qū)遺傳進化距離表明2011年新疆地區(qū)循環(huán)的8種EV-C均存在新的基因型或基因亞型,EV-C的基因復雜程度要高于EV-A和EV-B；對于17株EV-C代表株的重組分析,再次支持EV-C同EV-A和EV-B一樣存在廣泛的重組,并首次發(fā)現(xiàn)EV-C99存在型間重組,重組是HEV中非常普遍的現(xiàn)象；對于首次分離到的致RD細胞病變的CVA1的病毒受體進行初步探索的結果表明其可能利用一個新的未知的病毒受體在RD細胞上復制,對于CVA1受體需要繼續(xù)進行研究。
[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.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R512.5
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本文編號：2082853