肠道病毒71型分离鉴定及其致病机制的初步探讨

发布时间：2018-06-19 10:13

本文选题：肠道病毒71型 + 手足口病　；参考：《山东大学》2013年硕士论文

【摘要】：肠道病毒71型(enterovirus71, EV71)为小RNA病毒科肠道病毒属的成员,足引起婴幼儿手足口病(hand, foot and mouth disease, HFMD)的主要病原体之一。轻症患者只引起发热、疱疹等症状,重症患者会引起严重的中枢神经系统(central nervous system, CNS)症状如无菌性脑膜炎、脑干脑炎、脊髓灰质炎样麻痹等,病死率较高。目前EV71的致病机制尚不明确,不同临床症状的产生可能是由于病毒毒力不同,也可能是个体易感性的不同。本研究首先采集HFMD患儿的粪便标本,运用RD细胞分离肠道病毒并对其进行EV71的鉴定：其次对EV71的全基因组序列进行分析,寻找基因组序列的差异；最后分别在5'UTR、VP2、3D区的基础上对EV71的致病机制进行研究,以寻找EV71致神经系统病变的原因,为阐明EV71的致病机制提供重要基础。目的 1.从HFMD患者的粪便标本中分离EV71,为进一步的序列研究提供基础。 2.了解山东省临沂市分离的EV71病毒株的全基因组序列特征,寻找不同临床症状病毒株基因组序列上的差异,为进一步研究提供毒力候选位点 3.对FV715'UTR、VP2、3D区的毒力候选位点进行研究,探索EV71的致病机制。方法 1.将采集自200%2010年山东省临沂市HFMD患者的粪便标本进行处理,接科,于RD细胞进行初步分离,运用肠道病毒通用引物及EV71特异性引物对分离到的病毒RNA进行RT-PCR扩增,筛选出EV71。 2.根据GenBank中EV71的基因组序列设计引物,对SDLY11病毒株进行全基因组序列扩增并测序,运用DNAstar和MEGA4软件对EV71的全基因组序列进行分析。 3.根据SDLY01、SDLY11、SDLY48、SDLY96、SDLY107、SDLY153病毒株的全基因组序列设计引物,对8株EV71的5UTR区进行RT-PCR扩增并序列分析。 4.参考SDLY11、SDLY107全序列测序结果设计VP2、3D区扩增引物,以构建表达载体VP2-pBluescriptSK(+)、3D-Flag-pcDNA3.1,通过体外转染细胞来表达蛋白,并检测蛋白的致细胞病变作用。乳酸脱氢酶实验(LDH)检测不同临床症状病毒株的VP2蛋白、3D蛋白对细胞损伤的影响,Annexin-V与PI的联合使用可检测3D蛋白引起的细胞凋亡情况,通过MTT实验来检测3D蛋白对细胞增殖的影响。结果 1.本研究分离了2009～2010年的24份标本,经初步鉴定20份标本为EV71,4份标本为非EV71的肠道病毒,其中采集自2009年的22份样本中有18例为EV71(81.8%)。 2. SDLY11病毒株基因组全长7405nt,5'UTR包含742nt,3'UTR包含84nt,中间为6579nt的ORF,编码2193个氨基酸的多聚蛋白。经序列分析显示SDLY11属于V71的C4亚型。通过轻重症重症患者病毒株进行比对分析发现5UTR中的2处核苷酸突变(T40C、C575T)及VP2区的氨基酸突变(T144S)可能与病毒引起不同临床症状有关 3.8株EV71分离株的5UR区核苷酸序列为741nt～745nt,与C4亚型的同源性最高,同临床症状病毒株的5UTR进行序列比对发现有2处枋苷酸位点突变(G265A、(703T)。 4. SDLY11、SDLY107两个病毒株的VP2蛋白均可在Vero细胞中表达,LDH实验对VP2蛋白引起的细胞损伤程度进行分析,结果显示两组未出现明显不同。 5. SDLY11、SDLY107的3D蛋白可在RD细胞中表达,LDH实验结果显示SDLY11株3D蛋白转染组造成的细胞损伤程度大于SDLY107株3D蛋白转染组,细胞凋亡结果未有明显不同,而进一步的MTT实验结果显示,SDLY107株3D蛋白转染组的细胞增殖能力强于SDLY11株3D蛋白转染组。结论 1.初步判定2009年山东省临沂市流行的HFMD以EV71为主,SDLY11为C4亚型,为近年来中国大陆地区流行的EV71亚型。 2.5'UTR区的4处突变(T40C、G265A、C575T、G703T)可能与病毒产生的神经系统症状相关,这可为进一步的研究提供候选位点。 3.不同临床症状病毒株的VP2蛋白可能对细胞损伤的影响不大,但3D蛋白会引起细胞损伤情况、细胞增殖能力的不同,这为EV71致病机制的研究提供了重要基础。
[Abstract]:Enterovirus 71 ( EV71 ) is one of the main pathogens of small RNA virus family Enterovirus , which is one of the main pathogens of hand , foot and mouth disease ( HFMD ) .

In this study , the fecal samples of HFMD children were collected , and the EV71 was isolated by RD cells . The whole genome sequence of EV71 was analyzed to find out the difference of genomic sequence .
Finally , the pathogenic mechanism of EV71 was studied on the basis of 5 ' untranslated region , VP2 and 3D region respectively , in order to find out the cause of EV71 induced nervous system disease and provide an important basis for the pathogenesis of EV71 .

Purpose

1 . EV71 was isolated from stool specimens from HFMD patients , providing the basis for further sequence research .

2 . To understand the whole genome sequence characteristics of EV71 virus isolated from Linyi City , Shandong Province , and to find out the difference in the genome sequence of different clinical symptom virus strains , and provide the virulence candidate sites for further research .

3 . To study the virulence candidate sites of FV715 ' , VP2 and 3D regions , and explore the pathogenesis of EV71 .

method

1 . The stool specimens collected from HFMD patients in Linyi City of Shandong Province in 2010 were treated and isolated . The isolated virus RNA was amplified by RT - PCR using the general primers of Enterovirus and EV71 specific primers , and EV71 was screened out .

2 . According to the genomic sequence of EV71 in GenBank , the whole genome sequence of the strain SDLY11 was amplified and sequenced , and the whole genome sequence of EV71 was analyzed by DNAstar and MEGA4 software .

3 . RT - PCR amplification and sequence analysis were carried out on 8 strains of EV71 based on the whole genome sequence of SDLY01 , SDLY11 , SDLY48 , SDLY96 , SDLY107 and SDLY153 .

4 . The expression vector VP2 - pBluescriptSK ( + ) , 3D - Flag - pcDNA3.1 was designed by reference to SDLY11 and SDLY107 sequencing results .

Results

1 . This study has isolated 24 specimens from 2009 to 2010 , initially identified as EV71 and 4 specimens as non - EV71 enteroviruses , of which 18 of 22 samples collected from 2009 were EV71 ( 81.8 % ) .

2 . The genome of SDLY11 strain has a full length of 7405nt . The 5 ' untranslated region consists of the 742nt , the 3 ' untranslated region contains 84nt , the middle is 6579nt , and the polyprotein of 2193 amino acids is encoded . The sequence analysis shows that SDLY11 belongs to the C4 subtype of V71 . By comparison , it is found that amino acid mutation ( T40C , C575T ) and amino acid mutation ( T144S ) in VP2 region may be related to different clinical symptoms .

3 . The nucleotide sequence of the 5UR region of EV71 isolates was 741nt ~ 745nt , and the highest homology with the C4 isoforms was observed .

4 . VP2 protein of two strains of SDLY11 and SDLY107 could be expressed in Vero cells , and the degree of cell injury induced by VP2 protein in LDH experiment was analyzed .

5 . The 3D protein of SDLY11 and SDLY107 could be expressed in RD cells . The results of LDH experiment showed that the degree of cell damage caused by the 3D protein transfection group of SDLY11 strain was greater than that of SDLY107 strain . The results showed that the cell proliferation ability of SDLY107 strain was stronger than that of SDLY11 strain .

Conclusion

1 . It is preliminarily determined that the HFMD , which is popular in Linyi City in Shandong Province in 2009 , is EV71 , and SDLY11 is a C4 subtype , which is an EV71 subtype that has been popular in mainland China in recent years .

At 4 mutations ( T40C , G265A , C575T , G703T ) in the 2.5 ' - region may be associated with the neurological symptoms of the virus , which may provide candidate sites for further studies .

3 . VP2 protein of different clinical symptom virus strains may have little effect on cell injury , but 3D protein may cause cell injury and cell proliferation ability , which provides an important basis for the study of EV71 pathogenic mechanism .
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R373

【参考文献】