宁波地区手足口病病原学分型和肠道病毒71型的全基因组特征研究

发布时间：2018-04-22 15:08

本文选题：手足口病 + 病原学　；参考：《宁波大学》2012年硕士论文

【摘要】：目的： 1、通过对临床表现为手足口病(Hand-foot-and-mouth disease，HFMD)的生物样本进行检测，鉴定宁波地区引起HFMD病原种类，分析HFMD的病原学流行特征，为HFMD的综合防治提供科学依据。 2、在全基因组测序的基础上，了解宁波市EV71分离株的全基因特征，分区段分析肠道病毒71型(Enterovirus71，EV71)宁波株与其他地区代表株的同源性和亲缘进化关系，全面反映进化动态；分析VP1区潜在的B细胞抗原位点，以及计算毒株是否承受正向选择压力。方法： 1、依托宁波市疾病预防控制中心病毒研究室，收集2009~2010年宁波地区临床表现为手足口病的样本共446份，通过荧光定量RT-PCR和RT-PCR方法确定肠道病毒型别，序列拼接和比对使用DNAMAN和DNAStar软件，使用SPSS17.0软件进行数据统计，分析HFMD的病原学流行特征。 2、选择2010年的EV71阳性样本进行病毒分离和核酸提取，通过逆转录聚合酶链反应对EV71核酸进行全基因组序列扩增，同时测定其他30株EV71的VP1区序列，序列测定后使用相关生物信息软件进行基因片段的拼接、比对和分析，并将宁波株与GeneBank上不同时间、不同地点的EV71代表株序列进行同源性和亲缘进化分析，分析潜在抗原为点，以及计算毒株是否承受正向选择压力。结果： 1、2009~2010年共检测宁波市HFMD病例各类样本446份，其中有191份EV71(55.20%)；121份CoxA16(34.97%)；13份CoxA6(3.76%)；7份CoxA10；4份CoxA12；4份埃可病毒9型(Echovirus9，ECHO9)；2份CoxA2；CoxA4、CoxB1、CoxB4和其它肠道病毒各1份。在检测到的346份总肠道病毒阳性标本中，男性232份，女性114份，男女比例为2.04:1，发病人群主要集中在1~5岁年龄组，全年份均有发病，有较明显的季节性，发病主要集中在4~7月，12月有不明显小高峰，2010年检测人数较2009年有下降趋势，峰值也有所偏移，2009和2010年的发病高峰分别在4月和6月。 2、通过全基因组测序得到宁波4株EV71基因组序列，全长均为7406bp。基因组5'端和3'端各有长743bp和81bp的非编码区，中间为长6582bp的编码区序列，编码2193个氨基酸的多聚蛋白。与参考序列相比，宁波株编码区没有核苷酸的插入和缺失。同源性和亲缘进化分析推测宁波株EV71与中国大陆正在流行的C4a进化分支最为接近。分析VP1蛋白的亲水性，柔韧性，表面可能性及蛋白质二级结构，预测VP1蛋白以第39~40、159~167、212~220、287~290位氨基酸成为抗原表位的可能性较大。宁波株EV71编码区没有受到明显正向进化压力的作用，处于净化选择状态。小结： 1、病原学分型：2009~2010年共检测宁波市HFMD病例各类样本446份，其中有191份EV71(55.20%)；121份CoxA16(34.97%)；13份CoxA6(3.76%)；7份CoxA10；4份CoxA12；4份埃可病毒9型(Echovirus9，ECHO9)；2份CoxA2；CoxA4、CoxB1、CoxB4和其它肠道病毒各1份。 2、检测到总肠道病毒核酸阳性346份，男性232份，女性114份，男女比例为2.04:1，发病人群主要集中在1~5岁年龄组，全年份均有发病，有较明显的季节性，发病主要集中在4~7月，12月有不明显小高峰，2010年检测人数较2009年有下降趋势，峰值也有所偏移，2009和2010年的发病高峰分别在4月和6月。 3、宁波4株EV71全基因组核苷酸序列全长均为7406bp，GeneBank登录号为JF830007、JN864018~JN864020。 4、同源性分析：宁波株EV71与中国大陆流行的C4a基因亚型的同源性最高。 5、亲缘进化分析：从各区段进化树上看出，宁波株EV71与中国大陆流行的C4基因亚型的同源性最高，属于C4a进化分支。 6、正向选择压力：宁波株EV71编码区没有受到明显正向进化压力的作用，处于净化选择状态。 7、预测B细胞抗原位点：结合VP1蛋白的亲水性，，柔韧性，表面可能性的重合区域，并分析VP1的蛋白质二级结构，预测VP1蛋白以第39~40、159~167、212~220、287~290位氨基酸成为抗原表位的可能性较大。
[Abstract]:Purpose :

1 . By using the biological samples of Hand - foot - and - mouth disease ( HFMD ) , the pathogenic types of HFMD were identified and the epidemiological characteristics of HFMD were analyzed and the scientific basis was provided for the comprehensive prevention and treatment of HFMD .

2 . On the basis of whole genome sequencing , we know the whole gene characteristics of EV71 isolate in Ningbo City , and analyze the homology and genetic evolution relationship between the strains of Enterovirus 71 ( EV71 ) and other regions , and comprehensively reflect the evolution dynamics .
Potential B - cell antigen sites in VP1 region were analyzed , and whether the virulent strain was subjected to positive selection pressure was calculated .

Method :

1 . Based on the research room of the disease prevention and control center of Ningbo City , we collected 446 samples of hand - foot - mouth disease in Ningbo area from 2009 to 2010 . By means of quantitative RT - PCR and RT - PCR , DNA MAN and DNAStar software were used to analyze the epidemic characteristics of HFMD .

2 , selecting EV71 positive samples in 2010 to carry out virus isolation and nucleic acid extraction , carrying out complete genome sequence amplification on EV71 nucleic acid through reverse transcription polymerase chain reaction , simultaneously measuring VP1 region sequences of other 30 EV71 , carrying out homology and genetic evolution analysis on EV71 representative strain sequences at different times and different sites on the Ningbo strain and GeneBank , analyzing the potential antigen as a point , and calculating whether the virulent strain is subjected to positive selection pressure .

Results :

1 . 446 samples of HFMD cases were tested in Ningbo City from 2009 to 2010 , 191 of them were EV71 ( 55.20 % ) ;
121 parts CoxA16 ( 34.97 % ) ;
13 parts CoxA6 ( 3.76 % ) ;
7 parts CoxA10 ;
4 parts CoxA12 ;
4 copies of Ethyvirus 9 ( Echovirus 9 , ECHO9 ) ;
2 parts CoxA2 ;
CoxA4 , CoxB1 , CoxB4 and other enteroviruses were detected in 1 part . Among the 346 samples tested , 232 male and 114 female , and the male and female ratio were 2.04 : 1 .

Compared with the reference sequence , there were no nucleotide insertions and deletions in the coding region of Ningbo strain .

Summary :

1 . Pathogenic typing : 446 samples of HFMD cases in Ningbo were detected in 2009 - 2010 , 191 of them were EV71 ( 55.20 % ) ;
121 parts CoxA16 ( 34.97 % ) ;
13 parts CoxA6 ( 3.76 % ) ;
7 parts CoxA10 ;
4 parts CoxA12 ;
4 copies of Ethyvirus 9 ( Echovirus 9 , ECHO9 ) ;
2 parts CoxA2 ;
CoxA4 , CoxB1 , CoxB4 , and other Enterovirus 1 part .

2 . There were 346 positive nucleic acid positive in total enteroviruses , 232 male and 114 female , and the male and female ratio was 2.04 : 1 . The incidence of the disease was mainly in the age group of 1 - 5 years .

3 . The whole length of the whole genome of EV71 was 7406bp , and GeneBank accession number was JF8007 , JF84018 - JF84018 . The nucleotide sequence of EV71 was 7406bp .

4 . Homology analysis : The homology of EV71 between Ningbo strain EV71 and C4a gene in mainland China is the highest .

5 . Genetic evolution analysis : From the evolution tree of each section , the homology of EV71 between Ningbo strain EV71 and Chinese mainland is the highest , belonging to the evolutionary branch of C4a .

6 . Positive selection pressure : the EV71 coding region of Ningbo strain is not affected by obvious positive evolution pressure , and is in a clean selection state .

7 . Prediction of B cell antigen site : The overlapping region of the hydrophilicity , flexibility and surface possibility of VP1 protein was predicted , and the secondary structure of VP1 protein was analyzed .

【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.1

【参考文献】