人类肠道病毒B组山东地方株的基因型分布及其所致疾病分子流行病学研究

发布时间：2018-03-09 23:30

  本文选题：人类肠道病毒　切入点：B组　出处：《山东大学》2011年硕士论文　论文类型：学位论文

【摘要】：[背景] 人类肠道病毒(HEV)是一类在人类肠道中繁殖的常见病毒,根据生物学及遗传特性HEV被划分为A、B、C、D共4个基因组。HEV感染可引起多种疾病。无菌性脑膜炎(AM)主要是由各种病毒引起的一组以精神和意识障碍为突出表现的中枢神经系统感染性疾病,轻者能自行缓解,危重者可导致后遗症及死亡。文献报道AM全球每年发病率为11～27/10万。在所明确引起AM的病原体中,HEV约占85%～95%。 20世纪80年代以来,随着全球成功开展消灭脊髓灰质炎活动,许多国家已相继实现无脊髓灰质炎目标,与此同时,由非脊髓灰质炎肠道病毒(NPEV)引起的各类疾病如AM和手足口病(HFMD)的发病近年一直呈上升趋势。 本研究从1994～2008年山东省急性弛缓性麻痹病例(AFP)的常规监测中分离到大量的HEV～B,同时对部分引起疾病暴发的优势毒株进行了序列的测定和同源性分析,初步建立起HEV-B山东地方株的VPl区全基因数据库。本研究对于明确山东省HEV～B基因型的分布,以及通过分子流行病学方法阐明疾病的发生与病毒的遗传进化之间的内在联系均具有重大的理论和现实意义。 [研究的目的] 1.描述山东省1994～2008年HEV-B的分离情况,研究HEV-B山东地方株的基因型分布； 2.建立HEV-B山东地方株的VP1完整编码区基因数据库； 3.分析HEV-B部分优势毒株的VPl完整编码区基因序列的特征,并与国外和国内其他省份的分离株进行比较,分析其进化来源； 4.探讨HEV-B主要型别与其所致疾病之间的联系,分析遗传变异对其致病性和流行规律的影响,为相关疾病的预防控制提供理论依据。 [研究方法] 1.对1994-2008年山东省AFP监测系统中的病例标本以及2003～2008年3次AM暴发中的部分病例标本,采用细胞培养的方法进行病毒分离。对分离到的阳性分离物采用组合血清进行中和试验,确定其血清型。 2.按照毒株的分离年份及分离地点,选取具有代表性的部分毒株进行核酸的提取和VPl区序列的测定。 3.将测得的基因序列进行剪裁、拼接,利用NCBI提供的BLAST服务器进行序列比对,确定HEV-B山东地方株的基因型别。 4.将HEV-B山东地方株与GenBank中检索到的其它国家和地区分离株,采用BioEdit Sequence Alignment Editor software 5.0.9软件,对其VP1区核苷酸序列及其推导氨基酸序列进行同源性比较。 5.采用Mega4.1软件,以相邻连接方法(Neighbor-joining Method)构建VP1区系统发生树,对HEV-B山东地方株进行亲缘进化分析。 [主要结果] 1.山东省HEV-B的分离情况：1994～2008年山东省AFP病例的粪便标本共分离出NPEV 1021株,各年度的分离率在7.7%-17.2%之间；2003～2008年山东省3次AM暴发,其病原体均属于HEV-B,分别为CVB3、CVB5和ECH030。 2.HEV-B山东地方株基因型分布及其VPl区全基因进化树：HEV-B山东地方株分属于29个基因型,其中ECHO11、CVB3、ECHO6、ECHO14、ECHO25是分布最为广泛的HEV-B；各基因型HEV-B无明显的地区分布。 山东省HEV-B可以分为3个簇(Cluster), Cluster1包括ECH030在内的12种ECHO病毒,Cluster2包括CVB 1-5, Cluster3包括ECHO 12在内的另外11种ECHO病毒及CVA 9。每种血清型病毒单独成簇。 3.主要的HEV-B山东地方株遗传进化分析 (1)HEV-B山东地方株VPl区全基因数据库的建立及其同源性分析：根据VP1区全基因数据库毒株备选原则,从山东省AFP病例中分离到的HEV-B中选取171株,与HEV-B所致疾病暴发中的33株分离株,一起构建了山东地方株VP1区全基因数据库。 VP1区全基因序列同源性分析显示：山东地方株型内核苷酸差异最大的为ECHO1(6.7%-24.6%),差异最小的为CVB4(2.9%-5.6%)；CVB2与其原型株相比核苷酸差异最小(4.8%-7.7%),EV73型与其原型株相比核苷酸差异最大(24.6%)。 (2)CVB3山东地方株遗传进化分析：CVB3山东地方株与原型株之间的核苷酸同源性为77-8%-80.0%,氨基酸同源性为96.1%-97.5%。分离株177/2008TC/SD/CHN的核苷酸变异位点及变异结果与其余4株都有较大的差异；5株山东地方株氨基酸变异的数目为9或10。CVB3毒株可划分为A、B、C共3个基因型,山东地方株分布在A基因型内。A1-A3 3个基因亚型内的山东地方株分离年份依次为2004～2008年、1994～2002年、2004年和2008年。 (3)CVB5山东地方株遗传进化分析：CVB5山东地方株与原型株之间的核苷酸同源性为81.2%-81.6%,氨基酸同源性均为96.8%；山东地方株之间的核苷酸同源性为83.0%-94.6%,氨基酸同源性为96.4%-100.0%。在VP1完整编码区中核苷酸的变异位点分布较为均匀,也没有显示时间上的聚集性。相对于原型株,山东地方株发生了9个氨基酸变异。CVB5毒株划分为A、B、C、D 4个基因型,山东地方株分布在C和D两个基因型内。进化树中与山东地方株同源性最近的是浙江分离株Cox B5/Zejiang/12/02 (CSF)。 (4)ECH06山东地方株遗传进化分析：ECH06山东地方株之间的核苷酸同源性为77.6%～98.6%,氨基酸同源性为93.7%-99.6%,与原型株之间的核苷酸同源性为75.2%-78.8%,氨基酸同源性为92.7%-95.5%。VP1完整编码区核苷酸的变异显示出时间上的聚集性；氨基酸的变异存在三处变异较为密集的区域,变异数目为13-21。ECH06毒株划分为A、B、C共3个基因型,山东地方株分布在B和C两个基因型内。山东地方株来源于3个不同的进化分支。 (5)ECHO11山东地方株遗传进化分析：ECHO11山东地方株之间的核苷酸同源性为76.4%～100.0%,氨基酸同源性为91.4%-100.0%；ECHO11山东地方株与原型株Gregory之间的核苷酸同源性为77.7%-80.7%,氨基酸同源性为90.7%-94.8%。ECHO11山东地方株氨基酸变异数目大于20,存在时间上的聚集性以及变异较为密集的区域。ECHO11毒株划分为A、B、C、D 4个基因型,山东地方株分布在A和C两个基因型内。山东地方株构成了A1、A2和C1共3个新的基因亚型,A1基因亚型内的毒株均分离自1998年之后,A2和C1两个基因亚型内的毒株均分离自1994-1997年。 (6)ECHO19山东地方株遗传进化分析：ECHO19山东地方株之间的核苷酸同源性为83.3%～98.6%,氨基酸同源性为98.6%～99.3%。山东地方株与原型株之间的核苷酸同源性为78.6%～79.2%,氨基酸同源性为97.6%～99.3%。核苷酸与氨基酸的比对结果显示：氨基酸变异数目大于20,分离年份相近的毒株之间基因序列变异情况相似,毒株97206/SD/CHN/1997/E19基因序列变异与其他分离株相比差异较大。进化树显示9株国内毒株存在集聚现象,形成一个基因簇。 (7)ECHO30山东地方株遗传进化分析：ECHO30山东地方株与原型株之间的核苷酸同源性为81.9%～83.5%,氨基酸同源性为92.4%～93.1%；山东地方株之间的核苷酸同源性为86.0%～98.5%,氨基酸同源性为96.9%～98.9%。毒株06350/SD/CHN/2006/E30与其他山东地方株之间的同源性较小。核苷酸的变异情况显示：06530/SD/CHN/2006/E30和017/2008TC/SD/CHN 2株毒株与其他山东地方株的变异数目和变异位点均存在较大差异。山东地方株氨基酸变异数目大于20。ECHO30毒株划分为A～E共5个基因型,国内株全部分布在E基因型内,山东地方株分布在E1、E3两个基因亚型内。 4.部分HEV-B山东地方株引起的AM暴发：2003年章丘市AM暴发中各年龄别罹患率在7岁前随年龄增加而有所升高,7岁后随年龄的增加而减少。患者具有较为明确的接触史,神经系统症状较为明显。引起此次疾病暴发病原体为单一的ECHO30病毒,与同时期山东省泰安市、浙江省和江苏省AM暴发中的分离株之间存在较为密切的流行病学联系。 2005年山东省兖州市AM暴发以6岁以下儿童为主,2岁以下婴儿罹患率最高。病例及其看护人均无明确的接触史,患者的临床症状较轻。引起此次疾病暴发的病原体为CVB5毒株,主要分离自脑脊液标本,与2002的浙江分离株CoxB5/Zejiang/12/02 (CSF)流行病学联系密切。 2008年山东省郯城县AM暴发中1岁以下儿童发病数最多,各年龄别罹患率在随年龄增加而降低。大部分患者有AM病例接触史,临床症状相对较轻。引起此次疾病的病原体为CVB3,此次暴发由同一传播链的CVB3毒株所致,毒株与进化树中其他2008年的分离株同源性较近,为同一进化来源。 [主要结论] 1.HEV-B在山东省分布广泛,对人群健康存在着重要的影响,AFP病例监测系统每年可以分离到大量的NPEV；不同基因型毒株有不同的时间循环模式；某些优势基因型如CVB5、CVB3、ECHO30在山东省呈中度流行状态,优势毒株的变迁往往伴随着疾病的暴发。 2.VP1区全基因序列同源性分析显示,各基因型HEV-B山东地方株与其原型株相比均发生了较大的变异,相对于ECHO病毒,CV病毒核苷酸的变异大多为同义突变,并没有引起氨基酸的变异；ECHO病毒在进化中较为活跃。 3.相同基因型的HEV-B山东地方株内部,根据其遗传距离的远近可以划分为不同的基因亚型,进化树分析显示相同基因亚型毒株之间在地理和时间分布上存在着一定的聚集性。 4.在山东省,HEV-B引起的疾病暴发仍以AM为主,在HFMD的流行中偶见HEV-B组病毒,多数疾病暴发中分离到的HEV-B为单一的血清型,这与HEV-A病毒之间常见的协同流行略有不同。 5.对于部分HEV-B优势毒株的分子流行病学研究显示了其各自的流行特征：①CVB3山东地方株为原型株近似株而非变异株近似株,病毒在国内传播迅速,广泛流行,相同的年份有多个传播链的CVB3病毒在山东省流行；②CVB5毒株的变异进化速度相对较慢,进化树中D1基因亚簇内毒株2002-2005年在亚洲和欧洲的流行,导致了2002年浙江省和2005年山东省充州市AM的暴发；③ECH06毒株变异进化较为迅速,山东省近年流行株与往年流行株之间的核苷酸序列存在较大差异,可能与往年毒株为不同进化来源；④ECHO11病毒在进化上较为活跃,1994-1997年期间山东省存在两个差异较大的ECHO11基因亚型共循环,而A1亚型内的毒株为近年流行的基因亚型；⑤ECH019山东地方株的进化变异速度较慢,引起2003年泰安市疾病暴发的毒株在山东省流行广泛；⑥2003年章丘市AM暴发的ECH030分离株存在较大的抗原变异,感染性强,传播迅速,为山东省近几年的主要流行株,在2003-2008年山东省存在两个不同基因亚型的毒株流行。
[Abstract]:[background]
Human enterovirus (HEV) is a kind of breeding in the human gut the common virus, is divided into A, according to the biological and genetic characteristics of HEV B, C D, a total of 4 genomic.HEV infection can cause many diseases. Aseptic meningitis (AM) is the main disease caused by various viruses in a group of fine God and consciousness for the outstanding performance of the central nervous system infection, it can relieve itself, can lead to severe sequelae and death. The reported AM global annual incidence rate of 11 ~ 27/10 million. In the clear the causative agent of AM, HEV was about 85% ~ 95%.
Since 1980s, with the successful development of global polio eradication activities, polio free target, has been achieved in many countries at the same time, the non polio enterovirus (NPEV) caused by various diseases such as AM and hand foot mouth disease (HFMD) incidence in recent years has been on the rise.
In this study, 1994~2008 years from the Shandong province of acute flaccid paralysis (AFP) isolated from a large number of HEV ~ B in routine monitoring, at the same time on the part of the cause of outbreaks of dominant strains were determined and sequence homology analysis, initially established a whole genome database of HEV-B isolated in Shandong area VPl. This study to clarify Shandong province HEV ~ B genotype, the intrinsic link between genetic and molecular epidemiological methods to elucidate and by the viral diseases are of great theoretical and practical significance.
[the purpose of the study]
1. to describe the isolation of HEV-B in 1994~2008 years in Shandong Province, and to study the genotype distribution of HEV-B local strains in Shandong.
2. the VP1 complete coding region gene database of HEV-B Shandong local strain was established.
3., we analyzed the characteristics of VPl complete coding region gene sequence of some HEV-B dominant strains, and compared with the isolates from other provinces and other provinces in China, and analyzed their evolutionary sources.
4., we discussed the relationship between the main types of HEV-B and its diseases, and analyzed the effects of genetic variation on their pathogenicity and epidemic rule, so as to provide a theoretical basis for the prevention and control of related diseases.
[research methods]
1. in 1994-2008 years, the samples from AFP monitoring system in Shandong province and 2003~2008 cases of AM outbreak in 2003~2008 years were separated by cell culture. The positive isolates were neutralized by combinatorial serum, and their serotypes were determined.
2. the extraction of nucleic acid and the determination of VPl region sequence were selected according to the separation year and the separation site of the strains.
3. the gene sequence was tailored and spliced, and the sequence alignment of the BLAST server provided by NCBI was used to determine the genotypes of the local strains of HEV-B in Shandong.
4., we isolate the isolates from HEV-B Shandong and other countries and regions retrieved in GenBank. We used BioEdit Sequence Alignment Editor software 5.0.9 software to compare their nucleotide sequences and deduced amino acid sequences in VP1 region.
5. Mega4.1 software was used to construct a VP1 region phylogenetic tree with adjacent connection method (Neighbor-joining Method), and the phylogenetic evolution of local strains of HEV-B in Shandong was analyzed.
[main results]
1., the separation of HEV-B in Shandong Province: 1994~2008 years, 1994~2008 cases of NPEV were isolated from fecal specimens of AFP cases in Shandong province. The isolation rate in each year was between 7.7%-17.2%. In 2003~2008 years, 3 times AM outbreak in Shandong Province, all the pathogens were HEV-B, CVB3, CVB5 and ECH030. respectively.
2.HEV-B genotype distribution and VPl region gene evolution tree of Shandong local strain: HEV-B, Shandong local strain belongs to 29 genotypes, ECHO11, CVB3, ECHO6, ECHO14, ECHO25 are the most widely distributed HEV-B, and genotype HEV-B has no obvious regional distribution.
Shandong HEV-B can be divided into 3 clusters (Cluster), Cluster1 includes ECH030, including 12 ECHO viruses, Cluster2 includes CVB 1-5, Cluster3 includes ECHO 12, including 11 other ECHO viruses and CVA 9., each serotype virus is clustered separately.
Analysis of genetic evolution of 3. main HEV-B Shandong local strains
(1) establishment and homology analysis of whole genome database VPl HEV-B isolates from Shandong: according to the complete gene database VP1 strains isolated from the alternative principle, Shandong Province in the case of AFP HEV-B in the selected 171 strains, and HEV-B caused by disease outbreaks in 33 isolates, together to construct the whole gene database VP1 Shandong local strains.
VP1 gene sequence homology analysis showed that: Shandong local plant nucleotide difference maximum is ECHO1 (6.7%-24.6%), the smallest difference is CVB4 (2.9%-5.6%); CVB2 and its prototype strains compared to nucleotide differences (4.8%-7.7%), minimum EV73 and its prototype strain by nucleotide difference (24.6%).
(2) CVB3 analysis of Shandong local strains of genetic evolution: CVB3 isolates from Shandong and nucleotide homology between prototype strains of 77-8%-80.0%, amino acid homology of nucleotide variation and variation of the loci for 96.1%-97.5%. isolates of 177/2008TC/SD/CHN and other 4 strains are different; 5 strains of amino acid variation in the number of isolates from Shandong was 9 or 10.CVB3 strains can be divided into A, B, C a total of 3 genotypes, Shandong local strains of the year Shandong local strain distribution of.A1-A3 in the A genotype in 3 genotypes were within 2004~2008 years, 1994~2002 years, 2004 and 2008.
(3) CVB5 analysis of Shandong local strains of genetic evolution: CVB5 isolates from Shandong and nucleotide homology between the prototype strain 81.2%-81.6%, the homology of amino acid was 96.8%; Shandong local strains of the homology between 83.0%-94.6% and amino acid homology was 96.4%-100.0%. in VP1 complete nucleotide mutation sites in the encoding region is more evenly distributed, also show no aggregation time. Compared with the prototype strain, Shandong strain had 9 amino acid variant.CVB5 strains are divided into A, B, C, D 4 genotypes, Shandong local strain distribution in C and D two genotypes. And Shandong local strains recently isolated in Zhejiang is Cox B5/Zejiang/12/02 phylogenetic tree (CSF).
(4) ECH06 analysis of Shandong local strains of genetic evolution: Shandong ECH06 strains and the homology between 77.6% ~ 98.6%, the homology of amino acid was 93.7%-99.6%, and the nucleotide homology between the prototype strain 75.2%-78.8%, the homology of amino acid mutations for nucleotide 92.7%-95.5%.VP1 complete encoding region shows the time aggregation of the amino acid variation; there are three variants in more populated areas, the number of variant 13-21.ECH06 strains are divided into A, B, C a total of 3 genotypes, Shandong local strain distribution in B and C two genotypes. Shandong local strains derived from 3 different evolutionary branches.
(5) ECHO11 analysis of Shandong local strains of genetic evolution: Shandong ECHO11 strains and the homology between 76.4% ~ 100% amino acid homology to 91.4%-100.0%; ECHO11 between Shandong local strains and prototype strain Gregory nucleotide homology was 77.7%-80.7%, amino acid homology is the number of amino acid variation of 90.7%-94.8%.ECHO11 isolates from Shandong more than 20, the presence of time the aggregation and variation of relatively dense region of the.ECHO11 strain is divided into A, B, C, D 4 genotypes, Shandong local strain distribution in A and C two genotypes. Shandong local strains constitute the A1, A2 and C1 were 3 new genotypes, A1 gene subtype strains the separation since 1998, A2 and C1 strains of two genotypes were isolated from within 1994-1997 years.
(6) ECHO19 analysis of Shandong local strains of genetic evolution: Shandong ECHO19 strains and the homology between 83.3% ~ 98.6% amino acid homology is 98.6% ~ 99.3%. Shandong local strains and prototype strains nucleotide homology is 78.6% ~ 79.2%, the homology of amino acid was 97.6% ~ 99.3%. compared the nucleotide and amino acid showed that the amino acid the variation is greater than the number of 20 gene sequences similar to the separation between year similar strains, gene sequences of 97206/SD/CHN/1997/E19 strain compared with other strains. The phylogenetic tree showed large differences in strains of 9 strains of endotoxin agglomeration, forming a gene cluster.
(7) ECHO30 analysis of Shandong local strains of genetic evolution: ECHO30 isolates from Shandong and nucleotide homology between prototype strains ranged from 81.9% to 83.5%, the amino acid homology is 92.4% ~ 93.1%; Shandong local strains of the homology between 86% ~ 98.5% amino acid homology is 96.9% ~ 98.9%. 06350/SD/CHN/2006/E30 strain and other places of Shandong strains the homology of nucleotide variation is small. Display: 06530/SD/CHN/2006/E30 and 017/2008TC/SD/CHN 2 isolates and other isolates from Shandong the number variation and variation sites are quite different. The number of amino acid variations in Shandong local strains greater than 20.ECHO30 strains divided into A to E a total of 5 genotypes, the E genotype distribution in all strains in Shandong the local strain distribution in E1, E3 two subtypes.
The 4. part of the HEV-B Shandong strain caused by the outbreak of AM in Zhangqiu city in 2003: the outbreak of AM in each age incidence at the age of 7 with the increase of age increased, after 7 years of age decreased with the increasing age. Patients with definite contact history, nervous system symptoms are obvious. That caused the outbreak as a single pathogen the ECHO30 virus, with the same period in Shandong city of Tai'an Province, there is an epidemiological link more closely in Zhejiang province and Jiangsu Province in the outbreak of AM isolates.
Yanzhou City, Shandong province in 2005 outbreak of AM in children under 6 years old, infants under the age of 2 with the highest rate of patients and their carers. No clear history of exposure, the clinical symptoms of the patients were mild. Caused the disease outbreaks is the pathogen of CVB5 strains, mainly isolated from cerebrospinal fluid specimens, and 2002 strains of CoxB5/Zejiang/12/02 isolated in Zhejiang (CSF epidemiology) closely.
In 2008, Shandong County of Tancheng province AM in children under 1 years of age the largest incidence, prevalence rate in each age and decreased with the increasing of age. Most of the patients with AM cases of contact history, clinical symptoms are relatively mild. Caused by the pathogen of this disease is CVB3, by the outbreak of CVB3 strain caused by the same transmission chain, the other in 2008 strain and phylogenetic tree of isolates homology is near, for the same evolutionary origin.
[main conclusions]
1.HEV-B is widely distributed in Shandong Province, has an important impact on the health of the population, AFP surveillance system every year can be separated to a large number of NPEV; different strains have different circulation patterns; some dominant genes such as CVB5, CVB3, ECHO30 showed a moderate epidemic situation in Shandong Province, the dominant strains are often accompanied by changes an outbreak of disease.
2.VP1鍖哄叏鍩哄洜搴忓垪鍚屾簮鎬у垎鏋愭樉绀，

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