【作者】 徐爱强；

【导师】 赵仲堂；

【作者基本信息】 山东大学 ， 流行病与卫生统计学， 2012， 博士

【摘要】 [背景]人类肠道病毒(Human Enteroviruses, HEVs)属于小RNA病毒科(Picornaviridae)肠道病毒属(Enterovirus),目前有A、B、C、D4个组共109种血清型。HEVs抗原型别复杂,进化活跃,隶属不同组别的HEVs具有相同或相似的致病性,而导致不同疾病的HEVs在生物学特性和进化上非常相似和接近。根据HEVs抗原性及致病性的不同,最初被分为64个血清型：脊髓灰质炎病毒(Polioviruses, PV)1-3型,柯萨奇病毒A(Coxsackievirus A, CVA)1-22和24型,柯萨奇病毒B(CVB)1-6型和埃可病毒(Echovirus, Echo)1-7、9、11-21、24-27、29-33型,以及20世纪70年代后发现的新型肠道病毒(Newer Enteroviruses)EV68-71型。1999年,基于Oberste等提出的VP1基因序列的分子定型方法,许多使用传统血清学方法无法定型的HEVs(即新型HEVs)陆续被发现,目前已有50多个新型HEVs基因型,而且随着分子定型方法的推广,将来还会发现更多的新型HEVS。HEVs感染可以引起多种疾病,尤其对儿童健康危害大,已成为当前严重的公共卫生问题之一。HEVs隐性感染率高,人体感染病毒后通常不产生临床症状,但部分感染者可引起类感冒病症、永久性肢体麻痹、无菌性脑炎或脑膜炎、心肌炎、流行性肌痛、急性出血性结膜炎、手足口病(Hand Foot and Mouth Disease,HFMD)、疱疹性咽峡炎、Ⅰ型糖尿病等,严重者可导致死亡。新型HEVs是重要的人类致病病原,虽然发现较晚,但已有多起相关疾病的暴发和流行的报道,其感染严重威胁人群特别是儿童的健康与生命安全。EV71所致的手足口病暴发或流行已成为严重的公共卫生问题；EV70可导致急性出血性结膜炎流行；其他感染新型HEVs的表现有发热、消化道症状、呼吸系统疾病,严重者可伴有非特异性中枢神经系统疾病如无菌性脑炎、脑膜炎等病症。大多数新型HEVs基因型的致病性还有待进一步的认识。山东省是HEVs相关疾病的高发省份之一,曾发生过多起HEVs相关的无菌性脑膜炎、手足口病、急性出血性结膜炎等疾病的暴发或流行。国内对新型HEVs的研究起步较晚,田炳均等仅对云南分离的部分新型HEVs(EV75,80,81,83,96)进行了VP1部分区域的测定和型别鉴定。因而,现阶段开展新型HEVs分子流行病学研究,探索本地流行株的型别和分子特征,对于制订新型HEVs所致相关疾病的防控策略和建立预测预警机制,均具有重要的理论和现实意义。[研究目的]1.研究新型HEVs山东分离株的基因型分布特征；2.研究新型HEVs山东分离株的生物学特性和VP1基因的分子进化：3.研究新型HEVs山东分离株各基因型的全基因组序列特征与重组。[研究方法]1.对1989～2010年山东省急性弛缓性麻痹(Acute Flaccid Paralysis, AFP)监测系统中的病例标本、2007～2010年山东省手足口病病例标本,以及2010年临沂市无菌性脑膜炎病例标本,采用细胞培养的方法进行病毒分离。2.对病毒分离阳性但用HEVs组合血清中和试验无法定型的毒株,提取核酸并进行VP1完整编码区序列的扩增和测定。3.将获得的VP1完整编码区序列,在NCBI提供的BLAST服务器上,通过HEVs分子定型方法进行序列比对和型别鉴定。4.将鉴定出的新型HEVs山东分离株与GenBank中其它国家和地区分离株,采用BioEdit7.1.3软件,对其VP1区核苷酸序列及其推导的氨基酸序列进行同源性比较；采用Mega4.0软件,以邻位法(Neighbor-joining Method)构建VP1区系统发生树,对新型HEVs山东分离株进行亲缘进化分析。5.对部分新型HEV山东分离株血清型(EV71、EV75、EV76、EV90),采用BEAST1.6.2软件,进行进化遗传学分析,推算该血清型的起源和进化速度,探讨该HEVs血清型在山东或中国大陆的流行史。6.对部分新型HEVs山东分离株血清型(EV71、EV73、EV75、EV76、EV90、EV96、EV97)分离株,进行全基因组序列测定。使用BioEdit7.1.3软件对其与原型株基因组序列进行比对；使用Simplot软件,分析新型HEV山东分离株在基因组中的重组。[主要结果]1.新型HEVs山东分离株的基因型分布本研究鉴定出新型HEVs山东分离株共有10个血清型213株,分别是EV71(197株)、EV73(1株)、EV74(1株)、EV75(2株)、EV76(2株)、EV80(3株)、EV87(1株)、EV90(3株)、EV96(2株)、EV97(1株)。EV71分别源自AFP病例、HFMD病例和无菌性脑膜炎病例,其他9种血清型全部来源于AFP病例。2.EV71山东分离株VP1基因的分子进化本研究将59株EV71山东分离株的VP1完整编码区进行了序列测定。其中包括1996～2010年AFP监测病例分离株(13株)、2007～2010年HFMD病例分离株(35株),以及2010年临沂市无菌性脑膜炎病例分离株(11株)。2003年之后分离到的EV71山东分离株,均属于C4亚型的C4a分支,具有高度的同源性,在进化关系上与中国大陆地区1997年以来分离到的EV71同属于一个亚型。2003年之前,仅有1株EV71分离株,来自1996年的AFP病例分离株,属于C2亚型,这也是在中国大陆地区首次发现C2亚型的EV71。EV71C4亚型在山东省的流行具有时间动力学特征,即不同时期的分离株在系统发生树上位于不同的进化特征。通过使用Beast1.6.2程序的贝叶斯MCMC多动力学方法,推算出EV71C4亚型的起源时间(Time of Most Recent Common Ancestor,tMRCA)在1994年,进化速度为每年每个核苷酸位置变异5.199×10-3个碱基。C2亚型与C4亚型山东分离株的共同祖先在1985年,提示在该时间EV71开始分化出C2和C4亚型。3.其他新型HEVs山东分离株VP1基因的分子进化(1)全球EV73分离株可分为2个基因群(Genogroup):A和B。基因群B成员较多,分离年代跨度大(1964-2010),说明该群至今仍在流行。本研究鉴定出1株EV73山东分离株(04088),位于基因群B,但与其他分离株均无密切的亲缘关系(平均遗传距离为0.184,VP1同源性在75.9%-84.1%之间)。(2)本研究鉴定出1株EV74山东分离株(05293),与GenBank中的其他所有分离株的平均遗传距离为0.194,显示了山东分离株与其他分离株均无密切的亲缘关系。鉴于目前GenBank中的EV74基因序列较少,且大部分分离株之间无密切的亲缘关系,因此目前的研究数据上不支持进行型内基因群的划分。(3)本研究鉴定出2株EV75山东分离株(97102和97209),均来自1997年的AFP病例。GenBank中的全球EV75分离株可分为2个基因群：A和B。其中基因群B包括5个成员,全部来自中国大陆地区(山东省和云南省),均分离自AFP病例。山东分离株与云南分离株的平均遗传距离为0.128,而与其他分离株的平均遗传距离为0.247。EV75山东分离株之间的核苷酸同源性为100%,与国外其他病毒株VP1核苷酸同源性均较低,在71.0%-77.8%之间。通过基于贝叶斯MCMC方法的进化遗传学分析显示,全球EV75起源于1956年,以每年每个核苷酸位置变异1.647×10之个碱基的速度进化。(4)本研究共鉴定出2株EV76山东分离株(04360和05048),二者之间的核苷酸同源性很高(99.7%),属于同一个传播链；这两株病毒分别分离自济宁市汶上县和临沂市河东区,提示当时在这两个地区之间存在着远距离的传播。山东分离株与原型株FRA91-10369的同源性为86.0%,与云南分离株171-99的同源性为86.9%,与其他分离株的同源性为80.7%-94.7%。(5)本研究共鉴定出3株EV80山东分离株(97249,98323和04HZ+I),与原型株CA67-10387的VP1核菅酸同源性为76.4%-81.7%。98323和04HZ+I的核苷酸同源性分别为95.6%,显示了较高的亲缘关系,但97249株与其他两株的亲缘关系较远,VP1核苷酸同源性为80.3%-82.0%。(6)本研究共分离到1株EV87山东分离株(OOLY+2),这是国内首次分离到EV87。与原型株BAN01-10396的核苷酸和氨基酸同源性分别为80.3%和97.0%。(7)本研究共分离到3株EV90山东分离株(01336,01421和03446),属于基因群B,这是国内首次分离到EV90。3株EV90山东分离株之间核苷酸同源性为96.7%-98.0%,显示了密切的亲缘关系。与原型株BAN99-10399的核苷酸同源性为90.8%-91.4%,与国外其他分离株的核苷酸同源性为77.7%-92.3%。(8)本研究共分离到2株EV96山东分离株(05517和09228C1),核苷酸同源性分别为81.6%,显示了较远的亲缘关系。与原型株BAN00-10488的核苷酸同源性为78.1%-81.0%,与国外其他分离株核苷酸同源性为77.6%-87.9%。进化树上也位于独自的分支。(9)本研究共分离到1株EV97山东分离株(99188),这是国内首次分离到EV97。与国外其他6株EV97病毒株的亲缘关系均较远。VP1编码区3’端(nt,817-834)存在18nt的核苷酸的缺失。4.新型HEVs山东分离株各基因型的全基因组序列特征与重组EV71、EV73、EV75、EV76、EV90、EV96、EV97的全基因组序列分析显示,在新型HEVs山东分离株的非结构蛋白编码区,均发现了基因重组的证据：在该区域,EV71山东株序列与CVA14原型株G14和CVA16原型株G10同源性较高；EV73、EV75山东分离株与国内HEV-B分离株Echo30/Zhejiang/17/03/CSF同源性较高；EV76山东分离株与EV89和EV90原型株同源性最高；EV90山东分离株01421与芬兰株F950027的型内重组；EV96山东分离株09228C1和EV102原型株(EF555645)同源性最高；EV97山东分离株99188株与Echo9分离株DM和Echo30分离株Zhejiang/17/03/CSF同源性最高。EV90、EV96和EV97的全基因组序列分析还显示,在山东分离株的衣壳蛋白P1编码区,发现核苷酸的缺失,缺失的数目分别是3个或18个核苷酸,均不会导致蛋白质的移码突变。核苷酸插入缺失的位点,正是不同血清型间衣壳蛋白编码区长度变化的区域,如VP3或VP1的3t端等。提示HEVs衣壳蛋白在进化过程中,在点突变尚未积累到导致血清型发生改变的程度时,核苷酸的插入或缺失可能已经出现。[主要结论与建议]1.在山东省监测病例中发现多种新型HEVs,其中EV87、90、97血清型和EV71C2亚型为中国大陆地区首次发现。本研究共发现213株新型HEVs山东分离株,分为10种血清型,即EV71、EV73、EV74、EV75、EV76、EV80、EV87、EV90、EV96、EV97。EV71分别源自AFP病例、HFMD病例和无菌性脑膜炎病例,其他9种血清型全部来源于AFP病例。2.EV71是目前最为常见的致病新型HEVs,特别是导致自2007年以来中国大陆HFMD的广泛流行。本研究首次证实2007年在山东省临沂市发生的HFMD暴发的病原为EV71；在EV71C4亚型在我国大范围流行之前,我国存在着其他亚型的EV71,其在我国的流行范围和时期需更多的回顾性分子流行病学研究来阐明。此外,EV71是山东省无菌性脑膜炎病例的重要病原之一。3.除EV71之外,其他新型HEVs在山东省的分离率低,提示尚未在本地大规模地传播流行。4.各新型HEVs山东分离株与国内外分离株相比具有较大的核苷酸差异,亲缘关系较远；核苷酸的插入和缺失参与了新型HEVs衣壳蛋白编码区的进化。5.新型HEVs山东分离株的全基因组序列中均有重组发生的证据,确切的重组来源需要其他不同时期、不同地域分离株的基因组序列信息来阐明。6.鉴于新型HEVs已在山东省监测病例中广泛存在,建议今后我国应继续加强新型HEVs的监测及其地方分离株的分子流行病学研究工作。更多还原

【Abstract】 [Background]Human enteroviruses (HEVs) belong to the genus Enterovirus, family Picornaviridae. Genus Enterovirus comprises109serotypes which are grouped into four species:A to D. HEVs evolve actively. Different serotypes possess similar pathogenicity, and different diseases can be associated with a single serotype.According to the antigenicity and pathogenicity, HEVs are previously classified into64serotypes:polioviruses (PV) type1-3, coxsackievirus A (CVA)1-22and24, CVB1-6, echovirus (Echo)1-7,9,11-21,24-27, and29-33, and newer enteroviruses68-71. In1999, the molecular typing method based on VP1sequences was introduced by Oberste et al, and a lot of serologically untypeable HEVs were identified. More than50new serotypes were identified, and with the increasing application of molecular typing method, more new HEV serotypes will be identified in the future.HEVs can cause many diseases, especially in children. HEV associated diseases is an important public health problem. Their infection is known to be generally asymptomatic, but sometimes may cause illnesses such as summer cold, acute flaccid paralysis (AFP), aseptic encephalitis and meningitis (AM), acute myocarditis, epidemic myalgia, acute hemorrhagic conjunctivitis (AHC), hand, foot, and mouth disease (HFMD), herpangina, type1diabetes, and even death. Newer HEVs are important human pathogens. Although they were identified in recent years, there are several outbreaks and epidemics of associated diseases. EV71associated HFMD outbreak or epidemics are severe public health problem. EV70is pathogen of AHC. Other associated symptoms include fever, digestive indisposition, respiratory disease, and severe disease in central nervous system such as aseptic encephalitis and meningitis. Pathogenicity of other newer HEV serotypes is under further investigation.Shandong province is one of epidemic area of HEV associated diseases with several documented outbreaks or epidemics of aseptic meningitis, HFMD, and AHC. There is no intensive research on newer HEVs in China. Only Identification of EV75,80-83,96in Yunnan province was reported. Hence, to explore the molecular epidemiology, genotype distribution, and genetic characterization will contribute significantly to the prevention and control of related diseases and the establishment of precaution mechanism.[Objectives]1. To investigate the genotype distribution of newer HEVs in Shandong province.2. To explore the biological characterization and VP1molecular evolution of newer HEVs in Shandong province.3. To study the complete genome sequence characterization and recombination of newer HEVs in Shandong province.[Methods]1. Enterovirus isolation was performed on specimens from AFP cases in Shandong province in1989-2010, HFMD cases in Shandong province in2007-2010, and aseptic meningitis cases in Linyi city in2010.2. RNA extraction, RT-PCR and sequencing of entire VP1coding region were performed for serologically untypeable isolates.3. VP1sequence alignment and molecular typing were conducted on BLAST server provided by NCBI. 4. Homologous comparison on VP1nucleotide and amino acid sequences between Shandong isolates and global isolates was performed using BioEdit7.1.3. Phylogenetic trees on VP1sequences were constructed using neighbor-joining method in Mega4.0.5. Evolutionary genetics (origin and evolution rate) of partial newer HEV serotypes (EV71, EV75, EV76, and EV90) was inferred via BEAST1.6.2, and the epidemic history in Shandong province or China’s mainland was re-constructed.6. Complete genome sequencing was performed on partial serotypes (EV71, EV73, EV75, EV76, EV90, EV96, and EV97). Genome alignment was conducted using BioEdit7.1.3. Recombination in the genome in Shandong newer HEV strains was analyzed using Simplot.[Results]1. Genotype distribution of newer HEVsAltogether213newer HEV strains were identified. They belonged to10serotypes:EV71(197), EV73(1), EV74(1), EV75(2), EV76(2), EV80(3), EV87(1), EV90(3), EV96(2), and EV97(1). EV71came from AFP, HFMD and AM cases, and the other9serotypes came from AFP cases.2. Molecular evolution of EV71in Shandong provinceSequencing of VP1entire coding regions were performed on59EV71strains, including13strains from AFP surveillance in1996-2010,35strains from HFMD cases in2007-2010, and11strains from AM cases in Linyi in2010. Shandong EV71strains since2003had high homologies, and they all belonged to C4a lineage of C4subgenotype, phylogenetically closely related to the mainland strains since1997. Only1EV71was isolated before2003. It was isolated from AFP cases in1996, and belonged to C2subgenotype. This is the first report of C2subgenotype EV71in China’s mainland.Temporal dynamics of EV71in Shandong province was observed, as was reflected via the different clusters of viruses with different isolation years. By using the Bayesian MCMC method in BEAST1.6.2software, the time of most recent common ancestor (tMRCA) of C4subgenogroup of EV71was deduced in1994, and the evolution rate was5.199×10-3nucleotides per site per year. The tMRCA of Shandong C2and C4subgenogroups was1985, implying the divergence of EV71at that time.3. VP1molecular evolution of other Shandong newer HEV serotypes(1) Global EV73strains were divided into two genogroups:A and B. Genogroup B was a major group, and its members were isolated in1964-2010, revealing the continous circulation. Shandong EV73strain belonged to genogroup B, and it had long genetic distances with other strains (mean,0.184; VP1homomogies,75.9%-84.1%).(2) Shandong EV74strain,05293, had a mean genetic distance of0.194with other strains in GenBank, suggesting it had no close relationship with others. Because of the little genetic data of EV74available in GenBank, and the high divergence among global strains, the classification of genogroup could not be performed.(3) Shandong EV75strains,97102and97209, both came from AFP cases in1997. Global EV75strains segregated into two genogroups, A and B. Genogroup B contained5members, all from AFP cases in China’s mainland (Shandong and Yunnan). The mean genetic distance between Shandong and Yunnan strains was0.128, and the parameter between Shandong strains and other global strains was0.247. Shandong EV75strains had100%nucleotide homology with each other, and71.0%-77.8%homologies with other strains. The tMRCA of global EV75was dated back to1956with the evolution rate of1.647×10-2nucleotides per site per year.(4) Shandong EV76strains,04360and05048, had high VP1nucleotide homology (99.7%) with each other, and they belonged to the same transmission chain. They were isolated from AFP cases in Wenshang county and Hedong district, respectively, suggesting the long distance transmission between the two areas at that time. Shandong strains had86.0%VP1nt similarity with proto FRA91-10369,86.9%with Yunnan strain171-99, and80.7%-94.7%with other strains.(5) Shandong EV80strains,97249,98323and04HZ+1, had76.4%-81.7%VP1nt similarities with proto CA67-10387. A high similarity was observed between98323and04HZ+1, reflecting close relationship, while97249had relative low VP1 homologies with the other two Shandong strains (80.3%-82.0%).(6) Only one EV87strain was isolated in Shandong province, and this is the first report of isolation of EV87in China. The nucleotide and amino acid identities with proto BAN01-10396were80.3%and97.0%, respectively.(7) Three EV90strains,01336,01421and03446, were isolated in Shandong province. This is the first report of EV90in China. Shandong strains belonged to genogroup B, and had96.7%-98.0%VP1nucleotide similarities among themselves, reflecting close relationship. Shandong strains had90.8%-91.4%VP1nucleotide similarities with proto BAN99-10399, and77.7%-92.3%with other strains.(8) Two EV96strains,05517and09228C1, were isolated in Shandong province. They had81.6%VP1nucleotide similarities with each other, reflecting remote relationship. They had78.1%-81.0%nucleotide similarities with proto BAN00-10488, and77.6%-87.9%with other strains. They formed a distinct cluster in the phylogenetic tree.(9) Only1EV97strain was isolated in Shandong province, and this is the first report of EV97in China. It had remote genetic relationship with other6EV97strains available in GenBank. A deletion of18nt in3’end of VP1coding region was observed.4. Complete genome and recombination analysis of Shandong newer HEVsThe complete genome sequence analysis of EV71, EV73, EV75, EV76, EV90, EV96and EV97revealed the evidence of recombination in non-structural region of Shandong newer HEV. In this region, Shandong EV71strains had higher similarities with CVA14proto G14strain and CVA16proto G10strain than with EV71proto BrCr; EV73and EV75Shandong strains had highest similarities with domestic Echo30strain Zhejiang/17/03/CSF; EV76Shandong strain had highest similarities with EV89and EV90proto strains; EV90Shandong strain01421had highest similarity with Finland strain F950027; EV97Shandong strain99188had highest similarities with Echo9strain DM and Echo30strain Zhejiang/17/03/CSF.Complete genome sequence analysis of EV90, EV96and EV97revealed deletions of nucleotide in capsid protein coding region, and the number of deletion was3or18nucleotides, separately, resulting in no frame shift. The position of deletion was the hot region of length variance in different serotypes, such as VP3or3’ end of VP1region. This implyed the nucleotide insertion or deletion might happen before the serotype alteration caused by accumulation of point mutation.[Conclusions and suggestions]1. In this study, several newer HEV serotypes were identified from HEV associated diseases. This is the first report of EV87, EV90, EV97and C2subgenotype of EV71in China’s mainland.Altogether213newer HEV strains of10serotypes were identified, as were EV71, EV73, EV74, EV75, EV76, EV80, EV87, EV90, EV96, and EV97. EV71came from AFP, HFMD and AM cases, and the other9serotypes came from AFP cases.2. Compared to other serotypes, EV71was the most common newer HEV serotypes, and it had resulted in the HFMD epidemic in China’s mainland since2007.Our work demonstrated EV71was the causative agent of HFMD outbreak in Linyi in2007, and revealed the existence of other subgenotypes of EV71in China’s mainland before the broad epidemic of C4subgenotype. More retrospective study will provide a broad view of circulation of other subgenotypes in China’s mainland. Also, EV71was an important causative agent of AM in Shandong province.3. Except EV71, other newer serotypes had low isolation rate, suggesting they had not circulated extensively in China yet.4. Great nucleotide divergence was observed between Shandong newer HEVs and other strains available in GenBank, suggesting a remote relationship. Nucleotide insertions and deletions participate in the evolution of HEV capsid protein region.5. Evidence of recombination was revealed in the complete genome analysis of Shandong newer HEV strains. More genome sequence data is needed to identify the exact donator.6. On account of the fact that newer HEV strains were frequently isolated in surveillance cases, we recommend the surveillance and molecular epidemiology study on newer HEVs should be strengthened in the future.更多还原