内蒙古自治区手足口病主要病原分子生物学特征和人肠道病毒71型致病病理学研究
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摘要
研究背景：手足口病（Hand,foot,andmouthdisease,HFMD）是由人肠道病毒（Humanenterovirus,HEV）引起的常见儿童急性传染病，以发热和手、足、口腔和臀部等部位出现皮疹为主要临床特征，少数病例出现脑炎、心肌炎、肺水肿、急性弛缓性麻痹等严重并发症，极少数情况下可引起死亡。HFMD可由许多血清型的HEV引起，但最主要的病原体为人肠道病毒71型（HEV71）和柯萨奇病毒A组16型（CVA16）。自1981年HFMD在中国上海首次报道以来，全国各地均有HFMD暴发及散发的报道。内蒙古自治区自2007年首次报道HFMD以来，疫情基本呈逐年递增的趋势，而且HFMD的重症病例和死亡病例的发病率也呈逐年增加，截至2010年全年累计报告HFMD病例18944例，重症病例247例，死亡13例。然而到目前为止，HFMD的致病机理还不确定，而且还没有安全有效的疫苗和药物用于预防和控制本病，因此加强该病的流行病学、病原学及基因特征和病理学研究具有非常重要的意义。为了更好的预防和控制HFMD，我们开展了相关内容的基础研究。
研究目的：为了了解内蒙古自治区HFMD的流行情况，了解HFMD主要致病病原体的流行本底，更好地预防和控制HFMD，我们开展了相关内容的和应用基础研究，旨在阐明和揭示内蒙古自治区HFMD的流行病学特点、HEV71和CVA16分子流行病学特征和HEV71重症病例的病理学特点。
主要方法：
（1）采用描述性流行病学方法对2008～2011年内蒙古自治区HFMD的流行病学特征进行分析。
（2）应用Real-timePCR方法对2010年12个盟市和2011年部分盟市门诊疑似病例的标本进行病原鉴定，然后进行病毒分离，应用Real-timePCR方法对病毒分离物进行鉴定，对鉴定为其它HEV的阳性分离物进行VP4编码区基因扩增及核苷酸序列测定和分析。从临床诊断分别为普通型病例、重型病例的HFMD患者临床标本中分离到的HEV71随机选取代表株进行VP1编码区基因扩增及核苷酸序列测定和分析，应用MAGE5.0软件分析内蒙古自治区流行的HEV71代表株与HEV71其它各基因型和基因亚型的代表株的亲缘进化关系；从选取的HEV71代表株中按年代和症状不同随机选取12株进行基因组全长测序和分析，应用SimPlot软件分析和MAGE5.0软件分析内蒙古自治区流行HEV71代表株与A组其他肠道病毒的相似性和亲缘进化关系。
（3）对2010年在12个盟市和2011年在部分盟市门诊采集的疑似HFMD病例的临床标本进行病毒分离，应用Real-timePCR方法对病毒分离物进行CVA16鉴定。从临床诊断分别为普通型病例、重型病例的手足口病患者临床标本中分离到的CVA16随机选取代表株进行VP1编码区基因扩增及核苷酸序列测定和分析，与CVA16其它各基因型和基因亚型的代表株和内蒙古2007年和2008年的代表株构建亲缘性进化树；从选取的CVA16代表株中按照症状的不同选取6株进行基因组全长序列测定和分析，应用SimPlot软件和MAGE5.0软件分析内蒙古自治区流行CVA16代表株与其他肠道病毒的相似性和亲缘进化关系。
（4）对2010年河南省1例HEV71引起的死亡病例进行尸体剖检，并将多个组织分别进行福尔马林处理和液氮保存，并分别进行病理学和免疫组织化学检测，初步探讨HEV71的致病机理。
研究结果：
（1）2010～2011年内蒙古自治区HFMD的发病率不同年份、月份和盟市之间不同，统计学分析差异显著；发病高峰主要集中在6～8月，比南方城市的高峰期推迟一个月；发病人群主要集中在1～10岁,尤其是1～4岁，占总发病人的82.33%；男性发病率高于女性发病率，构成比为1.5:1，统计学分析差异显著，同时盟市人口的发病率比旗县人口的发病率高，统计学分析差异显著。
（2）内蒙古自治区HFMD的主要病原是CVA16和HEV71，同时从个别HFMD患者的临床标本中分离出10株其他肠道病毒，重症病例中以HEV71为主；内蒙古自治区流行的HEV71代表株属于C4基因亚型C4a进化分支，并且存在多个传播链；内蒙古自治区流行的HEV71代表株与2008年北京代表株亲缘关系比2007年内蒙古代表亲缘关系近，说明内蒙古自治区流行HEV71不是独立进化的，而是与中国流行的HEV71在共同进化；2010～2011年流行的HEV71代表株与2008台湾代表株来源于同一个原始株，经重组和亲缘进化关系树分析发现，2010～2011年流行的HEV71代表株与A组肠道病毒发生了重组，并以非编码区和非结构蛋白区为主。
（3）内蒙古自治区流行的CVA16代表株均属于B1基因亚型的两大进化分支B1a和B1b，并且存在多个传播链，有两条优势传播链；与2009年山东、北京和2010年河南的代表株亲缘关系很近，说明内蒙古自治区流行的CVA16不是独立进化的，而是与中国流行的CVA16共同进化；2010年B1b分支的CVA16与2009年上海代表株来源于同一个原始株，属于B1a进化分支的CVA16与2011年泰国的代表株来源于同一个原始株，同时B1b分支的CVA16进化的比较快；经相似性和亲缘进化关系树分析发现，2010年流行的CVA16代表株与A组肠道病毒发生了重组，并以非编码区和非结构蛋白区为主，而且非结构蛋白编码的核苷酸与HEV71原型株同源性很近，提示CVA16与HEV71发生重组。
（4）HEV71死亡病例尸体眼观病变主要表现为：肺淤血、水肿，支气管管腔内可见灰白色黏稠液体；脑水肿，蛛网膜下腔充血、出血。主要病理组织学病变为：脑脊髓发生水肿、管套形成、卫星现象、嗜神经元现象，推测HEV71引起了非化脓性脑脊髓炎；肺水肿、肺泡结缔组织增生、肺泡腔内透明膜形成、粉红色浆液渗出、巨噬细胞和淋巴细胞浸润、肺泡Ⅱ型细胞脱落，推测HEV71引起了病毒性肺炎。该病例的主要受害器官是脑脊髓和肺脏。免疫组化检测，不但在神经组织中发现了HEV71抗原信号，还在肺、支气管、阑尾、胃、十二指肠、肝、空肠、脾、胸腺、胰、肾组织细胞中发现阳性信号。据此推测HEV71是一种泛嗜性病毒；该病毒可通过多种细胞内增殖（包括血管内皮细胞增殖）经血液循环在机体内完成扩散；主侵器官是脑脊髓和肺脏，直接致死原因是神经系统功能紊乱和呼吸衰竭。
结论：
（1）本文阐明了内蒙古自治区HFMD的流行病学规律，阐明了HFMD的发病时间、易感人群和地区分布与全国其它地区的异同点，以及其自身的规律性。
（2）本文确定了内蒙古自治区HFMD的病原谱以HEV71、CVA16为主，和兼有其他肠道病毒如CVB4参与致本病。
（3）揭示了CVA16和HEV71的主要分子生物学特征（包括分子流行病学、病毒基因特征、进化关系等）。
（4）丰富了HEV71致病的病理学特征，并在神经组织、肺脏、支气管、胃肠道、肝脏、胰腺、肾脏和脾脏内发现了HEV71抗原信号。
BACKGROUND:Hand, foot and mouth disease(HFMD) is a common childhood acute infection,which is caused by Human enterovirus viruses, HFMD is characterized by fever, rash on the hands and feet, and mouth ulcers, occasionally accompanied by encephalitis, myocarditis, pulmonary edema, acute flaccid paralysis and other serious complications, and very few cases can lead to death. It is most commonly associated with the invasionof human enterovirus71(HEV71) and Coxsackievirus A type16. Since the first reported of HEV71in Shanghai in1981, there have been sporadic outbreaks in china. The first outbreak of HFMD was reported in Inner Mongolia Autonomous Region in2007. The prevalence of HFMD has increased greatly since2008,Up to2010, there were18944cases were reported in all, which contained247severe cases and13dead cases.But so far, the pathogensis of HFMD is uncertain, the safety and effective vaccine and drugs used for prevention and control HFMD has not been developed.
OBJECTIVE:In order to prevent and control HFMD more effectively in Inner Mongolia, the characteristics of epidemiology, molecular biology of major pathogen of HFMD and the pathologic changes in a serious case caused by HEV71were studied in this thesis.
METHODS:
(1)Descriptive epidemiology method was used to analyze the epidemiology characteristics of HFMD patients in2008-2011in the Inner Mongolia, and the statistical software and geographic information system.
(2)The clinical specimens including stools and throat swabs were collected from HFMD patients in outpatient service in2010-2011in Inner Mongolia, which were identified by using the real-time PCR method and then viral isolation was performed, the positive viral isolates were identified by using the real-time PCR method (detecting EV, HEV71and CVA16in a single tube), and VP4and VP1coding region amplification and sequencing was performed with the viral isolates that were identified as non-HEV71, non-CVA16HEVs. The VP1coding regions of representative HEV71isolates that selected from the patients presenting mild symptoms and the patients presenting severe symptoms randomly in2010-2011were amplified and sequenced, and then MAGE5.0software were used to analyze evolutionary relationship between representative HEV71strains and repredentative strains of each genotype/subgenotype. The full-lenth genome of12representative HEV71isolates that selected selected from the patients presenting mild symptoms and the patients presenting severe symptoms randomly in2010-2011were amplified and sequenced, and then MAGE5.0, BioEdit and Simplot software were used to analyze evolutionary and recombinztion relationship between representative HEV71strains and repredentative strains of A genotype.
(3)The clinical specimens including stools, throat swabs and vesicle fluids were collected from HFMD patients in outpatient service in2010-2011in Inner Mongolia and then viral isolation was performed, the positive viral isolates were identified by using the real-time PCR method detecting CVA16; The VP1coding regions of represented CVA16isolates that selected from the patients presenting mild symptoms, severe symptoms and the death patients randomly were amplified and sequenced. Finally the phylogenetic tree was constructed among the VP1coding regions of the different genotypes and subgenotypes of CVA16strains, and MAGE5.0software were used to analyze evolutionary relationship; The full-lenth genome of6representative CVA16isolates that selected selected from the patients presenting mild symptoms, severe symptoms and the death patients randomly in2010-2011were amplified and sequenced, and then MAGE5.0, BioEdit and Simplot software were used to analyze evolutionary and recombinztion relationship between representative CVA16strains and repredentative strains of A genotype.
(4) One serious dead case caused by HEV71was collected in Henan province in2010. The major organs were stored through formalin-fixation and liquid nitrogen respectively. An immunohistochemical techniques and histological section examination were performed to analyse the pathological features and HEV71antigen distribution.
RESULTS:
(1) The incidence of HFMD was sigenificant difference in difference years, months and cities during2008to2011in Inner Mongolia(P<0.05); The HFMD cases were reported in every month and the peak of incidence were abserved during June to July; the cases of HFMD were mainly scattered children and kindergarten children of10years old, especially children of5years old or younger; the male incidence was higher than female, the difference was significant(P<0.05); city incidence is higher than the incidence of towns, the difference was significant(P<0.05); CVA16and HEV71were the major pathogen of HFMD in Inner Mongolia, account for98.28%.
(2) HEV71and CVA16were the main pathogens of HFMD in Inner Mongolia during2010-2011,10HEVs isolation was isolated from the case of HFMD and most severe cases were caused by HEV71. The HEV71strains circulated in Inner Mongolia were all belong to C4a evolution branch within C4subgenotype. Phylogenetic analysis revealed that Inner Mongolia HEV71strains during2010～2011located in different lineages, and has larger nucleotide identity with2008Beijing HEV71strains than2007Inner Mongolia HEV71strains. This indicated that Inner Mongolia HEV71strains had not evolved independently, but co-evolved with the HEV71strains in other provinces in mainland China; The Inner Mongolia HEV71strains have the same ancestor to the Taiwan stains; Similarity plot and bootscan analyses revealed recombination between the Inner Mongolia HEV71strains and prototype stains in enterovirus A group, especially the non-encoding and non-protein region.
(3) CVA16strains isolated from different clinical symptoms of HFMD cases in the Inner Mongolia Autonomous Region during2010-2011belong to Bla and B1b evolution branch of B1genotype and the nucleotide acid of represented CVA16strains in Inner Mongolia were closed to CVA16strains isolated from mainland China since1998. This indicated that two evolutionary branch of CVA16in the Inner Mongolia Autonomous Region co-evolve and co-prevailed; The Inner Mongolia CVA16strains belong to the B1b evolution branch of B1genotype have the same ancestor to the Beijing stains, and has the Inner Mongolia CVA16strains belong to the B1a evolution branch of B1genotype have the same ancestor to the Thailand stains, Similarity plot and bootscan analyses revealed recombination between the Inner Mongolia HEV71 strains and prototype stains in enterovirus A group, especially the non-encoding and non-protein region, and there was a little difference in the nucleotide acid between the Inner Mongolia CVA16strains and the prototype HEV71stain, this revealed recombination between the Inner Mongolia CVA16strains and prototype HEV71stains.
(4) The dead case causing by HEV71was studied systematically. At necropsyt the changes mainly appeared in nervous system and respiratory system. Respiratory system showed congestion and edema of lungs,and the endotracheal white viscous liquid The nervous system showed brain swollen, subarachnoid hyperemia and hemorrhage. Histopathologically, the lesions were characterized by non-suppurative encephalomyelitis and viral pneumonia. The former included edema,lymphotic cuffing earound blood vessels, satellite phenomenon and neurotropic phenomenon. The later included pulmonary edeama, interstitial wideness of alveolar wall,hyaline membrane forming in alveolar cavity, a lot of macrophages, lymphocytes,pink serous exudation, and desquamated alveolar type II cells in alveolar cavity. Immunohistochemicaly,HEV71antigen signal was detected not only in nervous system,but also in lung, bronchus,stamoch, liver,duodenumjejunum, vermiform appendix,thymus, pancreas and kidney in this study.It is inferred that the HEV71is a pantropic virus; spread of the virus in the body was complicated through proliferation whithin cells(lnclede vascular enduthelium)and blood circulation;the main invasive organs were brain,spinal cord and lungs;the direct cause for death was disfunction of nervous system and respiratory failure.
CONCLUSIONS:
(1) Clarified the epidemiology of HFMD in Inner Mongolia during2008～2011, it was that the time of onset, susceptible populations and geographical distribution were regularity.
(2) The HFMD in Inner Mongolia pathogen spectrum was mainly HEV71, CVA16and other enterovirus.
(3) Revealed the major biological charateristics(including molecular epidemiology, genetic characteristics and evolutionary relationships) of HEV71and CVA16.
(4)Besides nervous system,HEV71antigen signal was detected in lung, bronchus,stamoch, liver,duodenum,jejunum, vermiform appendix,thymus, pancreas and kidney by immunohistochemical staining.
研究目的：为了了解内蒙古自治区HFMD的流行情况，了解HFMD主要致病病原体的流行本底，更好地预防和控制HFMD，我们开展了相关内容的和应用基础研究，旨在阐明和揭示内蒙古自治区HFMD的流行病学特点、HEV71和CVA16分子流行病学特征和HEV71重症病例的病理学特点。
主要方法：
（1）采用描述性流行病学方法对2008～2011年内蒙古自治区HFMD的流行病学特征进行分析。
（2）应用Real-timePCR方法对2010年12个盟市和2011年部分盟市门诊疑似病例的标本进行病原鉴定，然后进行病毒分离，应用Real-timePCR方法对病毒分离物进行鉴定，对鉴定为其它HEV的阳性分离物进行VP4编码区基因扩增及核苷酸序列测定和分析。从临床诊断分别为普通型病例、重型病例的HFMD患者临床标本中分离到的HEV71随机选取代表株进行VP1编码区基因扩增及核苷酸序列测定和分析，应用MAGE5.0软件分析内蒙古自治区流行的HEV71代表株与HEV71其它各基因型和基因亚型的代表株的亲缘进化关系；从选取的HEV71代表株中按年代和症状不同随机选取12株进行基因组全长测序和分析，应用SimPlot软件分析和MAGE5.0软件分析内蒙古自治区流行HEV71代表株与A组其他肠道病毒的相似性和亲缘进化关系。
（3）对2010年在12个盟市和2011年在部分盟市门诊采集的疑似HFMD病例的临床标本进行病毒分离，应用Real-timePCR方法对病毒分离物进行CVA16鉴定。从临床诊断分别为普通型病例、重型病例的手足口病患者临床标本中分离到的CVA16随机选取代表株进行VP1编码区基因扩增及核苷酸序列测定和分析，与CVA16其它各基因型和基因亚型的代表株和内蒙古2007年和2008年的代表株构建亲缘性进化树；从选取的CVA16代表株中按照症状的不同选取6株进行基因组全长序列测定和分析，应用SimPlot软件和MAGE5.0软件分析内蒙古自治区流行CVA16代表株与其他肠道病毒的相似性和亲缘进化关系。
（4）对2010年河南省1例HEV71引起的死亡病例进行尸体剖检，并将多个组织分别进行福尔马林处理和液氮保存，并分别进行病理学和免疫组织化学检测，初步探讨HEV71的致病机理。
研究结果：
（1）2010～2011年内蒙古自治区HFMD的发病率不同年份、月份和盟市之间不同，统计学分析差异显著；发病高峰主要集中在6～8月，比南方城市的高峰期推迟一个月；发病人群主要集中在1～10岁,尤其是1～4岁，占总发病人的82.33%；男性发病率高于女性发病率，构成比为1.5:1，统计学分析差异显著，同时盟市人口的发病率比旗县人口的发病率高，统计学分析差异显著。
（2）内蒙古自治区HFMD的主要病原是CVA16和HEV71，同时从个别HFMD患者的临床标本中分离出10株其他肠道病毒，重症病例中以HEV71为主；内蒙古自治区流行的HEV71代表株属于C4基因亚型C4a进化分支，并且存在多个传播链；内蒙古自治区流行的HEV71代表株与2008年北京代表株亲缘关系比2007年内蒙古代表亲缘关系近，说明内蒙古自治区流行HEV71不是独立进化的，而是与中国流行的HEV71在共同进化；2010～2011年流行的HEV71代表株与2008台湾代表株来源于同一个原始株，经重组和亲缘进化关系树分析发现，2010～2011年流行的HEV71代表株与A组肠道病毒发生了重组，并以非编码区和非结构蛋白区为主。
（3）内蒙古自治区流行的CVA16代表株均属于B1基因亚型的两大进化分支B1a和B1b，并且存在多个传播链，有两条优势传播链；与2009年山东、北京和2010年河南的代表株亲缘关系很近，说明内蒙古自治区流行的CVA16不是独立进化的，而是与中国流行的CVA16共同进化；2010年B1b分支的CVA16与2009年上海代表株来源于同一个原始株，属于B1a进化分支的CVA16与2011年泰国的代表株来源于同一个原始株，同时B1b分支的CVA16进化的比较快；经相似性和亲缘进化关系树分析发现，2010年流行的CVA16代表株与A组肠道病毒发生了重组，并以非编码区和非结构蛋白区为主，而且非结构蛋白编码的核苷酸与HEV71原型株同源性很近，提示CVA16与HEV71发生重组。
（4）HEV71死亡病例尸体眼观病变主要表现为：肺淤血、水肿，支气管管腔内可见灰白色黏稠液体；脑水肿，蛛网膜下腔充血、出血。主要病理组织学病变为：脑脊髓发生水肿、管套形成、卫星现象、嗜神经元现象，推测HEV71引起了非化脓性脑脊髓炎；肺水肿、肺泡结缔组织增生、肺泡腔内透明膜形成、粉红色浆液渗出、巨噬细胞和淋巴细胞浸润、肺泡Ⅱ型细胞脱落，推测HEV71引起了病毒性肺炎。该病例的主要受害器官是脑脊髓和肺脏。免疫组化检测，不但在神经组织中发现了HEV71抗原信号，还在肺、支气管、阑尾、胃、十二指肠、肝、空肠、脾、胸腺、胰、肾组织细胞中发现阳性信号。据此推测HEV71是一种泛嗜性病毒；该病毒可通过多种细胞内增殖（包括血管内皮细胞增殖）经血液循环在机体内完成扩散；主侵器官是脑脊髓和肺脏，直接致死原因是神经系统功能紊乱和呼吸衰竭。
结论：
（1）本文阐明了内蒙古自治区HFMD的流行病学规律，阐明了HFMD的发病时间、易感人群和地区分布与全国其它地区的异同点，以及其自身的规律性。
（2）本文确定了内蒙古自治区HFMD的病原谱以HEV71、CVA16为主，和兼有其他肠道病毒如CVB4参与致本病。
（3）揭示了CVA16和HEV71的主要分子生物学特征（包括分子流行病学、病毒基因特征、进化关系等）。
（4）丰富了HEV71致病的病理学特征，并在神经组织、肺脏、支气管、胃肠道、肝脏、胰腺、肾脏和脾脏内发现了HEV71抗原信号。
BACKGROUND:Hand, foot and mouth disease(HFMD) is a common childhood acute infection,which is caused by Human enterovirus viruses, HFMD is characterized by fever, rash on the hands and feet, and mouth ulcers, occasionally accompanied by encephalitis, myocarditis, pulmonary edema, acute flaccid paralysis and other serious complications, and very few cases can lead to death. It is most commonly associated with the invasionof human enterovirus71(HEV71) and Coxsackievirus A type16. Since the first reported of HEV71in Shanghai in1981, there have been sporadic outbreaks in china. The first outbreak of HFMD was reported in Inner Mongolia Autonomous Region in2007. The prevalence of HFMD has increased greatly since2008,Up to2010, there were18944cases were reported in all, which contained247severe cases and13dead cases.But so far, the pathogensis of HFMD is uncertain, the safety and effective vaccine and drugs used for prevention and control HFMD has not been developed.
OBJECTIVE:In order to prevent and control HFMD more effectively in Inner Mongolia, the characteristics of epidemiology, molecular biology of major pathogen of HFMD and the pathologic changes in a serious case caused by HEV71were studied in this thesis.
METHODS:
(1)Descriptive epidemiology method was used to analyze the epidemiology characteristics of HFMD patients in2008-2011in the Inner Mongolia, and the statistical software and geographic information system.
(2)The clinical specimens including stools and throat swabs were collected from HFMD patients in outpatient service in2010-2011in Inner Mongolia, which were identified by using the real-time PCR method and then viral isolation was performed, the positive viral isolates were identified by using the real-time PCR method (detecting EV, HEV71and CVA16in a single tube), and VP4and VP1coding region amplification and sequencing was performed with the viral isolates that were identified as non-HEV71, non-CVA16HEVs. The VP1coding regions of representative HEV71isolates that selected from the patients presenting mild symptoms and the patients presenting severe symptoms randomly in2010-2011were amplified and sequenced, and then MAGE5.0software were used to analyze evolutionary relationship between representative HEV71strains and repredentative strains of each genotype/subgenotype. The full-lenth genome of12representative HEV71isolates that selected selected from the patients presenting mild symptoms and the patients presenting severe symptoms randomly in2010-2011were amplified and sequenced, and then MAGE5.0, BioEdit and Simplot software were used to analyze evolutionary and recombinztion relationship between representative HEV71strains and repredentative strains of A genotype.
(3)The clinical specimens including stools, throat swabs and vesicle fluids were collected from HFMD patients in outpatient service in2010-2011in Inner Mongolia and then viral isolation was performed, the positive viral isolates were identified by using the real-time PCR method detecting CVA16; The VP1coding regions of represented CVA16isolates that selected from the patients presenting mild symptoms, severe symptoms and the death patients randomly were amplified and sequenced. Finally the phylogenetic tree was constructed among the VP1coding regions of the different genotypes and subgenotypes of CVA16strains, and MAGE5.0software were used to analyze evolutionary relationship; The full-lenth genome of6representative CVA16isolates that selected selected from the patients presenting mild symptoms, severe symptoms and the death patients randomly in2010-2011were amplified and sequenced, and then MAGE5.0, BioEdit and Simplot software were used to analyze evolutionary and recombinztion relationship between representative CVA16strains and repredentative strains of A genotype.
(4) One serious dead case caused by HEV71was collected in Henan province in2010. The major organs were stored through formalin-fixation and liquid nitrogen respectively. An immunohistochemical techniques and histological section examination were performed to analyse the pathological features and HEV71antigen distribution.
RESULTS:
(1) The incidence of HFMD was sigenificant difference in difference years, months and cities during2008to2011in Inner Mongolia(P<0.05); The HFMD cases were reported in every month and the peak of incidence were abserved during June to July; the cases of HFMD were mainly scattered children and kindergarten children of10years old, especially children of5years old or younger; the male incidence was higher than female, the difference was significant(P<0.05); city incidence is higher than the incidence of towns, the difference was significant(P<0.05); CVA16and HEV71were the major pathogen of HFMD in Inner Mongolia, account for98.28%.
(2) HEV71and CVA16were the main pathogens of HFMD in Inner Mongolia during2010-2011,10HEVs isolation was isolated from the case of HFMD and most severe cases were caused by HEV71. The HEV71strains circulated in Inner Mongolia were all belong to C4a evolution branch within C4subgenotype. Phylogenetic analysis revealed that Inner Mongolia HEV71strains during2010～2011located in different lineages, and has larger nucleotide identity with2008Beijing HEV71strains than2007Inner Mongolia HEV71strains. This indicated that Inner Mongolia HEV71strains had not evolved independently, but co-evolved with the HEV71strains in other provinces in mainland China; The Inner Mongolia HEV71strains have the same ancestor to the Taiwan stains; Similarity plot and bootscan analyses revealed recombination between the Inner Mongolia HEV71strains and prototype stains in enterovirus A group, especially the non-encoding and non-protein region.
(3) CVA16strains isolated from different clinical symptoms of HFMD cases in the Inner Mongolia Autonomous Region during2010-2011belong to Bla and B1b evolution branch of B1genotype and the nucleotide acid of represented CVA16strains in Inner Mongolia were closed to CVA16strains isolated from mainland China since1998. This indicated that two evolutionary branch of CVA16in the Inner Mongolia Autonomous Region co-evolve and co-prevailed; The Inner Mongolia CVA16strains belong to the B1b evolution branch of B1genotype have the same ancestor to the Beijing stains, and has the Inner Mongolia CVA16strains belong to the B1a evolution branch of B1genotype have the same ancestor to the Thailand stains, Similarity plot and bootscan analyses revealed recombination between the Inner Mongolia HEV71 strains and prototype stains in enterovirus A group, especially the non-encoding and non-protein region, and there was a little difference in the nucleotide acid between the Inner Mongolia CVA16strains and the prototype HEV71stain, this revealed recombination between the Inner Mongolia CVA16strains and prototype HEV71stains.
(4) The dead case causing by HEV71was studied systematically. At necropsyt the changes mainly appeared in nervous system and respiratory system. Respiratory system showed congestion and edema of lungs,and the endotracheal white viscous liquid The nervous system showed brain swollen, subarachnoid hyperemia and hemorrhage. Histopathologically, the lesions were characterized by non-suppurative encephalomyelitis and viral pneumonia. The former included edema,lymphotic cuffing earound blood vessels, satellite phenomenon and neurotropic phenomenon. The later included pulmonary edeama, interstitial wideness of alveolar wall,hyaline membrane forming in alveolar cavity, a lot of macrophages, lymphocytes,pink serous exudation, and desquamated alveolar type II cells in alveolar cavity. Immunohistochemicaly,HEV71antigen signal was detected not only in nervous system,but also in lung, bronchus,stamoch, liver,duodenumjejunum, vermiform appendix,thymus, pancreas and kidney in this study.It is inferred that the HEV71is a pantropic virus; spread of the virus in the body was complicated through proliferation whithin cells(lnclede vascular enduthelium)and blood circulation;the main invasive organs were brain,spinal cord and lungs;the direct cause for death was disfunction of nervous system and respiratory failure.
CONCLUSIONS:
(1) Clarified the epidemiology of HFMD in Inner Mongolia during2008～2011, it was that the time of onset, susceptible populations and geographical distribution were regularity.
(2) The HFMD in Inner Mongolia pathogen spectrum was mainly HEV71, CVA16and other enterovirus.
(3) Revealed the major biological charateristics(including molecular epidemiology, genetic characteristics and evolutionary relationships) of HEV71and CVA16.
(4)Besides nervous system,HEV71antigen signal was detected in lung, bronchus,stamoch, liver,duodenum,jejunum, vermiform appendix,thymus, pancreas and kidney by immunohistochemical staining.
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