中国华东地区基因Ⅳ型戊型肝炎病毒人畜共患与进化起源研究

【作者】 陆一涵；

【导师】 姜庆五； 郑英杰；

【作者基本信息】 复旦大学 ， 流行病与卫生统计学， 2009， 博士

【摘要】 戊型肝炎病毒（Hepatitis E Virus,HEV）主要通过粪一口途径传播,可以引起急性肝炎,人群普遍易感。HEV主要有两种流行模式:一种是由水源污染引起的大规模爆发流行,主要发生于卫生条件相对较差的发展中国家和地区,由基因Ⅰ型和Ⅱ型引起;另一种则是散发和小范围的爆发,在全球范围内广泛存在。其中,存在动物宿主的基因Ⅲ型和Ⅳ型HEV引起的散发病例较为普遍,多数怀疑与食源性感染有关。我国为戊肝高发区,迄今为止世界上最大的一次戊肝爆发发生于1986—1988年的我国新疆南部地区。2000年以来,虽然未再有大规模的爆发流行,但全国法定报告的肠道传染病中仅戊肝病例持续上升,表现为静默流行。近年来,基因Ⅳ型HEV已经逐渐成为我国大陆地区HEV的主导基因型。其宿主动物（主要为猪群）数量庞大、分布广泛,猪源性HEV进化速率亦较快。因此,基因Ⅳ型HEV在人群中的流行是否会因为其人畜共患性特征（例如种间传播）而“凸现”为更大的流行值得关注。本项研究将流行病学方法和生物信息学方法结合在一起,通过分析基因Ⅳ型HEV随着时间而自然进化和变异的客观规律,揭示其进化起源和流行趋势,既为HEV的流行预警提供科学依据,又可以为建立针对HEV的特异性防治措施积累资料,对于其它感染性疾病的相关研究也具有一定的借鉴作用。本项研究以华东地区上海、浙江德清、安徽安庆和江苏海门为研究现场,开展戊型肝炎流行病学调查和商品猪群HEV感染率横断面调查,分析人戊肝患者与感染HEV猪群的“三间分布”特征,比较两个群体中HEV RNA检测情况的时间分布一致性。在此基础上,分离人与猪HEV序列,分析华东地区HEV的主导基因型。然后进行同源性比对,分析同源性相似程度的“三间分布”特征。最后,结合“松散的”分子钟理论和基于MCMC算法的Bayesian统计推断方法,拟合核苷酸置换模型,计算人HEV和猪HEV的进化速率,推算各自的进化分歧时间,重建进化历史。同时,估算HEV理论感染人群数与猪群数随时间变化的趋势,掌握HEV的流行规律。华东地区戊肝临床流行病学调查显示,该地区的散发性戊肝主要由基因Ⅳ型HEV引起。男性多于女性;患者年龄集中于30—70岁年龄段,其中1/3集中于50—60岁之间。戊肝病例表现出明显的时间聚集特征,每年1月—4月是戊肝发病高峰期,占全部病例数的60%以上;5月—6月、11月—12月亦是戊肝多发时期。戊肝住院患者入院后的首份血清（采集时间中位数为9天）HEV RNA检出率最高（55%—64%）,第二份、第三份（采集时间中位数分别为19.5天、24.5天）血清的检出率则显著下降（28%—38%和4%—8%）。按照单份血清与系列血清的检测结果推算戊肝患者发病后的病毒血症持续时间,中位数分别为21天和29天,表明50%的戊肝患者在这段时间内病毒血症已消失;但其余50%的患者仍然存在病毒血症、可以成为传染源。对华东地区商品猪群HEV感染率的横断面研究证实,基因Ⅳ型HEV在猪群中常年广泛流行,从出生后约1月龄开始,一直到商品猪出栏、屠宰,以及母猪产仔时均能感染HEV。但各个月龄段的猪群感染率并不相同。其中,4—6月龄的商品猪HEV感染率最高,粪便HEV RNA检出率为33%—42%,胆汁检出率为4.8%—5.6%。另一方面,华东地区猪HEV感染率同时表现出地区性和季节性差异。各研究现场的胆汁HEV RNA两种片段检出率分别为3.7%—9%和2.7%—6%,沿长江地区研究现场的检出率较高,远离长江地区的则最低。季节性差异表现为9月—10月检出率最高（6%—8%）,5月—6月最低（3.7%—5%）,反映了猪HEV传播效力的季节变化规律。同时,猪HEV RNA检出率曲线与人戊肝病例季节分布的变化趋势相似,但前者的高峰与低谷均比后者提前约3个月,提示存在HEV从猪群向人群传播的可能。华东地区基因Ⅳ型HEV核苷酸同源性为84%—85%。其中,人HEV序列变异度较大,核苷酸置换主要为转换（C←→T）;而猪HEV序列变异度较小,颠换与转换发生概率间的差异也小。总体来看,华东地区分离的人HEV之间、猪HEV之间、以及人HEV与猪HEV之间同源性都较高,未表现出明显的宿主、时间和地区聚集特征。分离的Ⅳ型株划分为9个主要分支,组内和组间同源性分别为94%—98%、81%—90%。每一个分支均同时包含人HEV和猪HEV;部分人HEV与猪HEV划分于同一末端分支中,进化起源关系密切,支持HEV种间传播的观点。本项研究中,估算华东地区人与猪基因Ⅳ型HEV的进化速率分别为1.63×10^-3/位点/年、6.94×10^-3/位点/年,首次证明19世纪早期基因Ⅳ型HEV已在华东地区传播,这与中国近代沿海口岸开放时间一致,提示外源性传入我国的可能。沿长江地区人HEV进化分歧时间较早,远离长江的则较晚;但各地猪HEV分歧时间相近、且猪HEV进化速率远高于人HEV,提示最初的HEV感染者可能是人,通过人→猪→人的双向传播模式,使病毒在华东地区进一步扩散开来。重建HEV种群演变过程并分析理论感染宿主数量变化趋势后发现,华东地区基因Ⅳ型HEV理论感染人群数自20世纪90年代以来持续上升,感染猪群数则在近年来缓慢下降。由于人HEV与猪HEV的种群增长率均较快,同时基因Ⅳ型HEV还存在双向传播的风险,因此未来一段时间内人戊肝疫情仍将维持较高水平。更多还原

【Abstract】 Hepatitis E virus （HEV） is a pathogen of fecal-oral transmission which accounts for acute hepatitis E （HE） in the general population. HEV can lead to large-scale epidemics due to fecal contamination of drinking water in developing countries with poor sanitation as well as smaller outbreaks and sporadic cases over the world. Sporadic cases induced by zoonotic genotype-III and genotype-IV HEV are increasingly popular, which are maybe attributable to food-borne transmission.China is the area highly endemic for HEV. The largest outbreak of HEV worldwide occurred in Xinjiang Uygur Autonomous Region （XUAR） between 1986 and 1988. According to the bulletins on national epidemic situation issued by Ministry of Health of People’s Republic of China, HE is the only increasing one among legally-reported enterically-transmitted diseases domestically from 2000 to 2008. In China Mainland, genotype-IV HEV has been dominant and swine is proved to be a principle reservoir. The epidemic situation of genotype-IV HEV in human population could be possibly affected by zoonotic transmission with swine HEV, which is widespread in numerous herds and evolves rapidly. The present study is designed to explore the evolutionary and demographic history of genotype-IV HEV using the combination of epidemiology and bioinformatics. It would contribute to forecasting of HE epidemic and establishment of prophylaxis towards HEV. The study could also be used for reference to the evolutionary analysis of other pathogens.Based on the clinical epidemiological investigation of HE cases and cross-section study within swine herds, the present study was conducted in Eastern China, including Shanghai, Deqing （Zhejiang Province）, Anqing （Anhui Province） and Haimen （Jiangsu Province）. Initially, the distribution of time, region, and population within human HE cases and HEV-infected pigs was evaluated and the temporal identity of HEV prevalences between them was determined. Then human HEV and swine HEV were isolated for phylogenetic reconstruction. Finally, the nucleotide substitution rate and time to most recent common ancestor （tMRCA） was calculated using relaxed molecular clock theory and Bayesian inference based on Markov chain Monte Carlo （MCMC）. The host population （human and swine） dynamics was inferred using best-fit demographic model.Sporadic HE cases in Eastern China are mainly induced by genotype-IV HEV. There were more male patients than female ones （2.82:1）. The majority （84%） of the cases ranged from 30 to 70 years old, in which 1/3 was 50-60 years old. The temporal distribution was demonstrated by the concentration of cases （63%） between Jan and Apr. The positive rate of HEV RNA in the first-sampled serum （sampled within 0-45 days after the onset, median was 9 days） was 55%-64%. But it declined rapidly to 28%-38% in the second-sampled one （sampled within median time of 19.5 days after the onset） and 4%-8% in third-sampled one （sampled within median time of 24.5 days after the onset）, respectively. Employing Kaplan-Meier survival analysis, median detection time was estimated as 21 days and 29 days after the onset based on the temporal prevalence of HEV RNA in singly-sampled serum and serially-sampled ones respectively. It meant that the viremia in 50% of HE patients could remain detectable up to 21-29 days after the onset. It is implied that HE patient could still act as reservoir even after the duration of biochemical hepatitis.Genotype-IV HEV is also widely prevalent in swine herds in Eastern China. HEV RNA could be detected throughout the productive life of the pigs but the prevalence varied with time of life. It was the highest in the swine herds of 4-6 months, amounting to 33%-42% in feces and 4.8%-5.6% in biles respectively. Significant difference of HEV detection was observed in geographic distribution. The detection rates of Shanghai, Anqing and Haimen which all locate along the Yangtze River, were higher than that of Deqing which is far from the Yangtze River. Seasonal pattern was also demonstrated by the highest prevalence of 6%-8% between Sep and Oct and lowest one of 3.7%-5% between May and Jun. It could be interpreted as the seasonal pattern of swine HEV transmission. Interestingly, there was great similarity between the dynamics of swine HEV detection and human HE cases, but temporal distribution of the former is ahead of that of the latter by 3 months. Cross-species transmission of HEV was implied.In the present study, the HEV strains isolated in Eastern China were all grouped into genotype-IV and of 84%-85% identities with each other. The genetic diversity among human HEV isolates with great probability of C←→T transition was higher than that among swine HEV isolates with closer probabilities of transition and transversion. Regardless of isolation from different regions, human and swine genotype-IV HEV shared high similarity. All the isolates were clustered into 9 groups with identities of 94%-98% and 81%-90% within and between the groups. Each cluster was consisted of human HEV as well as swine HEV. It was noted that some human and swine isolates were clustered with bootstrap values of almost 100% and posterior probability of 1, in which cross-species transmission was demonstrated.Based on the general GTR model of nucleotide substitution and a relaxed molecular clock, Bayesian Skyline Plot （BSP） gave the evolutionary rate of 1.63×10^-3 substitutions/site/year and 6.94×10^-3 substitutions/site/year for human HEV and swine HEV respectively, which corresponded to estimated date for tMRCA of 1820s and 1970s. It was the first time to determine that genotype-IV HEV had been spreading since 1820s in Eastern China, which was consistent with the opening of local ports for trade with the outside world in Qing Dynasty. Exotic importation of HEV was implied. The tMRCAs of human HEV in the regions locating along the Yangtze River are earlier than that in the region far from the Yangtze River. But the tMRCAs of swine HEV in different regions were similar and much later than the tMRCA of human HEV. It suggested that original reservoir in Eastern China was maybe human beings. After a long time of circulation in human population the virus invaded pig herds, cross-species and bilateral transmission occurred consequently. The present study also estimated and reconstructed the demographics of genotype-IV HEV. The effective number of human population infected with genotype-IV HEV in Eastern China increased remarkably since 1990s but that of swine population infected decreased in recent years. Attributable to both of the fast growth rates of genotype-IV HEV isolated from human and swine and the risk of cross-species transmission, the epidemic of HEV locally will still remain considerable.更多还原