【作者】 施强；

【导师】 姜庆五； 居丽雯； 蒋露芳；

【作者基本信息】 复旦大学 ， 流行病与卫生统计学， 2010， 博士

【摘要】 流感长期以来一直是威胁人类健康的重要呼吸道病原体之一,流感的基因变异及重组所导致的抗原漂移和抗原转换是流感出现流行或者暴发的基础,虽然已有很多研究对流感的基因变异进行探讨,但是对流感病毒的进化情况和流行动力学的许多方面的认识仍然是未知的,特别对流感病毒的跨时空的变异缺乏精确的测量。在北半球流感发病存在如时钟般准确的流行季节高峰,是什么因素驱使流感出现明显的季节性高峰却众说纷纭,特别是关于基因变异和流感季节性高峰间的关系研究基本缺乏。本研究采用合理的流行病学和生物信息学方法,对流感的基因变异情况和流感季节性发病高峰的关系进行探讨,主要包括以下5个方面内容：1.在上海地区建立合适的流感监测点和网络,长期收集监测网络医院的类流感病例发病情况,利用时间序列分析流感的季节性发病高峰分布特征。结果显示：上海地区的流感监测病例每年存在2次季节高峰,两次高峰间的距离为6个月,和中国北方地区只有1次流行高峰且间隔为12个月不同。2.从研究现场的监测系统采集标本并分离流感病毒后并按照一定条件选择甲型流感病毒株进行HA、NA和PB2基因的全基因测序。利用基于MCMC抽样技术的Bayesian分析分别完成流感病毒HA, NA和PB2基因的跨时间变异情况分析,结合已有的流行病学资料,对人甲型流感3种重要基因变异情况进行全面的分析。结果显示甲型流感2个不同亚型的基因变异速度是不一致的,总的说来,H3N2亚型进化速度比较快,而H1N1的进化速度相对较慢。在同一个亚型中,一般是HA的进化速度比NA快,PB2最慢,这和病毒为了逃避宿主的免疫清除而进行的适应性变异紧密相关。在大部分时间中,H3N2亚型基因变异的程度相对较高,而H1N1亚型的基因变异程度相对较低,即使在占据优势的2004和2005年,其变异高峰也只有H3N2最高峰的三分之一不到。这从另外一方面证实H1N1亚型保存相对稳定,不容易突破人体的免疫屏障和H3N2亚型对其的压制。病毒基因变异程度高,并不意味着其传播能力的提高,需结合基因对流感发病造成的理论感染人数数据等数据进行综合的评估才能得到更准确的结果。3.选择典型的病毒株测试其对流感最有效药物是否出现耐受,间接证明病毒的基因变异是否有产生明显的抗原变异。发现基因变异并没有导致病毒出现抗原根本性的转变,以点突变和适应性变异为主要特点。4.对禽类相关从业人员血清进行检测,探索某些其他宿主病毒感染人可能的途径和人群对流感的易感性情况。结果表明人群对其他宿主来源的流感病毒普遍易感,一般人群和接触人群中均存在H9亚型流感病毒HAI抗体,接触人群H9抗体阳性率是17.9%,普通人群也达到2.6%。接触人群感染H9的危险性是普通人群的3.392倍。此外两个人群的H3N2和H1N1的抗体滴度并没有差别。在大部分受调查人群都没有接种疫苗的情况下,但是抗体滴度都相对较高,特别是变异速度比较快的H3N2亚型,抗体阳性率都为30%左右。5.探索甲型流感季节高峰分布和病毒主要基因变异间的关联情况,为流感的科学有效防治提供可靠的依据。甲型流感的基因变异在每个观察年的冬季出现一个明显的高峰,与监测病例每年2个高峰且冬春季高峰跨年不同,基因变异的高峰只有一个,且在流感监测病例的2个高峰间。结果表明,在一个相对稳定的环境中,病毒主要是在宿主的免疫压力驱动下产生适应性突变后造成一个新的流行。综合上述结果,可以认为上海地区存在流感发病的季节性高峰,流感的基因变异在短期内主要以基因的点突变和适应性变异为主,并没有出现明显的表型改变从而导致产生耐药的情形。在人群对流感普遍易感,其人口构成、生活状态和免疫水平也基本保存稳定、气候条件也没有很大的变化的情况下,流感的季节性高峰主要是受到甲型流感病毒适应性变异的影响。更多还原

【Abstract】 Influenza is one of the most important respiratory infections threatened the human health for a long time, epidemic or pandemic of influenza is based on antigenic drift and antigenic shift resulted from genetic diversity and reassortment. Despite the recent study for genetic diversity, many aspects of the evolutionary and epidemiological dynamics of influenza A virus remained opaque. In particular, there had been no rigorous measurement of viral diversity across time and among subtypes. The clock-like consistency of the winter incidence peaks of influenza virus represented one of the strongest examples of seasonality in infectious disease. However, the reasons that human influenza epidemics arise and then peak at winter in temperate regions of the northern hemispheres were unknown. Various theories had been proposed to explain which factor could help to shape influenza seasonality that remain largely untested and there was lack of study concern about correlation between genetic diversity and seasonality in influenza A. To help to resolve these issues, we quantify diversity of influenza A virus across time and among subtypes to explore the correlation using reasonable epidemiological methods and bioinformatics methods, it including five main contents as follow.1. Appropriate surveillance spot and website were founded in Shanghai area to collect influenza-like cases report in long period. The seasonal epidemic peak of influenza incident was analyzed by using time-series analysis method. It was revealed that there were 2 seasonal peaks of influenza-like cases in Shanghai with a consistent 6-month interval. It was different from the peek of influenza-like cases in northern China which only had one winter peak with 12-month interval.2. A/H3N2 and A/H1N1 isolations sampled from surveillance system and that was illustrative of large populations in Shanghai regions were selected and complete genome sequence was detected for the Hemagglutinin (HA), Neuraminidase (NA) and basic polymerase 2 (PB2) gene. The genetic diversity across time and among subtypes of three main segments was measured by employing Bayesian MCMC analyses. The evolutionary dynamics of influenza A virus main 3 segment was investigated comprehensively combined with concern epidemiological data. This analysis demonstrated that the genetic diversity for two different subtypes of influenza virus A was variance. In general, evolutionary speed of A/H3N2 was quicker than A/H1N1. The evolutionary speed of HA was faster than other and PB2’s was the lowest in same subtype, which was connected with the adaptive diversity of influenza A virus for escaping host’s immunity. The absolute amount of genetic diversity of A/H3N2 was higher than A/H1N1 in most study period. The amount of A/H1N1 was less 1/3 than the amount of A/H3N2 even though in its epidemiological dominant. This result demonstrated that A/H1N1 genetic diverse was more stable than A/H3N2 so it could not breakthrough human immunity shield and the pressure from A/H3N2. The high absolute amount of genetic diversity did not indicate that the improvement of transmission ability, which should combine the data of theory infection population result in genetic diversity and comprehensively assessment and then could get the more accurate conclusion.3. Typical influenza A virus was selected to find out resistant variant for oseltamivir carboxylate known as an effective drug to influenza. It could be indirectly demonstrated that genetic diversity had induced key point antigenic diversity. The result indicated that genetic diversity did not result in essential antigenic change, point mutation and adaptive diversity were key characters in genetic diversity.4. Sera from poultry associate workers were selected and examined to explore the potential infectious path from non-human host influenza A virus to human and investigate sensitivity of human infected non-human host influenza A virus. It was demonstrated that people in study area were all sensitive to non-human host influenza A virus. Hemagglutinin inhibitor antibody against poultry influenza virus subtype H9 had been found in poultry exposure and non-poultry exposure population. The antibody positive rate was 17.9% in poultry exposure worker and 2.6% in non-poultry exposure population. Comparing with non-exposure population, poultry exposure population had an adjusted odds ration (OR) of 3.392. The distribution of hemagglutinin inhibitor titer against influenza A virus subtypes H1N1 and H3N2 was no different between two groups, but hemagglutinin inhibitor titer was high within two population. If the seasonal influenza vaccine did not taken, especially for subtype H3N2 that had higher absolute amount genetic diversity, the antibody positive rate was a 30%.5. In this study, we explored the correlation between seasonality and genetic diversity in influenza A virus and gained some knowledge for the influenza prevention and control. The genetic diversity of influenza A virus had only one visible peak appeared in each winter. It was different from influenza-like case that had two peaks and its winter-spring peak went through calendar year. These results showed that influenza A virus adaptive diversity was forced under the host immunity pressure and induced a new epidemic.Our study suggested that there was seasonal epidemic of influenza incident in Shanghai area, stochastic point mutation and adaptive diversity were key determinants of short-term evolution in influenza A virus, there were not manifest phenotype change that resulted in drug-resistant strain. Seasonality of influenza A virus was mainly influenced by adaptive diversity under the circumstances that climate didn’t change frequently, population generally sensitive to influenza virus and its composition, life style and immunity level basically retained stable, etc.更多还原