山东省人感染H7N9禽流感流行病学与病毒全基因序列分析

发布时间：2018-08-02 15:58

【摘要】：研究背景人感染禽流感是禽流感病毒跨种传播引起的人类疾病。继H5N1、H9N2、 H7N7、H7N2、H7N3和H10N8禽流感病毒感染人类之后,2013年2月我国华东地区首次出现人感染新型H7N9亚型禽流感病例,因其引起的临床重症及高病死率,迅速引起全球关注。虽然病例呈散发态势,但国内已有部分省份数起家庭聚集性病例的报告,其流行的潜力,依然是严重的公共卫生问题。已确诊的H7N9禽流感病例中80%以上有禽或禽类相关环境(活禽市场)暴露史,传播途径主要是由禽到人的传播。若能在人感染H7N9禽流感疫情流行的早期及时发现被H7N9禽流感病毒污染的场所,并采取对应的防控措施如关闭活禽市场等,将最大可能减少病例的发生,达到有效的防控效果。禽流感病毒要突破种间屏障感染人必须发生受体结合偏好性改变,这种偏好性取决于病毒HA受体结合位点的关键氨基酸,病毒重要蛋白NA、聚合酶PB1/PB2/PA等关键功能位点上氨基酸的差异是决定病毒毒力和跨种传播的分子基础。目前抗流感病毒的药物主要为针对NA的神经氨酸酶抑制剂和M2离子通道抑制剂,这两种蛋白耐药位点关键氨基酸突变会导致耐药毒株的出现。本研究通过山东省2013年至2015年7例人感染H7N9禽流感病例流行病学调查资料和疫情发生地外环境污染状况调查资料,分析病例发病特点以及感染来源,寻找防控重点；通过毒株遗传进化及关键氨基酸位点分析,掌握毒株进化特征及其致病性,指导早期临床用药,也为进一步阐明H7N9禽流感病的致病机制提供科学依据。研究目的1、通过H7N9禽流感确诊病例个案调查资料,分析病例流行病学特点；对家庭聚集性病例的感染来源、传播方式和防控措施进行分析,为有效防控人感染H7N9禽流感疫情提供科学依据；2、通过对疫情发生地周围活禽市场及家禽养殖场的环境标本的调查资料分析,初步探讨外环境H7N9禽流感病毒污染情况,寻找防控重点,对早期发现、预防疫情的发生提供参考；3、通过病毒遗传进化及关键氨基酸位点分析,研究病毒来源、基因组特征以及相互间的分子差异,掌握山东省H7N9禽流感病毒进化特征及其致病性,并对早期临床用药进行指导,为进一步阐明H7N9禽流感病的致病机制提供科学依据。研究方法1、描述性流行病学：对病例流行病学资料整理分析,描述病例流行病学特征和发病特点；对疫情发生地周围活禽市场及家禽养殖场H7N9禽流感病毒污染状况调查资料整理分析,描述外环境H7N9禽流感病毒污染情况。2、禽流感病毒核酸提取与检测：应用实时荧光定量PCR对标本中H7N9禽流感病毒核酸进行检测,Ct值≤40.0的样本为阳性。3、禽流感病毒分离：对7例病人深部呼吸道标本及58份环境H7N9阳性标本用SPF鸡胚进行病毒分离,并鉴定。4、遗传进化及关键氨基酸位点分析：基因测序由国家流感中心帮助完成。利用NCBI提供的BLAST程序进行序列比对；分离株间8基因片段同源性使用DNAStar软件中MegAlign程序进行多序列同源性计算；使用Mega6.0软件进行基因进化树的建立；在ExPASy网站PROSITE数据库对HA和NA蛋白N-糖基化位点进行预测。结果1、人感染H7N9禽流感病例流行病学特征：(1)山东省7例人感染H7N9禽流感病例发病季节在冬春季(每年12月至次年5月)；病例以50岁以上中老年男性人群为主；(2)病例从暴露到发病最长时间间隔为9天,最短的每天都暴露,潜伏期为5天；从发病到确诊时间间隔1-16天,时间间隔中位数为7天；(3)山东省确诊的H7N9禽流感病例感染来源主要是禽暴露或者访问活禽市场；(4)2起家庭聚集性疫情,其中1起有足够的证据证明H7N9禽流感病毒的人间传播,但传播能力有限。2、H7N9环境污染状况：(1)山东省检出的H7N9禽流感病毒均来源于活禽市场,鸡养殖场禽类外环境标本中均未检出；(2)枣庄、烟台和泰安三地活禽市场中H7N9禽流感病毒检出率为12.8%,同时检测到H7N9、H5和H9亚型禽流感病毒。3、山东省H7N9分离株遗传进化与关键氨基酸位点分析：(1)2013-2015年分离的H7N9禽流感病毒与同期国内江西、浙江、江苏和湖南流行的H7N9禽流感病毒高度同源,同源性为99%-100%；本土病例分离到的病毒与活禽市场环境中的病毒基因高度同源；活禽市场环境中或病例的病毒基因与邻近省份病毒基因有较高的相似性；(2)山东省首起疫情分离的3株H7N9禽流感病毒H7血凝素基因来源于浙江鸭H7N3禽流感病毒的HA基因,NA基因来源于韩国野鸟的H7N9禽流感病毒,其余6个基因片段均来源于中国H9N2禽流感病毒；(3)2013-2015年H7N9禽流感病毒基因不断发生进化,并分化成不同的分支,部分H7N9禽流感病毒的6个内部基因片段与H9N2病毒发生了重组；(4)本研究涉及的15个毒力相关位点中,有7个毒力增强位点；山东省5株人感染H7N9禽流感毒株均对神经氨酸酶抑制剂仍然敏感,但耐离子通道抑制剂类药物。结论1、山东省病例季节分布以冬春季为主,中老年男性人群可能是该病的危险人群,传播途径为禽到人或环境(活禽市场/宰杀点)到人,存在有限的人与人的传播；2、活禽市场是人感染禽流感病毒的主要来源地,活禽市场外环境中存在多种亚型的禽流感病毒,不排除发生重组的可能性；3、山东省存在多个起源的H7N9禽流感病毒,H7N9禽流感病毒在传播中不断发生变异和进化,并分化成不同的小分支。山东省分离到的H7N9禽流感病毒来源于当地活禽市场,当地活禽市场的H7N9禽流感病毒来源于长三角地区。目前尚未获得经飞沫传播的充分条件,但同时病毒正在向适合感染哺乳类动物的方向发展。H7N9病毒尚未对神经氨酸酶抑制剂类药物(如达菲)产生耐药性,在病例发生早期使用达菲进行治疗可能减少重症发生、缩短病程、提高治愈率。
[Abstract]:Background human infection of avian influenza is a human disease caused by the spread of avian influenza virus. Following H5N1, H9N2, H7N7, H7N2, H7N3 and H10N8 avian influenza virus infection, the first human infection of the new avian influenza avian influenza cases in eastern China in February 2013 has been caused by the severe clinical severity and high mortality. Note. Although the cases are sporadic, there are reports of family aggregation cases in some provinces in China, the potential of which is still a serious public health problem. More than 80% of the confirmed H7N9 avian influenza cases have the exposure history of poultry or poultry related environment (live poultry market), and the transmission routes are mainly from poultry to human transmission. The early detection of the epidemic of H7N9 avian influenza epidemic and the timely detection of the sites contaminated by the H7N9 avian influenza virus and the corresponding prevention and control measures, such as closing the live poultry market, will most likely reduce the occurrence of cases and achieve effective prevention and control effects. This preference depends on the key amino acids of the binding site of the virus HA receptor, the difference in amino acids on the key functional sites, such as the viral important protein NA and the polymerase PB1/PB2/PA, is the molecular basis for determining the virulence and trans species transmission of the virus. The current anti influenza virus drugs are mainly anti NA inhibitors of neuraminidase and the M2 ion channel. The key amino acid mutation of these two protein resistance loci could lead to the emergence of drug resistant strains. This study analyzed the epidemiological investigation data of 7 cases of H7N9 avian influenza infection from 2013 to 2015 in Shandong province and the investigation data of the situation of the outbreak of the epidemic situation, analyzed the characteristics of the disease and the source of the infection, and looked for the key point of prevention and control. Through the genetic evolution and analysis of key amino acid sites, the evolutionary characteristics and pathogenicity of the strains were mastered, the early clinical medication was guided, and the scientific basis for further clarifying the pathogenesis of H7N9 avian influenza was provided. Objective 1, the epidemiological characteristics of cases were analyzed through the cases of H7N9 avian influenza, and the epidemiological characteristics of cases were analyzed. The source of infection, the mode of transmission and the prevention and control measures were analyzed to provide a scientific basis for the prevention and control of human infection of H7N9 avian influenza. 2, through the analysis of the environmental specimens in the live poultry market and poultry farms around the outbreak area, the preliminary study of the environment of the H7N9 avian influenza virus in the external environment was conducted to find the prevention and control of the virus. The key point is to provide reference for early detection and prevention of the occurrence of the epidemic. 3, through the genetic evolution of the virus and analysis of the key amino acid sites, the origin of the virus, the characteristics of the genome and the molecular differences between each other are studied, and the evolution characteristics and pathogenicity of the H7N9 avian influenza virus in Shandong province are mastered, and the early clinical medication is guided to further clarify the H A scientific basis for the pathogenesis of 7N9 avian influenza disease was provided. 1, descriptive epidemiology: an analysis of the epidemiological data of cases, the description of the epidemiological characteristics and the characteristics of the cases, the analysis of the contamination status of the H7N9 avian influenza virus in the live poultry market and poultry farm, and the description of the outer ring. H7N9 avian influenza virus contamination situation.2, avian influenza virus nucleic acid extraction and detection: using real-time fluorescent quantitative PCR to detect H7N9 avian influenza virus nucleic acid, Ct value less than 40 samples were positive.3, avian influenza virus isolation: 7 cases of deep respiratory tract specimens and 58 environmental H7N9 positive specimens were used to carry out the virus of SPF chicken. Separation, and identification of.4, genetic evolution and analysis of key amino acid sites: gene sequencing was completed by the National Influenza Center. The BLAST program provided by the NCBI was used for sequence alignment; the homology of the 8 gene fragment between isolated strains was calculated by the MegAlign program in the DNAStar software, and the Mega6.0 software was used to carry out the gene entry. The establishment of the tree and the prediction of the HA and NA protein N- glycosylation sites on the ExPASy PROSITE database. Results 1, the epidemiological characteristics of human infected H7N9 avian influenza cases were: (1) 7 cases of human infection with H7N9 avian influenza in Shandong province were in winter and spring (from December to May); the cases were dominated by middle-aged and elderly men over 50 years old; (2) the longest time interval from exposure to onset was 9 days, the shortest day was exposed and the incubation period was 5 days; the interval from onset to diagnosis was 1-16 days, the median of time interval was 7 days; (3) the main source of H7N9 avian influenza case infection in Shandong province was avian exposure or visitors to live poultry market; (4) 2 family aggregated epidemic situation, of which 1 There is sufficient evidence to prove the spread of H7N9 avian influenza virus, but the transmission capacity is limited.2, H7N9 environmental pollution status: (1) the H7N9 avian influenza virus detected in Shandong province all originate from the live poultry market, and the chicken breeding farm outside the poultry environment specimens are not detected; (2) the detection of H7N9 avian influenza virus in the live poultry market in three places in Zaozhuang, Yantai and Tai'an. The rate was 12.8%, and H7N9, H5 and H9 subtype avian influenza virus.3 were detected. The genetic evolution and key amino acid site analysis of H7N9 isolates in Shandong province were analyzed. (1) the 2013-2015 year isolated H7N9 avian influenza virus was highly homologous to the prevalent H7N9 avian influenza virus in Jiangxi, Zhejiang, Jiangsu and Hunan, and the homology was 99%-100%; local cases were separated. The virus is highly homologous to the virus gene in the live bird market environment; the virus gene in the live bird market environment or the case has a high similarity with the adjacent province virus gene; (2) the first H7N9 avian influenza H7 hemagglutinin gene from Shandong province is derived from the HA gene of the H7N3 avian influenza virus of Zhejiang duck and the NA gene source. In South Korea, the H7N9 avian influenza virus of wild birds, the remaining 6 genes are derived from China's H9N2 avian influenza virus; (3) 2013-2015 years of H7N9 avian influenza virus gene constantly evolved, and differentiated into different branches, some of the 6 internal gene fragments of the H7N9 avian influenza virus were reorganized with the H9N2 virus; (4) 15 poison involved in this study. Among the force related loci, there are 7 virulence enhancement sites, and 5 strains of human infected H7N9 avian influenza in Shandong province are still sensitive to neuraminidase inhibitors, but they are resistant to ion channel inhibitors. Conclusion 1, the seasonal distribution of the cases in Shandong province is mainly in winter and spring, and the middle aged and old men can be the dangerous population of the disease and the transmission route is bird to human. Or the environment (live poultry market / slaughtering point) to people, there is limited human and human transmission; 2, live poultry market is the main source of human infection of avian influenza virus, there are many subtypes of avian influenza virus in the outside environment of live poultry, and the possibility of reorganization does not exclude; and 3, there are several H7N9 avian influenza viruses, H7N9 avian influenza, in Shandong province. The H7N9 avian influenza virus isolated from Shandong province is derived from the local live poultry market, and the H7N9 avian influenza virus in the local live poultry market is derived from the Yangtze River Delta region. The development of.H7N9 virus in dairy animals has not yet produced resistance to neuraminidase inhibitors, such as Tamiflu. Early use of Tamiflu for treatment may reduce the incidence of severe disease, shorten the course of the disease, and improve the cure rate.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R511.7

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