【作者】 朱光剑；

【导师】 张树义；

【作者基本信息】 华东师范大学 ， 生态学， 2012， 博士

【摘要】 禽流感(Avian influenza, AI)是由正黏病毒科(Orthomyxoviridae)、流感病毒属(Influenzavirus)、甲型流感病毒引起的禽类的感染和/或疾病综合征,被国际兽医局确定为甲型烈性传染病。自1878年首次发现以来,已经造成全球范围内的多次爆发,感染的宿主范围不断扩大,已经不仅仅能够感染家禽、野鸟和部分哺乳动物,甚至在1997年,香港发生了人感染H5N1禽流感病毒并造成死亡的事件,严重威胁公共卫生健康,给公众生命财产带来了巨大的损失。禽流感病毒亚型众多,不同毒株间毒力差异很大,并不是所有亚型都能引起禽流感的爆发和流行,至今已发现的甲型流感病毒根据血凝素(HA)和和神经氨酸酶(NA)抗原的差异,将血凝素(HA)分为16个亚型(H1-H16),神经氨酸酶(NA)分为9个亚型(N1-N9), H9N2亚型禽流感病毒广泛分布于禽类、野鸟和一些哺乳动物之中,也能够感染人类,其中野鸟被认为是H9N2亚型禽流感病毒的自然宿主,H9N2亚型禽流感病毒广泛存在变异重组现象,对养殖产业和公众生命财产安全具有极大的威胁,被国际卫生组织(WHO)认为是潜在的、可能引起下一轮爆发的禽流感病毒,基于以上原因,本研究中对分离到的17株病毒中11株H9N2病毒的分子生物学特性、基因组序列和遗传进化关系进行了分析,旨在从分子水平上了解我国南方H9N2流感毒株的变异情况和导致其不同生物学特性的原因,研究其可能的来源及分子进化路径。在国内南方流行病调查期间,我们从野生赤颈鸭(Anas penelope)、雉鸡(Phasianus colchicus)、斑嘴鸭(Anas poecilorhyncha)和绿头鸭(Anas platyrhynchos)的拭子样品中分离并鉴定到17株H9N2亚型禽流感病毒,分别命名为：A/Anas penelope/Shanghai/23/11(H9N2), A/Phasianus colchicus/Jiangxi/30/11(H9N2), A/Phasianus colchicus/Jiangxi/31/11(H9N2), A/Anas poecilorhyncha/Jiangxi/32/11(H9N2), A/Anas poecilorhyncha/Jiangxi/33/11(H9N2), A/Anas poecilorhyncha/Jiangxi/34/11(H9N2) A/Anas poecilorhyncha/Jiangxi/35/11(H9N2), A/Anas poecilorhyncha/Jiangxi/36/11(H9N2)A/Anas poecilorhyncha/Jiangxi/37/11(H9N2), A/Anas platyrhynchos/Jiangxi/38/11(H9N2), A/Anas platyrhynchos/Jiangxi/39/11(H9N2), A/Anas platyrhynchos/Jiangxi/41/11(H9N2), A/Anas platyrhynchos/Jiangxi/43/11(H9N2), A/Anas platyrhynchos/Jiangxi/46/11(H9N2)。通过设计特异引物进行扩增、克隆和测序,得到17株分离株中11株的PB2、PB1、 PA、HA、NP、NA、M和NS8个片段的序列信息,进而对8个基因片段的分子进化关系进行了分析,分析结果表明这11株病毒和2009年在香港分离的2株人源H9N2病毒(A/HongKong/35820/2009和A/HongKong/33982/2009)具有很高的氨基酸同源性,具有相同的祖先来源,而且,该11株H9N2病毒为G1-like型、Ck/Bei94-like型和Y439-like型病毒的三重重组病毒,其中,NA基因来源于Ck/Bei94-like型H9N2病毒,PA基因来源于Y439-like型H9N2病毒,而其余6个基因来源于G1-like型H9N2病毒。在小鼠致病性研究实验中,用分离到的H9N2病毒(A/Anas platyrhynchos/Jiangxi/39/11)感染无特异病原体的小鼠(SPF),组织切片结果和Real-time PCR实验的结果表明我们所获得的H9N2亚型病毒能够感染小鼠并在小鼠的肺部和气管组织中进行自由复制,小鼠在感染H9N2病毒之后的3-6天内,引起20-40%的死亡率。表明野生鸭类作为宿主,在H9N2病毒重组中起到了重要作用,经过重组的H9N2病毒具有潜在的感染、致死哺乳动物的可能性,因此,必须对该型H9N2的防治引起广泛重视,加强对H9N2病毒变异演化的检测,防止造成新的禽流感亚型的爆发和流行,避免对公众健康和人民生命财产造成损失。更多还原

【Abstract】 Avian influenza is a kind of epidemic infectious disease which is caused by Orthomyxoviridae, type A avian influenza virus. It is listed as a fulminating infectious disease, type I by the World Organisation for Animal Health. Avian influenza viruses have broken out many times in a worldwide range since it was first detected in1878. It not only infected domestic poultry, wild birds, and some species of mammals (such as pigs), but also subtype H5N1infected humans in Hong Kong in1997, which resulted in the deaths of several people. It has caused huge economic losses to poultry industries as well as damages to human health.In total, there are16subtypes of the Hemagglutini gene (HA1-HA16) and9subtypes of the Neuramindase gene (NA1-NA9). Lethality in different subtypes of avian influenza viruses is very different; only some of them have caused outbreaks in birds and domestic poultry. The H9N2subtype of avian influenza virus is prevalent in many species. Its natural hosts vary among domestic fowls, many kinds of wild birds, some kinds of mammalians, and even human. Wild birds are considered to be the natural reservoirs for avian influenza A viruses(禽流感).This subtype is also regarded by the World Health Organization (WHO) as the next potential avian influenza subtype that may have high prevalence in the future, and which may bring huge damages upon global economies and public health. Based on those reasons, we discuss the characterization, sequence data and evolution of those isolated H9N2viruses in our study.During active influenza surveillance in southeast China, we isolated and characterized17H9N2viruses from four species of ducks, Anas penelope, Phasianus colchicus, Anas poecilorhyncha and Anas platyrhynchos, and named as:A/Anas penelope/Shanghai/23/11(H9N2), A/Phasianus colchicus/Jiangxi/30/11(H9N2), A/Phasianus colchicus/Jiangxi/31/11(H9N2), A/Anas poecilorhyncha/Jiangxi/32/11(H9N2), A/Anas poecilorhyncha/Jiangxi/33/11(H9N2),A/Anas poecilorhyncha/Jiangxi/34/11(H9N2), A/Anas poecilorhyncha/Jiangxi/35/11(H9N2),A/Anas poecilorhyncha/Jiangxi/36/11(H9N2), A/Anas poecilorhyncha/Jiangxi/37/11(H9N2), A/Anas platyrhynchos/Jiangxi/38/11(H9N2), A/Anas platyrhynchos/Jiangxi/39/11(H9N2), A/Anas platyrhynchos/Jiangxi/40/11(H9N2), A/Anas platyrhynchos/Jiangxi/41/11(H9N2), A/Anas platyrhynchos/Jiangxi/42/11(H9N2), A/Anas platyrhynchos/Jiangxi/43/11(H9N2), A/Anas platyrhynchos/Jiangxi/45/11(H9N2), A/Anas platyrhynchos/Jiangxi/46/11(H9N2).All8segments (HA, NA, M, NP, PB1, PB2, NS and PA) forming11of17isolated avian viruses were amplified, cloned and sequenced successfully. Phylogenetic analysis of the whole genome indicates that all isolates share high homology with each other and with two human H9N2isolates (A/HongKong/35820/2009and A/HongKong/33982/2009). All eleven isolates appear to belong to a novel reassortant H9N2genotype of G1-like viruses, containing Ck/Bei-like NA genes, Y439-like PA genes and six other G1-like genes (HA, M, NP, PB1, PB2, NS and PA).We chose one isolated H9N2virus (A/Anas platyrhynchos/Jiangxi/39/11) based on sequence data to infect specific pathogen free mice (BALB/c) to assess the pathogenicity of these isolates in mice. Data from histological section and real-time PCR demonstrated that these viruses could replicate efficiently in the lungs and tracheae of BALB/c mice and caused mortality in20-40%of infected groups after3-6days. Our results confirm that wild ducks play a role as reservoir hosts for the reassortant H9N2genotype. Because the results also suggest that these viruses are capable of establishing lethal mammalian infection, urgent attention should be paid to surveillance for these strains in humans.更多还原